Camille Lefebvre

Dan Ander- Sen

Larry Baack

Guillaume Calafat

Clélia Coret

Michel-Pierre Detalle

Violette Diaz

Jean Charles Ducène

Torbjørn Ødegaard

Joachim Östlund

Margarete M'hamed Oualdi

Enaam Ritz- Kowsky

Robin Sharfeddine

Rafaël Seignobos

Elena Thiebaut

Ismaïl Vezzadini

Toby Warscheid

Benedetta Green

Mercedes García-Arenal

Gerard Wiegers

Samuel Pallache

John-Paul Ghobrial

The Life of a Text: Carsten Niebuhr and ʿAbd al-Raḥmān Aġa's Das innere von Afrika

In July 1772, ʿAbd al-Raḥmān Aġa, a Tripolitan ambassador, and his entourage including two African slaves arrived in Copenhagen to renegotiate peace with the Nordic states of Denmark and Sweden. Muslim missions were a common and fairly regular phenomenon of the 18TH century Mediterranean cross-cultural diplomacy.1 Cases of Ottoman, Persian, Moroccan or Maghreb envoys coming to Europe to negotiate conditions for the maintenance of peace against a payment for their ruler were not uncommon.2 But there is more than this to the story of ʿAbd al-Raḥmān Aġa's four-month stay in Copenhagen. In the course of his trip, ʿAbd al-Raḥmān Aġa met and befriended the last living member of the Danish expedition to Arabia, Carsten Niebuhr. The conversations between the scientist, the Tripolitan ambassador, and his two African slaves that summer were transformed twenty years later into a printed account comprising five texts, which constitute one of the rare sources about the Central Sahara and Sahel in the second half of the 18TH century.3

This paper is a story of connectivity, of circulation of information, and of the cross-cultural co-production of knowledge. Connectivity in the early

co-production. The chapter examines the protagonists' previous individual experiences and looks at how these experiences enabled them to understand each other and discuss common interests. Moraes Farias analysed how Barth's views on African history took shape by considering the material conditions of Barth's travels -what copies of manuscripts he had access to in Gwandu; how he was hosted in Timbuktu; who he was able (or unable) to talk to; how he moved; where he could or could not go; and what were his language skills.6 Similarly, this chapter considers how communication occurred and information was exchanged in a particular African-European encounter.

What follows is necessarily an incomplete tale, because of the unbalanced nature of sources which consists, on the one hand, of abundant European diplomatic sources and, on the other hand, of almost inexistent data on African slaves. Carsten Niebuhr and ʿAbd al-Raḥmān Aġa's encounter happened at the intersection of several documentary traditions, including Mediterranean diplomatic correspondence, commercial record-keeping cultures, and multiple genres of scientific production from German orientalism to Tripolitan Arabic historical literature. Each tradition produced accounts in various languages (including Arabic, German, French, Italian, Lingua franca, and Danish) and various scripts such as Danish and German Fraktur and kurrentschrift (typographic script and hand-writing) or classical Arabic. Furthermore, some aspects of this encounter are forever lost to us because we cannot access directly the words of the two African slaves. There are no sources reporting verbatim ʿAbd al-Raḥmān Aġa's words and the testimonies of his slaves, which are always mediated through the reports of others. But by reading Niebuhr's' texts against the grain and looking for explicit references to information provided by his African informants, we can identify the contributions of ʿAbd al-Raḥmān Aġa and his slaves. We can thus attempt to interpret the political and intellectual projects of an African diplomat and his dependants travelling across the Mediterranean in the 1770s.

A Diplomatic Context

In the eighteenth century, in order to trade safely in the Mediterranean Sea, to guarantee the control of piracy and the security of their ships, European LEFEBvre states negotiated peace treaties with Maghrebi states.7 Diplomatic relations between European countries and the regencies of the Maghreb were organized by treaties, sealed and ratified by the two parties. This direct negotiation confirmed the relative autonomy of the regencies from Istanbul, even though Ottoman suzerainty was acknowledged in the treaties.8 But this new diplomatic sovereignty of the Maghrebi states was diminished by increasing military imbalance with the European states in the second half of the century. These treaties limited corsair operations in the co-signatory states, provided European merchants with trade outlets in the Maghreb; and supplied Maghreb regencies with cash, European products and trading opportunities with Europe.9 It is in this context, that peace between Denmark and Tripoli had been signed in 1757, followed by a first Tripolitan embassy in the Nordic countries. Maghreb rulers sent embassies to Europe regularly for many different official reasons, such as the enforcement of bilateral peace treaties, signing of trade agreements, ransoming of captives or participation in various ceremonial events (coronation, baptism, etc.).10

The idea of a new Tripolitan embassy in Denmark was first raised at the eve of the new regime in Copenhagen in 1770. In theory, from the 17th century onwards, the validity of treaties with European states was permanent and not anymore tied to the individual Monarch who signed the treaty in question. But in North Africa during 18TH century it was still current practice to consider the death of the beneficiary as putting an end to the validity of the treaty.11 In fact, in 18TH century Maghreb and Morocco, Islamic jurists saw diplomacy with non-Muslims as an approved instrument of policy as long as the agreements reached were temporary and short term.12 In the European context, these embassies were financed by European monarchies, which tried to avoid incurring excessive expenditures in connection to diplomatic missions. Official correspondence between the courts and their consular representatives in Mediterranean coastal countries reveal constant attempts to prevent the organisation of diplomatic embassies. Indeed, the acting Danish consul in Tripoli, the trader Lochner, tried to prevent ʿAbd al-Raḥmān Aġa's departure for Copenhagen for over two years.13

The purpose of the embassy of Tripoli seemed to have been both political and economic. Since the beginning of the reign of the Qaramānlī dynasty in 1711, Tripoli's politics had been characterized by increasing autonomy from Istanbul and a strategy of territorial unification, leading to a series of campaigns towards the interior of the country.14 In this context, diplomatic missions to Europe can be seen as part of a political strategy of independence and sovereignty, which started with embassies to Paris in 1719 and London in 1728.15 Dealing directly with European chancelleries was a way to be recognized as a sovereign state independent from Istanbul, even if the legal status of these relations was ambiguous. It was also a way for the Pasha to strengthen his power in an unstable internal political environment. During Ali Pasha's reign the influence of Tripoli's separatist factions had increased so much that it challenged the authority of the Diwan, thereby creating a tense situation.16 In addition, a series of bad harvests and droughts since 1765 had led to an economic crisis culminating in 1769.17 In this same year Russian Empress Catherine II SENT a fleet in the Mediterranean to open up a second naval front against the Ottomans. The impact on the Regency's trade was disastrous, as Russians considered Tripoli an Ottoman province and did not hesitate to stop French, English, and other neutral ships from trading with them. In his despatch of April 1771, Lochner described this critical situation, with trade and navigation almost completely ʿAbd al-Raḥmān Aġa had already twice accomplished the pilgrimage to Mecca, once in 1760 and then again with his family in 1768.26 Like the Pasha, his ministers, the rāyis and most of the high-ranking officials during the Qaramānlī period in Tripoli, he was also an active trader with extensive foreign connections.27 As a merchant, he was involved in the Caravane maritime and rented European ships to carry goods and passengers between ports in Europe, the Ottoman Empire and North Africa.28 From the charter contracts signed by him, copies of which are kept in London and Nantes, we can see that he traded with British, French, Italian and Spanish captains to ship goods to Naples, Marseille, Leghorn, Smyrna, Venice, Malta, Tunis, Bône Benghazi, Alexandria, Istanbul and the Levantine ports.29 He traded a broad range of goods including grain (corn, bran, barley), plants (beans, alizaris/ʿurūq fuwwa, barils or prickly saltwort), cattle, fabrics, furniture and also African slaves. One third of his contracts contain a clause discharging the captain's liability in case of death of the 'negroes,'30 proving he was involved in the slave trade, which explains his relations with traders in the interior of Africa.31 The diversity of products and outlets in his trade is consistent with the practice of other great Tripolitan traders of this period, such as Ḥasan al-Faqīh Ḥasan.32 It would appear that his diplomatic career also helped him expand his business, as he used his missions to Europe to load ships and to try to avoid paying taxes.33 As was usual for this kind of mission, the Ambassador was not travelling alone. He had with him a retinue including his nephew Ḥağği Musṭafā acting as his secretary, a cook and two black men designated sometimes as his valets, sometimes as his slaves, accompanying him on his journeys. ʿAbd al-Raḥmān's life was intertwined with the lives of his black servants or slaves. In all his travels as a diplomat or during his private trips, for example in his second pilgrimage to Mecca, or in his daily life in Tripoli, he and his second wife always had with them several African slaves.34 From Miss Tully's narrative this seems to have been a customary practice in Tripoli's high society. ʿAbd al-Raḥmān also spoke some Kanuri (the language from Bornu), which he had learned from his nanny as a child and from playing with Kanuri children in Tripoli.35 When his first wife died, he decided for several years not to marry again but instead to take a black concubine; he said he thought this would be better for his children.36 In the archives of his second stay in Copenhagen, there is a list detailing his retinue, in which we see three individuals designated by the term neger (black): the cook Hach Belgcer, Hach Marrub, and Hach Barka. Interestingly all three are indicated as having done the pilgrimage which, in all likelihood, they would have accomplished by accompanying ʿAbd al-Raḥmān during his second trip which was made with a large party. ʿAbd al-Rahman and his family members therefore seem to share their existence with black slaves, and trusted slaves accompanied them everywhere and participated in all of their daily lives.

The archives are almost completely quiet about the two sub-Saharan Africans in Copenhagen in 1772. What we know from them we learn from Carsten Niebuhr. Without naming them he describes two young men -one from Timbuktu, and the second born near the Niger River speaking Afnu/Hausa as his national language.37 Niebuhr describes him as a 'young well-grown Kafr with thick lips and a flat nose' , who 'had on each cheek several large scars from stab wounds. He himself and his master assured me that it was used to recognize

Everyday Interaction and Communication

In 18TH-century Copenhagen, as elsewhere in Europe, the reception of Maghreb or foreign diplomats followed a strictly codified protocol. The ambassador was accompanied at all times by an interpreter, his accommodation was arranged by the king, and social events were organised for his entertainment. The visit culminated in the envoy's audience with the king. As was customary in early modern diplomacy, daily activities of the ambassador were recorded by the interpreter in a journal, written in this case for the Danish authorities.39 Andreas AEreboe, former consul in Algeria from 1763 to 1771, was appointed to serve as ʿAbd al-Raḥmān's interpreter because he was supposed to be fluent in Arabic.

Carsten Niebuhr and ʿAbd al-Raḥmān's first meeting is described by Niebuhr's son in his biography of his father. According to Barthold Georg Niebuhr, AEreboe's Arabic skills were very weak and ʿAbd al-Raḥmān rapidly felt the need to speak with other people. As Arabic-speaking people were few in Copenhagen at this time, Niebuhr was called upon to assist. He was eager to help and 'to have the opportunity to hear and speak Arabic, to indulge again in the habit so long laid aside and to make himself acquainted with a region of the Arab world that he had not himself visited.'40 Reading AEreboe's Journal, it is unlikely that it happened this way. AEreboe records the first visit of Niebuhr to the Ambassador's house on July 5th, two days after his arrival, which implies their encounter must have been programmed ahead.

