Pascal Yan

Sayegh Phd

Cultural Hybridity and Modern Binaries: Overcoming the Opposition Between Identity and Otherness? Draft paper "Cultures in Transit" conference Liverpool, 2008

Introduction

German sociologist Ulrich Beck argues that we have entered "late modernity", or in other words, "reflexive modernity". It is not the aim of the paper to discuss Beck's considerations but to set a general context for the following reflection. In his dramatic account of risk society, Beck argues what reflexive, radicalised modernity puts to the foreground: a paradigmatic change in which "we" have no other choice than to reflect on and even think beyond dualisms. It is a paradigmatic change as these dualisms or binaries are "modern mental habits" [START_REF] Ulrich | Risk Society: Towards a New Modernity[END_REF][START_REF] Ulrich | Risk Society: Towards a New Modernity[END_REF]. One particular habit of thought of the "modern imaginary", is the binary opposition between the Self and the Other. Beck argues that "we" (the Self) can no longer imagine "ourselves" in such a binary opposition: the Other is among "us", and even more, "we" are the Other. [START_REF] Ulrich | Risk Society: Towards a New Modernity[END_REF] This relates of course to what can be described as the postcolonial condition of the contemporary world -a condition that is not new, but which is, or so it seems, more and more experienced in everyday life. (Nederveen Pieterse 2001:238) There are many academic approaches which study and express this condition. We can mention one of the most common metaphors for describing what is considered its main attributes, changeability and uncertainty. The metaphor is "fluidity" or "liquidity". "Fluid identities" or "boundaries", "liquid modernity" or even "liquid life" are but a few of the metaphorical expressions extensively used in the past decade. (See e.g. [START_REF] Bauman | Liquid Modernity[END_REF][START_REF] Bauman | Liquid Modernity[END_REF][START_REF] Bauman | Liquid Modernity[END_REF]Longhurst 2001) It is from this very general setting that I wish to address the debate on cultural hybridity. Hybridity, as it is understood in postcolonial theory, is perceived as having the potential to go beyond the sort of modern binaries from which, as Beck suggests, contemporary social imaginaries have to find a way out. According to Jan Nederveen Pieterse, hybridity is precisely that: "Hybridity is to culture what deconstruction is to discourse: transcending binary categories." (Nederveen Pieterse 2001:238).Yet, as it is pointed out in many works discussing cultural hybridity, the term and the vast array of concepts it encapsulates has raised already long-running discussions and debates.

The term refers notably to discourses and ideologies of racism. Robert C. Young traces the origins of the term back to the early seventeenth century. Derived from Latin, it was seldom found until the nineteenth century but in biological or botanical descriptions. "Its first recorded use in the nineteenth century to denote the crossing of people of different races is given in the OED as 1861." But Young points out that it had also been previously used in the context of race mixture (and especially in relation to human fertility). He also relates the definition found in the OED to other previously used terms, like "mixed" or "intermediate races". The significant point in Young's account of the increasing use of the terms "hybrid" and "hybridity" (and their eventual inscription) in the nineteenth century, is that it "marks the rise of the belief that there could be such a thing as a human hybrid." (Young 1995:6) The belief, in other words, that there are objective different human races (or "species").

Another significant point is hinted with the idea of fertility in the context of nineteenth century racial hybridity. It is a narrative that runs throughout the colonial experience and maybe even beyond. It was believed that the "hybridisation" of different human races would eventually cause the downfall of the different "pure" species. A downfall caused by supposed sexual lust and infinite fecundity of hybrids or to the contrary by their perceived biological inferiority, which made them barren. [START_REF] Brun | Transgressive Hybridity? The Historical Association of Hybridity and Transgression[END_REF] One particular aspect that emerges from this very brief overview of the colonialist ideas around hybridity, is how the mixing of races was considered in a negative way. The negative aspect of human mixing can be found throughout human history, and it is best defined through the perception of its transgressive potential. [START_REF] Brun | Transgressive Hybridity? The Historical Association of Hybridity and Transgression[END_REF] It is also this transgressive, revolutionary aspect that runs through the opposing narrative on hybridity (biological, racial, cultural or simply as a process). Terms like bricolage (or simply colage), métissage, creolisation and indeed hybridity used in cultural studies (and related fields) do not necessarily have the negative take on the concept(s). To the contrary, it is in fact the particularity of the hybrid space to be able to transgress, or better, to transcend, to go beyond. The hybrid position is no longer seen by some as a "badge of failure or denigration, but as a part of the contestational weave of cultures." (Mudrooroo 1990:24) One of the major contemporary theorists recently associated with cultural hybridity is Homi Bhabha. In most of his works, Bhabha considers the interrelations and interdependence between the colonisers and the colonised. Through the colonial experience, the social categories exerted on the colonised (the ideas of superior and inferior human races and cultures for instance) imprints an imaginary, which collides with their own, "displacing" or "disjuncting" it. This "encounter" eventually creates new "hybrid" expressions (of culture, of belonging), which in turn challenge the beliefs and experience of the colonisers. Bhabha argues that these colonial -and postcolonialcultural systems and statements are constructed in a "liminal space": the "Third Space of Enunciation" (Bhabha 1995:209). The aim of his argument is the deconstruction the colonisers' (and more generally Western and modern) essentialist claims of an inherent purity of culture.

Although ground breaking, Bhabha's theory is not without contradictions and has regularly been the subject of criticism. A particularly strong criticism is expressed in the work of anthropologist Jonathan Friedman. One of the main points of Friedman's critique is the elitist approach of, among others, Bhabha's work. He eventually defines postcolonial theory of hybridity as the ideology of a new elite: a "postmodern" cosmopolitanism. (1997:75) In this paper, I would like to address the question whether "cultural hybridity" can indeed transcend modern binaries and whether is not something more (or else) than simply a theoretical construction for the selfidentification of the postmodern elite Friedman points a finger at.

The paper will overview Bhabha's definition of cultural hybridity and Friedman's critique. In spite of what will appear to be rather simplistic