In AEreboe's Journal, we see eighteen visits by Niebuhr to the ambassador's house listed between July 5th and November 26th 1772. They usually lasted half a day and most of them involved long, one-on-one discussions. Both Niebuhr and ʿAbd al-Raḥmān refer afterwards to the other as a friend. Niebuhr in the third volume of his travel account, published posthumously, recalls in a footnote coming to the ambassador's home almost every day and becoming his close caravans. In Yemen he noted differences in the language and pronunciation of the mountain dwellers and the Imam's courtiers in Sanaa, as well as in today's Iraq between the inhabitants of the city of Basra and the surrounding 'tribes. '49 In Das Innere von Afrika, Niebuhr recalls the difficulty he had in understanding the dialect of a Sheikh from Lamu (modern Kenya) with whom he had spoken in Bombay in 1763.50 He does not mention any problems in communicating with ʿAbd al-Raḥmān. Niebuhr had mainly learned Arabic on his own in situ, which helped him develop a sense for dialectal diversity which he shared with ʿAbd al-Raḥmān. As we have seen, the latter had completed the pilgrimage twice, which in those days meant long stays in the Arabian Peninsula, which probably accustomed him to the Arabic of this area. However, in a letter to Michaelis written on 30 September 1773, Niebuhr reports that while he communicated easily with the Ambassador who dealt regularly with foreign Arabs, when ʿAbd al-Raḥmān was talking with his entourage, or when they were talking together, he understood less than half of what was said.51 Most likely ʿAbd al-Raḥmān made a special effort to be understood, an attitude he would have developed as a diplomat and a merchant. He had conducted business and negotiations in Istanbul, Venice, Livorno, Marseille and Prussia, with people ranging from friends and family to strangers, such as Ottoman and European court officials or merchants and sea captains in the Mediterranean. Europe was neither strange, nor new, nor marvelous to him. In this he differed from other Maghreb ambassadors travelling for the first time in Europe.52 He spent several months in Venice and he signed letters written in Italian. The letters may have been also written by his secretary, but he was surely familiar with the language or with the Lingua franca.

Although Niebuhr had an excellent command of spoken Arabic, there is evidence in the archives that he could not read it easily. Asked repeatedly by the Danish authorities to translate letters from the Pasha, he replied that beyond the nomia propria and several insignificant words he could not understand a text, insisting on the difference between oral and written Arabic.53 Niebuhr and ʿAbd al-Raḥmān had more in common than one would be inclined to think. Both had had cross-cultural experiences, and both were accustomed to crossing boundaries of language and religion. Niebuhr had used Arabic to communicate and connect with people in Bombay, Iran, Aleppo or Sanaa during his five years of travel. ʿAbd al-Raḥmān too was accustomed to negotiating with European, Ottoman or North African traders based in Africa. Their shared experience of a long stay in Arabia certainly helped them understand each other. An anecdote told by Niebuhr shows us how much they knew about cultural differences and could find ways to act appropriately in a different cultural context.54 During his stay in Copenhagen, ʿAbd al-Raḥmān did not stand up to greet high ranking officials coming to visit him in his house. When Niebuhr noticed this, as a friend he felt entitled to tell him that Christians in Europe were gentlemen as much as 'Mohammedans' in Barbary, and that he should behave politely. ʿAbd al-Raḥmān argued that in Tripoli it would not be seen as appropriate for a person of high rank and an Ambassador as himself to stand up to receive a Christian. To this Niebuhr answered that birth was equally important in Europe and the men visiting him were of high rank just like him. Niebuhr's point was convincing. ʿAbd al-Raḥmān did not want to be seen as impolite, and thereafter he made sure that high-ranking visitors would find him standing already so that he would not have to get up for them, but still would not be considered rude. This solution was made possible by their relationship, or as Niebuhr wrote: 'Since he was assured that I was his friend, he did not take offence with this discussion.'

A Scholarly Encounter

The scholarly conversation between ʿAbd al-Raḥmān and know about the making of Das Innere von Afrika comes from Niebuhr's writings and from a draft of this work, in which he regularly mentions his sources of information.

In the introduction to a draft of Das Innere von Afrika retained in Niebuhr's personal archives in Kiel, Niebuhr describes what appears as his project and his philosophy towards informants and second-hand information.57 He argues that as Europeans would not be able to visit all parts of Africa in the near future, they should try to learn about the region from the traders coming to Cairo or from slaves in the European or American colonies, especially as some of these slaves were literate in Arabic and were 'mollahs' or traders in their native countries, and therefore good informants. People should, he said, 'make the effort to win their friendship and to let themselves be informed by them.' Not every African is competent and willing to do so, he insists, but people should constantly question those they trust and soon they will find valuable informants. This is consistent with his fieldwork methodology in Arabia and his scientific writing practices. During his journey, Niebuhr was an indefatigable and natural conversationalist.58 He preferred to work without an interpreter, and constantly interrogated all kinds of people about various subjects, from everyday life, to local names, plants, religions, political structures, or vocabularies. He verified his information with local inhabitants, regularly acknowledging his debt towards his informants.59 In the published version of Das Innere von Afrika, we see the same concern to identify sources. Niebuhr quotes ʿAbd al-Raḥmān as his source nine times, four times the Afnu/ Hausa slave and once the Timbuktu slave. Moreover, in this text's manuscript he includes two additional references -one to ʿAbd al-Raḥmān and another one to both Africans.60

Both the published text and the manuscript start with the Niger River. The discussion about the interior of Africa seems to have started around what was going to become in the next decades a major geographical question: the 57 Folio noted 1, Preliminary version Noch etwas über die mohammedanischen Freistaaten in der Barbarei, Christian-Albrechts-Universität zu Kiel. Universitätsbibliothek, Vorarbeiten zum 3. Band der Reisebeschreibung; 2, http://dibiki.ub.uni-kiel.de/viewer/resolver?u rn=urn:nbn:de:gbV:8:2-1668126 consulted in november 2016. Despite his title the drafted manuscript concerns simultaneously the articles about Maghreb namely Über die mohammedanischen Freistaaten in der Barbarei and the ones about Africa such as Das Innere von Afrika. 58 Baack,Undying Curiosity,305. 59 Ibid.,374. 60 Folio noted 21, Preliminary version Noch Etwas Über die Mohammedanischen Freistaaten in der Barbarei.

determination of the source of the Niger, its direction of flow, its course and its end. But what comes next does not seem to be specifically set by European knowledge or questions: patriotism of Africans, ' Afnu' and 'Bernu' kingdoms, religious practices in this area, scarification, languages, war and military tactics, slavery, flora, fauna, system of production, lists and descriptions of places.

If we look closely at the structure of the part of Das Innere von Afrika dedicated to central west Africa we see that it is shaped around ʿAbd al-Raḥmān's knowledge. For example the list of cities in the second half of the text is taken from ʿAbd al-Raḥmān's account, even though other sources are invoked to enlighten, enhance or question his statements. Niebuhr's interest in Africa grew as a direct consequence of his meeting with ʿAbd al-Raḥmān and the discussions they had.61 In his 1803 paper, in which he compares Horneman's book with the information he had collected from ʿAbd al-Raḥmān, Niebuhr describes a part of his work with the former. He insists on the fact that he recalls ʿAbd al-Raḥmān saying Afnu and not Asnu, and he remembers asking him to write down the name in Arabic script to check the spelling.62 In the preliminary version all place-names are written in Arabic showing, that he did so systematically. By comparing Niebuhr's use of sources in the draft manuscript with the published version, it is possible to identify patterns revealing his hierarchy of authorities. In the draft he mentions that the Timbuktu slave had spoken about a country named Flata with white inhabitants, but the Ambassador believed that these Flata weren't the same as the ones he previously mentioned.63 In the published version Niebuhr follows ʿAbd al-Raḥmān's opinion, listing two different 'Flata,' one in the Ambassador's list of cities and one connected to the information given by the Timbuktu slave, carefully adding that the slave couldn't say anything noteworthy except that they were white because he had been abducted when he was too young.64 The statement of the slave was thus cross-checked by interrogating the Ambassador to whom was given more credit. One reference to the fact that the information had been given by the 'Africans' disappeared in the published version, where the information is still quoted but without attribution.65 However, on several matters, the knowledge of the slave from Afnu/Hausa was specifically sought, as with regards to the and what he had been told, which certainly further enhanced his credibility for Niebuhr. ʿAbd al-Raḥmān's knowledge about the interior of Africa derived, as stated by Niebuhr, from his interactions as a trader with North Africans based in the interior of Africa. In the preliminary version there is a longer description of them: 'some traders of [Tripoli] spent several years there and came back rich. Others never came back because they found the country better than theirs and because justice is better served there than in white "Mohammedan" countries.'66 Niebuhr never says anything about the goods that ʿAbd al-Raḥmān and those traders exchanged, but they were most likely slaves. Regarding ʿAbd al-Raḥmān's credibility, Niebuhr notes twice that his description of things could have been influenced by his fidelity to his Pasha. This raises the issue of the projects behind the texts.

Intellectual Agendas and Writing Genres

To try and grasp the projects and cultural viewpoints embodied in this text we need, as Moraes Farias taught us, to think about Niebuhr's text on Africa as a whole and not a collection of parts. We need to understand how this text was constructed and how it integrated both Niebuhr's and ʿAbd al-Raḥmān's visions and aims.67 Only by looking at the five texts and their drafts does it become possible to identify the political and intellectual agendas that nourish them.

From the draft, it seems that Niebuhr had a project to write a piece on all the information he had collected from various sources about Africa, including what he had learned about the west coast of the Arabian Gulf, about Habbesch and the surroundings area, about the Arabs of the south east coast of Africa and the lands of the blacks.68 The various pieces were at some point scattered and published in distinct articles and chapters. In the preliminary version and in the various published papers we see two main arguments -the validity of second hand information in regions that have not been visited by Europeans and the opportunity to explore Africa with the right method. Niebuhr's approach towards scientific travel was that travellers should speak the local language, and learn about the culture and customs of the place they are visiting. For Niebuhr the planning of an exploration mission required the preliminary gathering of second-hand information, to develop a travel plan and gain at least some familiarity with the languages, religions, and topography of the areas to be visited. Tully,Narrative,[191][192][193][194] Rigsarkivet, 1770-1777 Tripolis Sager verdr De Tripolitanske gesandtskaber.

followed by a praise of his host, the Danish King, and his lineage. The Ambassador was careful about the accuracy of the details he provides: he transliterated the names and the titles, and collected the genealogy of the Danish kings.81 In this preamble, honorific titles and genealogy are mentioned to project a positive image of the European potentate reflecting back on the authority of the Ambassador and his ruler. The second part of the text describes how the Ambassador was received in Copenhagen. It is designed to impress the elite in Tripoli, insisting on the royal carriage reserved for the Ambassador, the horses for his retinue, the crowd who came to praise him and the joy of the King when he met the Ambassador, all of it making it a 'great day in scale and importance.'82 ʿAbd al-Raḥmān brought sixty printed copies of this text back to Tripoli, at a time when printing was rarely used in the Ottoman Empire and Maghreb. This text was widely circulated, discussed and admired within the court in Tripoli.83 Ahead of his time, and fifteen years before the creation of the African Association, Niebuhr's text represented Africa as an object of geographical and scientific interest. But his project of exploration of central west Africa went unheeded for years and his work on Africa remained almost unknown for centuries. By studying how he composed his texts, we can reconstruct the process through which oral and written information became intertwined and shaped his ideas; and we can reveal his perceived hierarchy of sources. These texts are the result of information circulating broadly across linguistic, geographic and cultural boundaries. This information had been gathered through the rich cross-cultural experiences of Niebuhr, ʿAbd al-Raḥmān and the two slaves. Das Innere von Africa can be summed up as a Tripolitan notable's observations on the interior of Africa, crosschecked against the testimonies of two African servants or slaves, and analyzed and put into narrative form by a German explorer and specialist of Arabia. It sheds light not only on the discourses of Tripolitan traders about the Central Sahara and Sahel but also on the answers of two sub-Saharan African slaves asked to provide information about their homeland. It is an example of co-produced knowledge across cultures, languages, and status groups, a complex text that reveals -most of all -an ongoing intellectual dialogue and collaboration between African and European authors.

of Denmark and Norway.21 Born in 1720, to an influential and re- nowned family from Tripoli, he was related to the Šaiḫ al-Bilad of the city.22

  Venice in 1763-64 to negotiate peace; in Florence in 1765 to congratulate the Grand Duke of Tuscany for his accession to the throne; and in Prussia to congratulate the Kaiser for the same reason.23 He was acting consul of Tuscany in Tripoli at the time he left for Copenhagen.24 Miss Tully who frequented ʿAbd al-Raḥmān and his family between 1783 and 1790, described him as 'a most enlightened man, having been repeatedly to the chief courts of Europe' , who was in Tripoli 'universally beloved by Christians as well as Moors, and adored by his family' .25

	stopped and starvation looming.18 That same year an epidemic decimated the Well-trained diplomat, he had already conducted embassies in Istanbul in
	slave caravan from Fezzan and the Awlad Sulayman refused to pay taxes. Fur-1746; in
	thermore, serious grain shortage was aggravated by the inability to trade with
	Levantine markets. The loss of tax revenue was seriously depleting the Regen-
	cy's revenues and as a result Ali Pasha was buried in debt.19 In this context, re-
	negotiating peace meant potentially gaining access to payments in cash from
	European Monarchies to the Regency and possibly new trade opportunities,
	such as selling salt to the Nordic countries.
	2	A Most Enlightened Man
	In the 18TH century Maghreb, being chosen as an envoy was a mark of
	distinction, and diplomatic appointment was eagerly sought. Maghrebi states
	had no permanent embassies abroad. Negotiations were carried out by special
	envoys appointed by the ruler who travelled to Europe on specific missions
	and came back as soon as they completed their assignment. However, it
	seems that in the 17TH-18TH century Maghreb, the ambassadorial function was
	professionalized. Rulers selected ambassadors who had travelled widely and
	had become familiar with the societies of the naṣārá.20 In Tripoli during the
	Qaramānlī period most Tripolitan ambassadors worked with European consuls
	in Tripoli and were sent on missions to Europe on a regular basis.
		The Barshaw's ambassador to Europe in 1772 had already travelled to Europe
	on several occasions. He designated himself as al-Sayyid al-Ḥāǧǧ ʿAbd al-
	Raḥmān Aġa ambassador (anbāšadūr) to the master (ṣāhib) of the province
	(wilāyat) 18 TNA FO-160-3, Letter from Lochner to Osten, April 13th 1771, folio 49 verso.
	13 19 14 20 21 15 22 16 17	Anne-Charles Froment de Champlagarde, Histoire Abrégée de Tripoly de Barbarie, ed. Rigsarkivet, Copenhagen, 1771-1789 Tripolis depecher 1771, file 1, Letter from Lochner to Alain Blondy (Saint-Denis: Bouchène, [1794] 2001), 27; Marc-Frederick Dyer, The Foreign Bernstorff, August 1771; The National Archives, London, FO-160-3, Letter from Lochner, Trade of Western-Libya 1750-1830 (Unpublished PhD diss., Boston University Graduate June 5th 1770, folio 46 verso. School, 1987), 252-253. Kọla Fọlayan, Tripoli During the Reign of Yusuf Pasha Qaramanli (Ife: University of Ife Matar, In the Lands of the Christians, 95-96. Press, 1979), 3; Sharfeddine, La Classe Marchande, 69; Budeeb, Zeddan, "Les Relations Di-In the Arabic one page travelogue he engraved in Copenhagen, Rigsarkivet, 1770-1777 plomatiques Entre la Régence de Tripoli et la France (1711-1832)," (unpublished PhD diss., Tripolis Sager verdr De Tripolitanske gesandtskaber. Université de Provence Aix-Marseille I, 1995), 1-2. Anonymous, "44. Unkosten bei der Gesandtschaft von Tripoli nach Schweden, 1780," Brief-Sharfeddine, La Classe Marchande, 179; Seton Dearden, A Nest of Corsairs: The Fighting wechsel meist Historischen und Politischen Inhalts, Siebender Theil, XXXVII-XLII (1780), Karamanlis of the Barbary Coast (London: John Murray Publishers, 1976), 62. 237. On his appointment as Sheikh al Bilad I diverge from Nora Lafi's analysis; Nora Lafi, Ibid., 82. Une Ville du Maghreb entre Ancien Régime et Réformes Ottomanes: Genèse des Institutions Laurent-Charles Féraud, Annales Tripolitaines (Saint-Denis: Bouchene, [1927] 2005), 250; Jean-Claude Zeltner, Tripoli, Carrefour de l'Europe et des Pays du Tchad 1500-1795 (Paris: Municipales à Tripoli de Barbarie. 1795-191, (Paris: L'Harmattan, 2002), 146.
		L'Harmattan, 1992), 242-243.

  country.'38 This sentence is a rare illustration of scarification practices in the second half of the 18TH century. Let's now look at the everyday life of ʿAbd al-Raḥmān Aġa and his party during their stay in Copenhagen in the summer 1772.

	32	Sharfeddine, La Classe Marchande, 379.
	33	TNA, FO-76/3, folio 242; Rigsarkivet, 1770-1777 Tripolis Sager verdr De Tripolitanske ge-
		sandtskaber, file 2, August 20 and 21ST reports 1773, signed Cartsen Niebuhr.
	34	Letter from September 20th 1787, Tully, Narrative, 152.
	35	Niebuhr, "Das Innere von Afrika," 982.

36 Letter from July 24 1784, Tully, Narrative, 56. 37 Niebuhr, "Das innere von Afrika," 987 and 1003; Niebuhr, "Bemerkungen," 433. his

  .41 Likewise ʿAbd al-Raḥmān in 1773, advises the Danish authorities to ask 'his friend Mister Niebuhr who speaks perfect Arabic' to translate a letter from the Pasha.42 During their meetings they read and discussed Arabic manuscripts and inscriptions, talked about geography and went together to visit the court painter Pilo and the Kunstkammer, the art collection of the Danish king.43 To entertain ʿAbd al-Raḥmān, AEreboe regularly read and translated for him the Gazettes, and parts of Niebuhr's travelogue Beschreibung von Arabien.44 Niebuhr lent him an edition of an Arabic geographer's text, certainly Ibn al-Wardī's Cosmography written around 1428, that he had brought with him from Arabia. ʿAbd al-Raḥmān was especially fascinated with his description of Denmark.45 AEreboe also saw the Ambassador write down in a notebook the names of the Ministers and the genealogy of the King of Denmark.46 Niebuhr also gave him a copy of the first volume of his narrative Reisebeschreibung and ʿAbd al-Raḥmān told him he was going to order an Arabic translation of it inTripoli.47 Discussions between Niebuhr and ʿAbd al-Raḥmān took place in Arabic. Niebuhr had started studying the language in 1758 in Gottingen with the Biblical scholar and specialist in ancient eastern languages Johann David Michaelis, while preparing for his expedition to Arabia. But he was, by his own admission, slow in making progress and it was only during his stay in Cairo at the beginning of his journey that his fluency in Arabic grew dramatically as he was using it every day in the streets.48 He left for Arabia with a basic knowledge of classical Arabic, and came back with a good command of spoken Arabic. During his years in the Middle East, he conversed in Arabic with his acquaintances. In his narrative he describes the variety of dialects he encountered travelling with

	41	Carsten Niebuhr, Carsten Niebuhrs Reisebeschreibung nach Arabien und andern umliegen-
		den Länder (Copenhagen: Nicolaus Möller, 1837), 127-128. I am thankful to Michel-Pierre
		Detalle for drawing my attention to this footnote.
	42	Rigsarkivet, 1771-1789 Tripolis depecher 1771, file 2, Letter from Lochner to Bernstorff,
		January 8th 1775.
	43	Rigsarkivet, 1770-1777 Tripolis Sager verdr De Tripolitanske gesandtskaber, AEreboe's Jour-
		nal, July 19th, August 3rd, August 11TH, September 8th.
	44	Idem., AEreboe's Journal, August 11TH, October 14TH.
	45	The manuscript is part oft he collection of Arabic manuscripts acquired by participants
		of the Arabian Journey, 1761-1767 kept and digitized by the Royal Danish Library, manu-
	38	Niebuhr, "Das innere von Afrika," 981. script Harīdat al-'aǧā'ib wa-farīdat al-ġarā'ib, Cod. Arab 93, folios 70r-70v. http://www
	39	Rigsarkivet, 1770-1777 Tripolis Sager verdr De Tripolitanske gesandtskaber. The Journal .kb.dk/manus/ortsam/2009/okt/orientalia/da/object59528/ consulted in January 2017. I
		is written in manuscript old Danish black letter, I am thankful to Dan Andersen for deci-am grateful to Jean Charles Ducène for identifying the manuscript.
	46	phering and translating it for me. Idem., AEreboe's Journal, August 14TH.
	47	AEreboe, Journal, November 10.

40 Barthold Georg Niebuhr, The Life of Carsten Niebuhr, the Oriental Traveller, 3:1-4, (Edinburgh: T. Clark, 1836), 42. friend48 Lawrence J. Baack, Undying Curiosity: Carsten Niebuhr and the Royal Danish Expedition to Arabia (1761-1767) (Stuttgart: Franz Steiner Verlag, 2014), 51-52 and 119.

  Niebuhr started on July 19th, when on his third visit to the North African diplomat Niebuhr brought with him Arabic books, manuscripts and copies of inscriptions that he had done in Arabia. They read the inscriptions together and ʿAbd al-Raḥmān identified quotations from the Qurʾān.55 On August 3rd, Niebuhr arrived again with Arabic documents and drawings done in Medina and Mecca about which they discussed at length.56 The same day they talked about Tripoli and its surroundings. AEreboe's journal does not mention a discussion about Africa, or a discussion between ʿAbd al-Raḥmān, Niebuhr, and the 'Africans.' What we

54

Niebuhr, Carsten Niebuhrs Reisebeschreibung, footnote, page 127-128. 55

Idem., AEreboe's Journal, July 19th. 56

Idem., AEreboe's Journal, August 3.

  Preliminary version Noch Etwas Über die Mohammedanischen Freistaaten in derBarbarei; Niebuhr, "Das Innere von Afrika," 986. various names of the Niger River, the Afnu/Hausa language or on the use of cowries. Niebuhr's hierarchy of trustworthiness thus seems to place direct observation by an established European scientist on top; information acquired from a literate or experienced adult male first-hand observer (Tripolitan or Hausa) in the middle; and information provided by an African enslaved at a young age at the bottom. More than social or racial hierarchy, what discredited the Timbuktu slave's information to Niebuhr's eyes, was his young age when he was abducted and forced to leave the region.ʿAbd al-Raḥmān appears to have considered his slaves (and especially his Afnu/Hausa slave) as potentially valuable informants. Accordingly, he encouraged Niebuhr to question them. He also made a careful distinction between what he had witnessed himself,

	61	This is confirmed by his son, Barthold Georg Niebuhr, The Life of Carsten Niebuhr, 43.
	62	Niebuhr, "Bemerkungen," 432.
	63	Folio noted 29, Preliminary version Noch Etwas Über die Mohammedanischen Freistaaten
		in der Barbarei.
	64	Niebuhr, "Das innere von Afrika," 988 and 990.
	65	Folio noted 21,

  69 Das Innere von Afrika can be seen as a collection of information intended to assist future European travellers developing projects for expeditions.70 ʿAbd al-Raḥmān, understood this agenda and even supported the idea of a scientific exploration of Tripoli. In his speech at the Swedish Academy of Science in 1773, he offered to support and help fund a European exploration of the Regency. Indeed when he returned to Tripoli from Sweden he brought with him the naturalist Göran Rothman and helped him during his travels, as he did later with Simon Lucas.71To understand what ʿAbd al-Raḥmān had in mind when he worked with Niebuhr, we need to consider the social and intellectual context of Tripoli. During the Qaramānlī period the country witnessed an intellectual growth, characterized by the intellectual leadership of an Arabicised, local and independent literate elite.72 The Pasha lavished attentions on the ʿulamāʾ (learned men), promoted the resumption of the riḥla (travel narrative) and the number of zāwiyas increased. Aḥmad al-Qaramānlī, the founder of the dynasty, sponsored the work of Muḥammad b. Ḫalīl Ibn Ġalbūn (dead in 1763-1764?), a 68 Folio noted 1, Preliminary version Noch Etwas Über die Mohammedanischen Freistaaten in commissioned to write the story of Tripoli.73 His book was the first attempt to present the history of the Regency in a unified narrative. Indeed, the description of the history and geography of Tripoli provided by ʿAbd al-Raḥmān to Niebuhr seems to have been influenced by the emergence of a scholarly discourse that emphasised political autonomy and intellectual pride. ʿAbd al-Raḥmān insists on the dynastic and hereditary nature of the power of the Qaramānlī, describes a regency controlling Fezzan and Cyrenaica and a Pasha with absolute powers comparable to the Ottoman Sultan.74 ʿAbd al-Raḥmān's description of the advent of the Qaramānlī dynasty and of Aḥmad Qaramānlī's takeover is clearly derived from the official history of the Qaramānlī, as written by Ibn Ġalbūn who was an eye witness of its establishment.75 Niebuhr is not wrong in considering this part of ʿAbd al-Raḥmān report as influenced by the latter's proximity to the Pasha. Proud diplomat, his discourse can be seen as shaped by a desire to glorify the Qaramānlī dynasty and the Regency of Tripoli.The issue of national pride is also raised when discussing 'Africans.' Niebuhr wrote in one of his classical attempts to correct European prejudice, that while European scholars were still uncertain whether Africans were men or monkeys, these Africans considered themselves to be the finest and most important of all nations.76 Miss Tully in her narrative also recalled that the Prince of Fezzan who came to Tripoli at the end of 1788, considered his country as the 'most fertile and beautiful in the world.'77The Maghreb tradition of the riḥla was definitely part of ʿAbd al-Raḥmān's cultural repertoire, as for most of his Maghreb learned contemporaries, providing the logical form in which to cast travel experiences.78 We do not know if ʿAbd al-Raḥmān wrote a riḥla hijāzīya of his two pilgrimages, but he did deliver a long oral narrative of his second pilgrimage to MissTully.79 Regarding what he thought about his travels to Europe, we only have a one-page travelogue he wrote and had printed in Copenhagen, thanks to Niebuhr's help.80 The text has a formal tone and follows the rhetorical conventions of Arabic diplomatic writing. It starts with a preamble about the Ambassador's ruler and his lineage,

		der Barbarei.
	69 73	This argument is better expressed in Niebuhr, "Bemerkungen," 429-430. Ibid.
	70 74	We know that at some point Niebuhr thought he could be this traveller, Barthold Georg Niebuhr, "Noch Etwas Über die Mohammedanischen Freistaaten in der Barbarei. Fortset-
		Niebuhr, The Life of Carsten Niebuhr, 43. zung," 119-120; Niebuhr, "Das Innere von Afrika," 994.
	71 75	A Swedish translation of his speech by Baage is kept in the archives of the Royal Academy Ibn Ghalbun, al-Tidhkār fīman malaka Ṭarāblus; quoted in Féraud, Annales Tripolitaines,
		of Science in Stockholm, Sekreterarens arkiv k. 10:3. A Dutch version was published in De 204-205.
	76	Maandelykse Nederlandische Mercurius, March 1773, 90-91. I am thankful to Rafaël Thie-Niebuhr, "Das innere von Afrika," 979-980.
	77	baut for his translation. Concerning ʿAbd al-Raḥmān regarding's role in Lucas's journey, Tully, Narrative, 183.
	78	see TNA, FO-76/05, letter 162. Miller, Disorienting Encounter, 52.
	79

72 Ammar Guhaydar, "Sources Pour l'Étude de la Vie Intellectuelle en Libye à l'Époque des Karamanli," Revue d'Histoire Maghrébine 59-60 (October 1990): 165.

chronicler

  81Which is not unusual; Matar, In the Lands of the Christians, 95. 82 ʿAbd al-Raḥmān's text kept in Rigsarkivet, 1770-1777 Tripolis Sager verdr De Tripolitanske gesandtskaber. I am thankful to Robin Seignobos and Ismail Warsheid for helping me with the translation of the Arabic text. 83 Rigsarkivet, 1771-1789 Tripolis depecher 1771 file 2, letter from Lochner to Bernstorff, January 8 1775.

Moraes Farias, Arabic, XLVI-LI; Paulo Fernando de Moraes Farias, "Barth, Fondateur d'une Lecture Réductrice des Chroniques de Tombouctou," in Mamadou Diawara, Paulo Fernando de Moraes Farias, Gerd Spittler, eds., Heinrich Barth et l'Afrique (Cologne: R. Köppe, 2006), 215-224.

This research has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, ERC-STG 2017 Grant agreement n° 759390


Chloé Maurizy

Claire Abeza

Valérie Pinet

Marina Ferrand

Conception Paul

Francina Langa

François Gerbe

Philippe Jay

Céline Verheggen

Nicola Tinari

Chloé Maurizy1

Claire Abeza1

Marina Ferrand1

Conception Paul1

Julie Bremond1

Francina Langa3

François Gerbe4

Philippe Jay4

Céline Verheggen1

Nicola Tinari6

Dominique Helmlinger7

Rossano Lattanzio6

Edouard Bertrand1

Michael Hahne1

Bérengère Pradet-Balade2

The R2TP chaperone assembles cellular machineries in intestinal CBC

come

Introduction:

The R2TP complex was first discovered in Saccharomyces cerevisiae as an HSP90 cochaperone [START_REF] Zhao | Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone[END_REF]. HSP90 folds several hundreds of substrate proteins (also called "clients") into their native, active state [START_REF] Schopf | The HSP90 chaperone machinery[END_REF]. The chaperone activity of HSP90 is coupled with its ATPase cycle, and is regulated by a number of co-factors called cochaperones. These co-chaperones aid client loading and regulate HSP90 ATPase cycle [START_REF] Schopf | The HSP90 chaperone machinery[END_REF]. R2TP is an unusual HSP90 co-chaperone for two reasons: first, it is composed of four different subunits; second, it is specialized in quaternary protein folding, i.e. it enables the incorporation of clients into multisubunit complexes (for review [START_REF] Coulombe | PAQosome: An R2TP-based chaperone for quaternary structure formation[END_REF]). In mammals, R2TP is composed of a heterodimer between PIH1D1 and RPAP3, which associates with a heterohexamer of RUVBL1 and RUVBL2 (Figue 1A). PIH1D1 is involved in substrate recognition [START_REF] Hořejší | Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex[END_REF] while RPAP3 recruits the chaperones HSP90 and HSP70 [START_REF] Pal | Structural Basis for Phosphorylation-Dependent Recruitment of Tel2 to Hsp90 by Pih1[END_REF], [START_REF] Henri | Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex[END_REF]. RUVBL1 and RUVBL2 are related AAA+ ATPases that also have chaperone activity [START_REF] Zhou | Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones[END_REF], [START_REF] Zaarur | RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils[END_REF]. Multiple contacts between the PIH1D1:RPAP3 heterodimer and RUVBL1/2 heterohexamer allow a regulation of their ATPase activity [START_REF] Hořejší | CK2 phospho-dependent binding of R2TP complex to ℡2 is essential for mTOR and SMG1 stability[END_REF]. Importantly, the RPAP3:PIH1D1 heterodimer is specific to R2TP whereas RUVBL1/2 are also part of other complexes such as the chromatin remodelers INO80 and SRCAP [START_REF] Jha | RVB1/RVB2: Running Rings around Molecular Biology[END_REF]. In mammals, R2TP associates with a set of six prefoldins and prefoldin-like proteins that are believed to prevent client aggregation.

Altogether, R2TP with prefoldins have been termed the PAQosome for Particle for Arrangement of Quaternary structure [START_REF] Coulombe | PAQosome: An R2TP-based chaperone for quaternary structure formation[END_REF].

The first documented R2TP clients were the small nucleolar ribonucleoparticles (snoRNPs), which are required for the maturation of ribosomal RNAs [START_REF] Zhao | Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation[END_REF], [START_REF] Boulon | The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery[END_REF]. These particles can be grouped into two families, the C/D and H/ACA snoRNPs, and each family is characterized by a set of four proteins, which assembles with a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted December 19, 2019. ; https://doi.org/10.1101/2019.12.19.882712 doi: bioRxiv preprint variety of small nucleolar RNAs. Later on, other R2TP substrates have been identified, including the U4 and U5 spliceosomal snRNPs [START_REF] Bizarro | NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins[END_REF], [START_REF] Malinová | Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones[END_REF], [START_REF] Cloutier | R2TP/Prefoldin-like component RUVBL1/RUVBL2directly interacts with ZNHIT2 to regulateassembly of U5 small nuclear ribonucleoprotein[END_REF], the nuclear RNA polymerases [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF], [START_REF] Cloutier | New insights into the biogenesis of nuclear RNA polymerases?[END_REF], and the PI3K-like kinases (PIKKs) mTOR, ATR, ATM, DNA-PK and SMG-1 [START_REF] Hořejší | CK2 phospho-dependent binding of R2TP complex to ℡2 is essential for mTOR and SMG1 stability[END_REF]. R2TP clients thus include multimeric cellular complexes with crucial roles in transcription, ribosome biogenesis and cell growth [START_REF] Coulombe | PAQosome: An R2TP-based chaperone for quaternary structure formation[END_REF], [START_REF] Boulon | HSP90 and the R2TP co-chaperone complex: building multi-protein machineries essential for cell growth and gene expression[END_REF], yet the role of R2TP in tissue homeostasis has not been studied. Most of our knowledge about R2TP originates from studies in S.cerevisiae, mammalian cell lines or in vitro studies with recombinant proteins. RPAP3 knock-down does not affect cell line proliferation [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF]. One study in D. melanogaster showed that knock-down of the RPAP3 ortholog Spag hinders early development, but not somatic organogenesis nor homeostasis, suggesting that R2TP could be specialized for stress conditions [START_REF] Benbahouche | Drosophila Spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries[END_REF]. In fact, the role of HSP90 on tissue development is also poorly documented.

Constitutive knock-out (KO) murine models showed that one cytosolic paralog of HSP90 was necessary for spermatogenesis while the other one was essential for early development [START_REF] Grad | The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse[END_REF], [START_REF] Voss | Mice lacking HSP90beta fail to develop a placental labyrinth[END_REF]. Conditional models to address the role of HSP90 and R2TP in specific tissues are still missing.

In this study, we chose to address the role of R2TP in the intestine, the most dynamically self-renewing tissue in adult mammals. The intestine has a well-defined architecture which is particularly amenable to study cell proliferation and differentiation [START_REF] Vermeulen | Stem cell dynamics in homeostasis and cancer of the intestine[END_REF]. Briefly, active stem cells called Crypt Base Columnar (CBC) stem cells reside at the bottom of intestinal crypts where they divide to either self-renew, or give rise to progenitors (see schematic Figure 3C). Progenitors migrate through the transient amplifying (TA) compartment to undergo several rounds of division and differentiate. Differentiated cells then migrate towards the tip of the villus, where they ultimately undergo apoptosis and are shed off [START_REF] Barker | Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration[END_REF]. Progenitors can differentiate into enterocytes, which are responsible for water and nutrient absorption, and compose most of the small intestine epithelium. Alternatively, progenitors differentiate into one of the secretory subtypes. Among these, the goblet cells are the most represented. These cells produce a lubricant protective mucus layer, and their density increases from the duodenum to the colon. In the small intestine, CBC stem cells also generate long-lived Paneth cells (3-6 weeks), which secrete anti-microbial peptides and maintain crypt niche conditions (for review [START_REF] Barker | Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration[END_REF]).

To study the role of R2TP in intestinal homeostasis, we generated murine models bearing a conditional knock-out allele of Rpap3. Indeed, Rpap3 is central to R2TP, bridging together HSP90, HSP70, PIH1D1 and RUVBL1/2 [START_REF] Henri | Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex[END_REF], [START_REF] Martino | RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex[END_REF], [START_REF] Maurizy | The RPAP3-Cterminal domain identifies R2TP-likequaternary chaperones[END_REF][START_REF] Muñoz-Hernández | Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM[END_REF]. Our work uncovers a crucial role of R2TP in CBC stem cells and progenitors, by coupling the assembly of key cellular machineries with proliferation. In agreement, we found that high RPAP3 expression in the human colorectal cancer (CRC) tissue is associated with a shorter disease-free survival in patients.

Results

Rpap3 is an essential gene in mice

To generate Rpap3-deficient mice, we tested different recombined murine Embryonic Stem cells (ES cells) available from the KOMP consortium (for a schematic explanation of the genomic constructs and their products, see Supp. Figure 1A,B). In the Rpap3wtsi allele, a strong acceptor splice site is introduced before exon 7. The resulting mRNA encodes a Rpap3wtsi protein truncated after the first TPR domain, at amino acid 223, followed by a selection cassette that includes the bacterial -galactosidase gene (Supp. Figure 1.A, B). Site-specific recombination at the Rpap3 locus was confirmed by Southern blot in clone H10 (Supp. Figure 1C). Injection of H10 ES cells into blastocysts generated 3 chimeras with germinal transmission. Intercrossing of Rpap3wtsi/+ animals did not yield any homozygous Rpap3wtsi/wtsi animal, suggesting that this truncation of Rpap3 is homozygous lethal (0/34 pups; Supplemental Figure 1B).

We then took advantage of the -galactosidase cassette to characterize Rpap3 promotor activity. In the small intestine of Rpap3wtsi/+ animals, we detected -galactosidase activity exclusively at the bottom of the crypts, intercalated between lacZ-cells (Figure 1B). This location could correspond to either CBC stem cells or Paneth cells (see Figure 3C for a schematic representation of small intestinal crypts). In the colon, the signal was weaker and diffuse throughout the epithelium (Figure 1B). These results show that Rpap3 promoter is active in the intestinal crypts.

Rpap3 is required for small intestine maintenance

To generate a conditional knock-out strain, Rpap3wtsi/+ mice were crossed with mice carrying a transgene encoding the Flipase (Flp) [START_REF] Kranz | An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase[END_REF]. In the progeny, excision of the Flpin cassette produced a Rpap3 allele with exon 7 flanked by loxP sites (Supp. Figure 1A).

Rpap3flox mRNA encodes a protein predicted to be identical to wild-type Rpap3 and indeed, there was no bias in transmission nor any obvious phenotype in Rpap3flox/flox mice. Excision of exon 7 by Cre recombinase produces a Rpap3∆7 mRNA with a premature termination codon in exon 8. This is far upstream of the wild-type termination codon in exon 17, predicting Rpap3∆7 mRNA degradation by the nonsense-mediated decay pathway (Supp. Figure 1A,B).

To obtain a constitutive deletion of Rpap3 in the intestinal epithelium, Rpap3flox/flox mice were crossed with villin-Cre (VilCre) transgenic mice. VilCre transgene encodes a constitutively active Cre that is specifically expressed in all cells of the epithelium of the small and large intestine [START_REF] El Marjou | Tissue-specific and inducible Cre-mediated recombination in the gut epithelium[END_REF]. We were unable to generate homozygous VilCre; Rpap3flox/flox animals (0/100 pups; Supplemental Figure 1A), showing that the absence of Rpap3 in the intestinal epithelium is lethal. To bypass this problem, we turned to an inducible CreERT2 under the control of the same promotor (VilCreERT2). This Cre-recombinase is also specifically expressed in the intestinal epithelium, but it needs to be activated by tamoxifen [START_REF] El Marjou | Tissue-specific and inducible Cre-mediated recombination in the gut epithelium[END_REF]. VilCreERT2; Rpap3flox/flox animals were healthy and did not present any obvious phenotype. We then injected intraperitonealy two doses of tamoxifen separated by an interval of 24 hours, in eight-week-old mice because the intestine is fully developed at this age (Figure 1C). This yielded an efficient recombination, already detectable one day after the first injection (Supp. Figure 1D). The Rpap3 protein was no longer detected in epithelia from both jejunum and colon, 5 days after the first tamoxifen injection (Figure 1D).

VilCreERT2; Rpap3flox/flox mice showed no obvious phenotype during the first days following tamoxifen injection but suffered from an important weight loss from day 6 onwards, such that most females and males had to be sacrificed at day 8 or 10 (Figure 1 E). The small intestines from these animals were shorter than those of controls and displayed a massive swelling with a transparent liquid or blood (Figure 1 F,G). Histological analysis revealed no alteration in the small intestine of VilCreERT2; Rpap3fox/flox mice within the first 6 days (Figure 2A, B red square). At day 7, however, epithelial cells in the villi presented an altered morphology with surface enterocyte disorganization and focal crowding (Figure 2B). At day 8, there was a severe destruction of the small intestinal architecture with villous atrophy and tufts of extruding epithelium (Figure 2B). In contrast, the intestines of the VilCreERT2; Rpap3flox/+ mice did not show any phenotype (Supp. Figure 2 A), suggesting that under normal conditions, one allele of wild-type Rpap3 is sufficient to maintain tissue homeostasis.

R2TP promotes cell proliferation within the epithelium

To understand the basis for this rapid degeneration of the intestinal epithelium, we measured Ki67 expression. Ki67 marks proliferative cells which are, in the intestine, the CBC stem cells and progenitors from the transient amplifying (TA) compartment. Immunostaining revealed a loss of Ki67 in the crypts of VilCreERT2; Rpap3flox/flox animals starting 7 days after tamoxifen injection (Figure 2B and Supp. Figure 2D for a zoom of the crypt). To directly monitor cell cycling, BrdU was injected into the mouse peritoneum two hours before sacrifice. BrdU is a nucleoside analog incorporated into the DNA during S-phase. BrdU staining was comparable from day 1 to day 6 in the crypts of control and VilCreERT2; Rpap3flox/flox animals. At day 7 however, there were significantly less BrdU+ cells (Figure 2C and Supp. Figure 2E for quantification). This is coherent with the loss of Ki67 staining and confirms cycle arrest in the crypts and the TA compartment. At day 8, the atrophic small bowel mucosa showed remnants of crypt glands with Ki67+ cells (Figure 2B, black arrowheads). Such remnants of crypt glands have also been reported upon TCF4 ablation [START_REF] Van Es | A Critical Role for the Wnt Effector Tcf4 in Adult Intestinal Homeostatic Self-Renewal[END_REF].

To verify if CBC stem cells were affected, we performed immunostaining for Olfm4, a trans-membrane protein commonly used for CBC identification [START_REF] Van Der Flier | OLFM4 Is a Robust Marker for Stem Cells in Human Intestine and Marks a Subset of Colorectal Cancer Cells[END_REF] (Figure 3A and Supp. Figure 3A). Olfm4 pattern was similar between control and VilCreERT2; Rpap3flox/flox mice from day 4 to day 6 after tamoxifen injection. At days 7 and 8 however, Olfm4 expression was undetectable in many VilCreERT2; Rpap3flox/flox crypts (Figure 3A,B and Supp. Figure 3A), in agreement with the loss of Ki67+ staining (Supp. Figure 2 C,D). In the crypts, CBC stem cells intercalate between larger differentiated cells, called Paneth cells. These cells are Ki67-and recognizable with the lysozyme marker (Figure 3C,D) or by pink cytoplasmic dots stained by Hematoxylin/Eosin (HE), (Supp. Figure 2 C,D). In marked contrast to CBC stem cells, Paneth cells were detected in a comparable manner in the crypts of control and VilCreERT2; Rpap3flox/flox mice until day 8 (Supp. Figure 2D and Figure 3D). Altogether, these observations confirmed that loss of Rpap3 induces the rapid disappearance of proliferating stem cells and progenitors, but not of differentiated Paneth cells. Interestingly, we observed cells with round, dense nuclei, resembling apoptotic cells, in the crypts of small intestines deficient for Rpap3. We verified this by immunostaining for cleaved caspase 3, a well-established marker of apoptosis. In control mice, few caspase 3 positive cells were visible at the tip of some villi, but we detected significantly more cleaved caspase 3+ cells in the crypts of tamoxifen-injected VilCreERT2; Rpap3flox/flox mice (Figure 3E). These apoptotic cells were observed in the crypts and TA compartment, but not at the tip of villi, as observed in normal epithelia because of cellular turnover. Thus, Rpap3 deletion eventually induces apoptosis of CBC stem cells and progenitors.

Under stress, cells stabilize p53, which induces cell cycle arrest and/or apoptosis. Since we observed both events, we analyzed its expression. By IF and WB, we saw an increase of p53 at day 6 in the crypts and the TA compartment (Figure 3F and Supp. Figure 3B). Altogether, these results show that inactivation of Rpap3 affects proliferative cells by inducting p53 expression, cell cycle arrest and apoptosis.

Rpap3 stabilizes clients of diverse structural families

Misfolded HSP90 clients are usually degraded 32, and we thus determined the expression levels of R2TP clients in VilCreERT2; Rpap3flox/flox animals 6 days after the first tamoxifen injection, when the epithelial architecture was still preserved. R2TP has first been characterized as an assembly factor for the box C/D snoRNPs by recruiting NOP58, one of the four core proteins in these particles [START_REF] Boulon | The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery[END_REF]. Western blot analysis of crypt epithelial cell lysates showed a near two-fold reduction of NOP58 levels in the extracts from VilCreERT2; Rpap3flox/flox animals, as compared to controls (Figure 4A, quantification in Supp. Figure 4A). PRP8 and EFTUD2 are two core components of the U5 snRNP, a splicing ribonucleoparticle assembled by R2TP in HeLa cells [START_REF] Malinová | Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones[END_REF], [START_REF] Cloutier | R2TP/Prefoldin-like component RUVBL1/RUVBL2directly interacts with ZNHIT2 to regulateassembly of U5 small nuclear ribonucleoprotein[END_REF]. Both proteins were also reduced by about two-fold (Figure 4A et Supp. Figure 4A,B). HSP90 and R2TP are thought to stabilize clients only before their assembly, and the extreme stability of snoRNPs and snRNPs may thus explain the moderate but consistent decrease observed here for their components [START_REF] Lemm | Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies[END_REF].

In mammals, PIKKs consist of six structurally related proteins (ATR, ATM, DNA-PK, mTOR, TRRAP and SMG1). PIKKs are stabilized by the trimeric co-chaperone TTT [START_REF] Takai | Tel2 regulates the stability of PI3K-related protein kinases[END_REF], [START_REF] Izumi | Heat shock protein 90 regulates phosphatidylinositol 3-kinase-related protein kinase family proteins together with the RUVBL1/2 and Tel2-containing co-factor complex[END_REF], [START_REF] Hurov | A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability[END_REF], [START_REF] Kaizuka | Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly[END_REF] and in murine fibroblasts, TTT recruits R2TP to assemble PIKKs with their partners [START_REF] Hořejší | CK2 phospho-dependent binding of R2TP complex to ℡2 is essential for mTOR and SMG1 stability[END_REF]. We analyzed by Western blot the expression of PIKKs in extracts enriched for crypt cells. We observed a strong diminution of ATR and ATM, the primary sensors of DNA damage, as well as for mTOR, which activates translation and cell proliferation (Figure 4A and Supp. Figure 4 C, D). These results show that R2TP participates in the stabilization of ATR, ATM, and mTOR.

In contrast, TRRAP levels did not vary, either because R2TP does not chaperone TRRAP or because it is a very stable protein.

Another R2TP client is Rpb1, the biggest subunit of RNA polymerase II (RNA PolII) [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF], [START_REF] Cloutier | New insights into the biogenesis of nuclear RNA polymerases?[END_REF]. R2TP incorporates neo-translated Rpb1 within RNA PolII in the cytoplasm, after which it is addressed to the nucleus [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF]. As expected, IHC for Rpb1 displayed a nuclear staining in control intestines.

However, in Rpap3-deficient animals, Rpb1 accumulated in the cytoplasm of CBCs and TA progenitors. Interestingly, it remained nuclear in differentiated epithelial cells, including Paneth cells (Figure 4B,C). This revealed that Rpap3 is necessary for the assembly and nuclear import of RNA pol II in the proliferative compartment (Figure 4D).

Rpap3 activity correlates with cellular turnover in small intestine and colon

Tamoxifen treated VilCreERT2; Rpap3flox/flox animals displayed comparable depletion of Rpap3 protein in either small intestines or colons (Figure 1D). Nevertheless, none of the phenotypes affecting the small intestine were observed in the colon: the size of the organs did not vary between control and Rpap3 KO mice (Figure 1F), nor did their architectural organization, as observed by staining with Periodic Acid Schiff (PAS) or Ki67 (Supp. Figure 5A). To verify whether Rpap3 deletion affects colonic crypts, we generated Lgr5-EGFP-IRES-CreERT2; Rpap3flox/flox animals (Figure 5A). In these mice, GFP and the tamoxifen-inducible Cre are both under the control of the Lgr5 promoter, which is only expressed in CBC stem cells [START_REF] Barker | Identification of stem cells in small intestine and colon by marker gene Lgr5[END_REF]. This driver has a mosaic expression, which allows to analyze Rpap3-deficient andexpressing crypts in parallel. Tamoxifen treated Lgr5-EGFP-IRES-CreERT2;Rpap3flox/flox mice did not show any visible physiological alteration and could survive for at least 3 weeks.

To analyze the activity of R2TP in the Lgr5-EGFP-IRES-CreERT2;Rpap3flox/flox mice, we performed a double staining for Rpb1, and GFP to identify crypts with an active Cre. Seven days after the first tamoxifen injection, Rpb1 was localized in the nucleus of GFP-crypts of the small intestine but accumulated in the cytoplasm of CBC stem cells and progenitors from GFP+ crypts (100% of GFP+ crypts; Figure 5B). In Lgr5-EGFP-IRES-CreERT2; Rpap3+/+ control mice, Rpb1 was nuclear in both GFP-as well as in GFP+ crypts (Figure 5B). At day 10, we did not detect any GFP+ crypt in the small intestine of Lgr5-EGFP-IRES-CreERT2; Rpap3flox/flox mice, possibly because recombinant CBC stem cells had been eliminated and replaced by GFP-CBC-derived crypts. In the colon, Rpb1 localization was affected but with a delay, as compared to the small intestine (Supp. Figure 5B). At day 7, Rpb1 was nuclear in GFP-and GFP+ colonic crypts from Lgr5-EGFP-IRES-CreERT2; Rpap3flox/flox mice (Supp. Figure 5C). At day 10, however, Rpb1 accumulated in the cytoplasm of CBC stem cells and progenitors of GFP+ crypts (Figure 5B). In colons of control animals, Rpb1 was nuclear in GFP-and GFP+ crypts (Supp.

Figure 5C). Thus, Rpap3 is also required for Rpb1 assembly in the colon but cytoplasmic accumulation of Rpb1 is detectable with a delay of three days, as compared to the small intestine.

We then decided to compare the cellular turnover in both tissues. For this, we injected the nucleoside analog BrdU as a cell tracer into wild type mice and sacrificed them subsequently at different time points (Figure 5C). Two hours after injection, BrdU was incorporated exclusively by CBC stem cells and progenitors in both the small intestine and the colon (Figure 5C). After one day, BrdU+ cells were detectable above the TA compartment in the small intestine, while some remained at the bottom of the crypt. BrdU+ cells then migrated to the tip of the villus at day 3, and were eliminated at day 4, in accordance with the kinetics previously described [START_REF] Tetteh | Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters[END_REF]. In contrast, in the colon, migrating BrdU+ cells did not reach the tip of the crypt before day 4 and were still detectable at day 5 (Figure 5C). This illustrates a slower cellular turnover in the colon than in the small intestine, correlating with our finding that neo-synthesized Rpb1 accumulates more slowly in the cytoplasm of colonic cells.

High expression of RPAP3 correlates with poor prognosis in colorectal cancer patients

Our previous findings suggest a strong link between R2TP activity and cell proliferation during intestinal homeostasis. To test if R2TP is also involved in pathogenic proliferation, we took advantage of the CODREAD dataset (available at https://xenabrowser.net/). This showed a significant enrichment of mRNAs encoding RUVBL1, 2 and RPAP3 in human primary colorectal tumors (n= 380) as compared to normal tissue (n=51), in line with a previous report on a smaller cohort [START_REF] Kim | Metabolic Stress Controls mTORC1 Lysosomal Localization and Dimerization by Regulating the TTT-RUVBL1/2 Complex[END_REF] (Fig. 6A).

This prompted us to test the expression of RPAP3 by immunohistochemistry on Tissue Microarrays (TMAs) sections from colorectal cancer (CRC) patients, employing customary anti-RPAP3 antibodies (unfortunately, this antibody detects human RPAP3 but not its murine homolog [START_REF] Machado-Pinilla | Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs[END_REF]). We analyzed TMAs containing core tissues from patients diagnosed with CRC without pathological evidence of nodal involvement and distant metastasis. One hundred and fifty-seven out of 177 (88.7%) cases expressed RPAP3 in the tumor cell cytoplasm. The proportion of RPAP3-positive cells was in the range of 4-100%, with a mean ± S.E. of 62.6% ± 2.6. To dichotomize the RPAP3 expression level in RPAP3high and RPAP3low, an optimal cut-off value of 26% of positive tumor cells was chosen based on the Receiver Operating Characteristic (ROC) analysis (AUC = 0.593). Examples of low and high expression of RPAP3 are shown in Figure 6B. The relationships between RPAP3 expression and clinico-pathological parameters were investigated by Pearson's 2 test: RPAP3 expression negatively correlated with the tumor stage of CRC (P = 0.049) (Supplemental Table 1, 2).

Thirty-nine out of 123 (31.7%) patients with RPAP3high tumors and 6 out of 54 (11.1%) patients with RPAP3low tumors had disease relapse. Analysis of Kaplan-Meier curves showed that patients with RPAP3high tumors had a lower Disease-Free Survival (DFS) rate than patients with RPAP3low tumors (P = 0.037; Figure 6B). Multivariate analyses of DFS adjusted for other prognostic factors confirmed that RPAP3 expression was a significant prognostic parameter influencing disease relapse (HR = 2.7: 95% CI, 1.2-6.5; P = 0.023) (Supp. Table 3), but not the overall survival of patients. These results provide evidence that high RPAP3-expression levels in CRC tissues are associated with poor patient prognosis.

Discussion

R2TP inactivation leads to client dysfunction, p53 activation and apoptosis

Since its discovery in 2005, structural studies of the R2TP chaperone have provided substantial knowledge [START_REF] Zhao | Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone[END_REF], [START_REF] Coulombe | PAQosome: An R2TP-based chaperone for quaternary structure formation[END_REF]. It is now clear that RPAP3 is a central factor within R2TP. It recruits the chaperones HSP70/90 via its TPR domains, PIH1D1 through a short peptide domain and the AAA+ ATPases RUVBL1/2 via its C-terminal domain [START_REF] Henri | Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex[END_REF], [START_REF] Martino | RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex[END_REF], [START_REF] Maurizy | The RPAP3-Cterminal domain identifies R2TP-likequaternary chaperones[END_REF], [START_REF] Muñoz-Hernández | Structural mechanism for regulation of the AAA-ATPases RUVBL1-RUVBL2 in the R2TP co-chaperone revealed by cryo-EM[END_REF].

Nevertheless, how R2TP ultimately assembles neo-synthesized proteins into active complexes is still unknown. Most of the R2TP clients have been identified in mammalian cell lines [START_REF] Cloutier | New insights into the biogenesis of nuclear RNA polymerases?[END_REF], [START_REF] Boulon | The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery[END_REF], [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF], [START_REF] Hořejší | Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex[END_REF], [START_REF] Cloutier | R2TP/Prefoldin-like component RUVBL1/RUVBL2directly interacts with ZNHIT2 to regulateassembly of U5 small nuclear ribonucleoprotein[END_REF], [START_REF] Malinová | Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones[END_REF]. For several of them, we validate here their dependence on RPAP3 in a mammalian tissue. This includes PRP8, part of the splicing snRNP U5, and NOP58, a core component of box C/D snoRNPs. Another class of R2TP substrates are PIKKs [START_REF] Hořejší | CK2 phospho-dependent binding of R2TP complex to ℡2 is essential for mTOR and SMG1 stability[END_REF]. Several studies have documented that these large proteins are stabilized by a trimeric co-chaperone called TTT, which itself interacts with R2TP [START_REF] Hurov | A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability[END_REF], [START_REF] Kaizuka | Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly[END_REF], [START_REF] Izumi | Heat shock protein 90 regulates phosphatidylinositol 3-kinase-related protein kinase family proteins together with the RUVBL1/2 and Tel2-containing co-factor complex[END_REF], [START_REF] Takai | Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes[END_REF]. We show here that mTOR, ATR and ATM depend on Rpap3 in intestinal crypts. Finally, a major R2TP client is RNA polII. This key enzyme is assembled in the cytoplasm and its Rpb1 subunit remains cytoplasmic when assembly is deficient [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF]. The observed cytoplasmic accumulation of Rpb1 in Rpap3 knock-out cells thus likely results from a defective assembly of RNA polII.

Invalidation of Rpap3 triggers p53 stabilization and cell cycle arrest, ultimately leading to apoptosis and destruction of the intestinal epithelium (Figure 6C). Transcription inhibition is well known to activate p53 [START_REF] Ljungman | Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress[END_REF]. In addition, while neither mTOR nor ATM loss affect intestinal homeostasis [START_REF] Sampson | mTOR disruption causes intestinal epithelial cell defects and intestinal atrophy postinjury in mice[END_REF], [START_REF] Brandt | mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis[END_REF], [START_REF] Barlow | Atm-deficient mice: a paradigm of ataxia telangiectasia[END_REF], defective ribosome biogenesis or ATR inactivation both induce a degradation of the intestinal epithelium through p53 activation, similar to the one described here [START_REF] Stedman | Ribosome biogenesis dysfunction leads to p53-mediated apoptosis and goblet cell differentiation of mouse intestinal stem/progenitor cells[END_REF], [START_REF] Ruzankina | Tissue regenerative delays and synthetic lethality in adult mice after combined deletion of Atr and Trp53[END_REF]. The alterations that we observed for snoRNPs, ATR and RNA PolII might thus concur to induce the phenotype observed in the Rpap3 knock-out intestine.

R2TP is active in the proliferative compartment

We found that differentiated cells in the small intestine are barely affected by Rpap3 removal, in contrast to the rapidly dividing CBC stem cells and TA progenitors. Indeed, Rpb1 accumulates in the cytoplasm of proliferating cells, while it remains fully nuclear in the differentiated cells of the epithelium. This correlation between proliferation and sensitivity to Rpap3 removal further extends to the colon. Rpb1 also accumulated in the cytoplasm of colonic recombinant crypts, but this occurred several days later than in the small intestine, in line with its slower proliferation rate. Highly proliferative cells appear to be very sensitive to Rpap3 depletion. We thus propose that basic cellular machineries are assembled by R2TP in the proliferative compartment: CBC stem cells and progenitors.

Several additional data support this model. First, -galactosidase knock-in models showed that Rpap3 mRNA is expressed in the crypts where CBCs reside. Second, some R2TP clients are themselves preferentially active in the intestinal proliferative compartment: (i) under homeostatic conditions, ATR is required to signal defective DNA replication in the intestinal proliferative compartment [START_REF] Ruzankina | Deletion of the Developmentally Essential Gene ATR in Adult Mice Leads to Age-Related Phenotypes and Stem Cell Loss[END_REF]; (ii) markers of RNAPolI activity have been detected preferentially in the crypts and TA compartment with decreasing levels along the crypt-villus axis [START_REF] Stedman | Ribosome biogenesis dysfunction leads to p53-mediated apoptosis and goblet cell differentiation of mouse intestinal stem/progenitor cells[END_REF]; (iii), a recent transcriptomic analysis along the cryptvillus axis detected mRNAs encoding ribosomal, splicing and transcription components at the very bottom of the villus, but not along the villus or at the tip [START_REF] Moor | Spatial Reconstruction of Single Enterocytes Uncovers Broad Zonation along the Intestinal Villus Axis[END_REF]. All this supports a model in which ribosomal biogenesis, transcription machineries and PIKK complexes are built in the rapidly proliferating stem cells and progenitors. These assembled complexes would suffice for the relative short lifespan of differentiated cells, until they are shed off the villus (Figure 6C).

In Drosophila, the RPAP3 ortholog, Spag, is required for ovarian Germline Stem Cell (GSC) maintenance [START_REF] Chen | Spaghetti, a homolog of human RPAP3 (RNA polymerase II-associated protein 3), determines the fate of germline stem cells in Drosophilaovary[END_REF]. Ovarian germline stem cells divide asymmetrically to self-regenerate and give rise to an oocyte. Knock-down of Spag induces a loss of GSC but not of the non-dividing somatic cells in the ovary. This exemplifies another case where RPAP3/Spag affects dividing stem cells, and suggests a general role of R2TP in proliferation, especially of active stem cells, across Metazoans.

R2TP is linked with pathological cell proliferation

HSP90 can sustain tumor growth by folding numerous proteins, some of which are involved in oncogenic signaling pathways. In addition, an enhanced stability of the HSP90 interactome has been observed in some tumors and called the "epichaperome" [START_REF] Rodina | The epichaperome is an integrated chaperome network that facilitates tumour survival[END_REF]. All this explains why some tumor cells are particularly sensitive towards HSP90 inhibition [START_REF] Fierro-Monti | Dynamic Impacts of the Inhibition of the Molecular Chaperone Hsp90 on the T-Cell Proteome Have Implications for Anti-Cancer Therapy[END_REF], [START_REF] Echeverría | The sensitivity to Hsp90 inhibitors of both normal and oncogenically transformed cells is determined by the equilibrium between cellular quiescence and activity[END_REF], [START_REF] Pillarsetty | Paradigms for Precision Medicine in Epichaperome Cancer Therapy[END_REF]. In a mouse model, invalidation of a cytoplasmic HSP90 paralogue (only essential for spermatogenesis [START_REF] Grad | The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse[END_REF]) diminishes lung metastasis [START_REF] Vartholomaiou | Cytosolic Hsp90alpha and its mitochondrial isoform Trap1 are differentially required in a breast cancer model[END_REF]. Yet, in clinical trials, HSP90 inhibitors have been deceptive, showing strong cytotoxicity. This has been attributed to the broad range of HSP90 substrates, which also includes tumor-suppressors [START_REF] Vartholomaiou | Unusual Suspects in the Twilight Zone Between the Hsp90 Interactome and Carcinogenesis[END_REF], (Suman Chatterjee and Timothy Burns, 2017). To bypass this problem Neckers et al.

suggested to target HSP90 co-chaperones that are specific for a given class of clients [START_REF] Neckers | Hsp90 molecular chaperone inhibitors: are we there yet?[END_REF]. We propose to consider R2TP for alternative strategies, as a role for R2TP in intestinal carcinogenesis is supported by several lines of evidences: (i) a higher expression of mRNAs encoding R2TP subunits in colorectal tumors, as compared to matching controls; (ii) high RPAP3 protein levels in biopsies of CRC patients with poor diagnostic, (iii) a strong dependency on RPAP3 in the proliferative compartment, (iv) the induction of p53 upon R2TP inactivation. R2TP is interesting not only as a therapeutic target but also for diagnostic purpose, since R2TP components RPAP3 and PIH1D1 were both identified as part of the epichaperome of HSP90-sensitive tumours [START_REF] Rodina | The epichaperome is an integrated chaperome network that facilitates tumour survival[END_REF].

In conclusion, we show that the R2TP chaperone plays a crucial role in the homeostasis of the intestinal epithelium, by regulating proliferating stem cells and progenitors. facility. Naive mice were minimum 6 weeks old and are euthanized by CO2 and isoflurane. To activate the CreERT2, controls and animals of interest received two intra-peritoneal (IP) injection of 2mg tamoxifen each. For BrdU incorporation assays, mice were intraperitoneally injected with 100 µg Bromodeoxyuridine (BrdU) per gram body weight. The entire small intestine (cut in 3 parts) and colon were flushed with PBS, then with neutral buffered formalin (4% formaldehyde) and fix in 24h, dehydrated, and embedded in paraffin.

Histology and immunostainings

For histological analysis, tissue sections (4μm thick) were deparaffinized and rehydrated. They were stained with hematoxylin and eosin (HE), Periodic Acid Shiff staining (PAS) for preliminary analysis.

For immunostainings, tissue sections were deparaffinized, rehydrated and subsequently subjected to heat induced antigen retrieval by immersing them, depending on the antibody, either in a water bath with a sodium citrate buffer (pH 6) or an EDTA buffer (pH 9).

Immunohistochemistry was performed using a Dako autostainer (Dako, Glostrup, Denmark).

After neutralization of the endogenous peroxidase activity, the sections were incubated with the primary antibodies. Antibody was visualized using the Envision® system (Dako). Diaminobenzidine (Dako) was used as the chromogen and the sections were lightly counterstained with hematoxylin. Slides were prepared by RHEM platform and visualized with a NanoZoomerslide scanner controlled by the NDP.view software (MRI platform).

For p53 immunofluorescence, paraffin-embedded tissue was cut into 3-µm-thick sections, mounted on slides, then dried at 37°C overnight. It was performed on the Discovery Ultra Automated IHC staining system from Roche Ventana. Following deparaffination with Discovery EZ Prep solution at 75°C for 24 minutes, antigen retrieval was performed at 95°C for 16 minutes with Discovery CC1 buffer. After blocking with (TBS-10%goat serum-5%BSA-5%milk-0, 3%triton), the slides were incubated after rinsing at 37°C for 60 minutes with a rabbit anti-P53 antibody (Leica, P53-CM5P-L, 1∶250). Signal enhancement was performed using the OmniMap anti-Rabbit HRP (Roche, 760-4457) then Cy5 Kit (Roche, 760-238). Slides were then counterstained for 8 minutes with DAPI and manually rehydrated before coverslips were added.

For Rpb1/GFP immunofluorescence, antigen retrieval with 1mM EDTA was performed for 30 min at 99°C. Slides were blocked with ( 5%goat serum-PBS-0,3%triton X-100 ) for 1h at RT then with primary antibodies overnight at 4°C. Samples were incubated with Dapi+ Secondary antibodies solution for 1h at RT. Slides were mounted with coverslip and ProLong Gold Antifade Mountant.

For immunohistochemistry (Rpb1, Lysozyme, Olfm4), tissue slides were deparaffinized and rehydrated by performing the following washes: xylene, ethanol and dH2O. Tissue slides were incubated with 10 mM sodium citrate pH6.0 (T0050, DiaPATH) for 20 min at 100°C or Tris10 mM-EDTA 1mM pH9.0, for antigen retrieval, depending on the antibody. Endogenous peroxidases were inactivated with PBS-0,3% hydrogen (#H1009, Sigma). After blocking in 2.5 % blocking serum-5% BSA-5% non-fat milk for 30 min at room temperature, tissue slides were incubated with primary antibody overnight at 4°C. Then corresponding secondary antibody reagents (ImmPRESSTM kit, Vector Laborarories), directed against rabbit, rat or mouse antibodies were used for detection.

Incorporation of BrdU in proliferating intestinal epithelial cells was detected using an anti-BrdU antibody (Biolegend, 1:100) after deparaffinization of the tissues, antigen retrieval in citrate buffer, as described above, and DNA denaturation using 2NHCl for 1H at 37°C followed by an incubation in 0.1M borax buffer pH9. Revelation was performed using the Avidin/Biotin Vectastain System kit (Vectorlab, USA) according to the protocol. cells were collected by centrifugation at 1410 rpm for 7 min. They were washed in 10ml wash buffer. Collected cells were split in different tubes and stored at -80°C.

To enrich for crypt-cells portion, before the incubation with the lysis buffer, the samples are scrapped with a slide to remove the villi part. These villi cells are collected by pipeting in 1 ml of cold PBS. The rest of organ is incubated with 10 ml lysis buffer to dissociate crypt cells.

Western blots

Proteins were prepared from cell preparation by lysis in cold RIPA buffer (50 mM Tris pH 8, 150 mM NaCI, 1% NP40, 0.5% deoxycholate) supplemented with inhibitors. Protein lysates were separated by gradient 4%-15% SDS-PAGE (BioRad) and transferred to a nitro-cellulose membrane (Amersham Protran 0.2µm NC). Membranes were blocked with 2, 5% non-fat milk (w/v) in TBS with 0.05% Tween and incubated with the appropriate primary antibodies at the appropriate dilution and followed by incubation with secondary antibodies conjugated to a fluorophore. Fluorescence was revealed using a scanner (Amersham Typhoon).

Primary antibodies from Cell Signaling: ATM D2E2 (CST 2873S) rabbit at 1/1000, ATR E1S3S (CST 13934S) rabbit at 1/1000, mTOR (CST 2972S) rabbit 1/1000, p53 1C12 (CST2524S) mouse 1/1000; PRP8 (sc-30207, Santa Cruz) rabbit 1/200; from ABCAM: EFTUD2 (ab72456) rabbit 1/2000, GAPDH (ab8245 6C5) mouse 1/10000; and from Sigma: NOP58 (HPA018472) rabbit 1/100, RPAP3 (SAB1411438) rabbit 1/1000, Tubulin (I2G10) mouse 1/500, TRRAP 2TRR-1B3 (MABE1008) mouse 1/1000. All antibodies have been validated by manufacturers.

DNA extraction.

Samples are incubated in 100 µl of alkaline lysis buffer (NaOH 25mM, EDTA 0,2mM) with at 92°C for 20 min. Then, they are mixed with 100 µl of neutralizing reageant (Tris-HCl-pH 5, 40mM). 2 µl were used for PCR. PCR products are respectively 400bp and 300bp for floxed and wild type allele.

Southern blot

Briefly, ES cells were incubated o/night at 65°C in 100mM Tris HCL pH8.8 -5 mM EDTA -0.2% SDS -200 mM NaCl with 0, 1 mg/ml proteinase K in a humid chamber. On the following morning, the lysates were extracted twice with phenol pH= 7, 0-8, 0 and once with chloroform and precipitated with NaCl/EtOH. Following recovery, 10 -15 g genomic DNA were digested for 24h to 48h with appropriate highly concentrated restriction enzymes (40-50 U/l -10 U/g gDNA). The analysis was performed by Southern blot as described in [START_REF] Brown | Southern blotting[END_REF].

PCR for genotyping

Initatial denaturation:95°C ( 2min), Denaturation 95°C (30sec), Annealing 55°C (45sec), Extension 72°C( 45sec), Final extension : 72°C ( 2min) 35 cycles.

Name

Sequence

F5 GGTGCCACAGTGTGAGTG R5 TGCCTCCTGACTCACTACAG R6 ACCGTGTCGCTACGTCACTGCG CREERT2-F CREERT2-R TTACGGCGCTAAGGATGACT AAGGCCAGGCTGTTCTTCTT

Statistical analysis

For the outcome endpoints, the disease-free survival (DFS) was defined as the measure of time after treatment during which one of the following events occurred: relapse at local or distant

Figure 1 :

 1 Figure 1: Rpap3 deletion compromises the small intestine and mouse survival.

Figure 2 :

 2 Figure 2: Rpap3 is necessary for proliferation in the small intestine.

Figure 3 :

 3 Figure 3: Rpap3 invalidation induces CBC stem cells loss, p53 stabilization and apoptosis

Figure 4 :

 4 Figure 4: Rpap3 deletion decreases expression of R2TP clients and leads to cytoplasmic

Figure 5 :

 5 Figure 5: Cytoplasmic accumulation of Rpb1 in RPAP3 KO crypts correlates with

Figure 6 :

 6 Figure 6: R2TP expression correlates with pathological cell proliferation.

  sites, or intercurrent death without recurrence. Overall survival (OS) was defined as the time between surgery and death from any cause. Survival curves were analyzed by the Kaplan-Meier method and compared using the log-rank test. Cox's proportional hazards model, adjusted for other prognostic factors (i.e., gender, tumor location, tumor grade, tumor stage, and RPAP3 status), was used to evaluate the association of RPAP3 expression with outcome.

	Transcriptomic analysis of R2TP subunits in human samples were extracted from the
	CODREAD cohort available at UCSC Xena platform for public and private cancer genomics
	data visualization and interpretation https://xenabrowser.net/ (Goldman et al., 2018). One-way
	ANOVA test was performed using GraphPad Prism 5.0.
	Statistical analysis was performed employing the following two statistical software
	packages: GraphPad Prism 5.0 and SPSS 15.0 (SPSS Inc., Chicago, IL); p < 0.05 was
	considered as statistically significant. For group comparisons, normality was first tested to
	choose a pertinent statistical test, as described in the text and Figure legends.

Acknowledgments

We thank Solange Morera and EMBL facility for help with anti-RPAP3 antibody generation. The ES cells used for this research project were generated by the trans-NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org). NIH grants to Velocigene at Regeneron Inc (U01HG004085) and the CSD Consortium (U01HG004080) funded the generation of gene-targeted ES cells for 8500 genes in the KOMP Program and archived and distributed by the KOMP Repository at UC Davis and CHORI (U42RR024244). We thank the CIGM team in Institut Pasteur for technical support in microinjection experiments and animal husbandry. We thank the in-house animal facility, RAM, MRI for their excellent support. We acknowledge the "Réseau d'Histologie Expérimentale de Montpellier" (RHEM) facility supported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, the European Regional Development foundation and the Occitanian Region for processing our animal tissues. We thank the SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553. The work was supported by La Ligue Nationale Contre le Cancer (équipes labelisées to EB, PJ) and the INCa grants PLBIO 2016-161 to DH, EB, MH, BPB and PLBIO 2018-158 to PJ. CM and CA Ph.D. fellowships were from la Ligue Nationale Contre le Cancer.

Data and sample availability statement

Mouse sperm for the different strains is available upon request to pradet@crbm.cnrs.fr.

Transcriptomic analysis of R2TP subunits in human samples (Figure 6A) used the UCSC Xena platform for public and private cancer genomics data visualization and interpretation https://xenabrowser.net/ [START_REF] Goldman | The UCSC Xena platform for public and private cancer genomics data visualization and interpretation[END_REF],

The data concerning the patient biopsies (Figure 6B) are not publicly available because the database contains sensitive information that could compromise patient privacy.

Details about the experimental procedures are available upon request to pradet@crbm.cnrs.fr.

Authors contributions

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Methods

Human colorectal sample collection and analysis, and Tissue Microarray (TMA) construction

A total of 177 CRCs were collected from patients surgically treated at the University "G. D 'Annunzio", Chieti, Italy, between 1996 and2010. Only the CRC patients who did not receive adjuvant systemic therapy were included in the study. The median follow-up was 53 months (range 3-238 months). During the follow-up, 25.4% of CRC patients (45 out of 177) had a disease relapse, while deaths were observed in 18.6% of CRC patients (33 out of 177). Tumor stage was determined according to the American Joint Committee on Cancer (AJCC) TNM staging system (8th edition). Histologically, each CRC case was graded according to the criteria of the WHO classification of tumors of the digestive system (4th edition). Patients and tumor characteristics are summarized in Supplemental Table 1&2. The study was reviewed and approved by Institutional Research Ethics Committee and written informed consent was obtained from all patients.

TMAs were constructed by extracting 2-mm diameter cores of histologically confirmed neoplastic areas from the 177 CRC cases using a manual Tissue Arrayer (MTA, Beecher Instuments, WI), as previously detailed [START_REF] Lattanzio | Overexpression of activated phospholipase Cγ1 is a risk factor for distant metastases in T1-T2, N0 breast cancer patients undergoing adjuvant chemotherapy[END_REF].

Mice generation and treatments

Mouse experiments were performed in strict accordance with the guidelines of the European Community (86/609/EEC) and the French National Committee (87/848) for care and use of laboratory animals and comply the ARRIVE guideline. RPAP3wtsi/+mice were generated from ES cells generated by the trans-NIH Knock-Out Mouse Project (KOMP) and obtained from the KOMP Repository (www.komp.org). Genotyping was performed by PCR amplification using primers F5, R6, on the mouse tail genomic DNA (gDNA -Supplemental Figure 1A). Mice were bred in an SOPF animal facility and maintained during the experiment in an SPF animal preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

All antibodies used for immunostainings are described in Supplemental Table 4 and validated by the manufacturers, except for 19B11 [START_REF] Boulon | HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II[END_REF], [START_REF] Machado-Pinilla | Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs[END_REF].

-galactosidase activity

Small intestines and colons cryo-preserved were cut to slices of 10 μm thickness. Samples were treated with 0.5% glutaraldehyde for 10 min at room temperature, followed by 24h incubation at 37°C in a staining solution containing 1 mg/ml X-gal, 5 mM potassium ferricyanide (K3Fe(CN)6), 5 mM potassium ferrocyanide (K4Fe(CN)6) and 2 mM MgCl2, 0.1% Triton in PBS.. Samples were washed in PBS for 5 min, counterstained with Nuclear Fast Red and briefly rinsed with dH2O before mounting on aqueous mounting agent Aquatex (108562; Merck).

Microscopy and imaging

Histological slides were scanned using Nanozoomer 2.0 HT scanner with a 40x objective, and visualized with NDP.view2 viewing software (Hamamatsu). Fluorescent images were acquired on a brightfield microscope (Leica) using Metamorph software or inverted Confocal SP5 (Leica) using the Leica LAS AF software. Images were processed with ImageJ. Images were assembled and adjusted with Adobe Photoshop/ Illustrator.

Isolation of epithelial cells from the intestine

Small intestines and colons were isolated and flushed with cold PBS. Colon and the 3 intestine fragments were cut open length-wise, then kept in 10 ml cold wash buffer (PBS, 2% FBS and antibiotics). Tubes were shaken several times by hand to wash the fragments and put into 15 ml falcon tube containing 10 ml CE buffer (PBS, 1% BSA, 1mM DTT, 1mM EDTA, 5, 6 mM glucose). Tubes were placed on a vertical shaker at 37°C for 30 min. After removing tissue, preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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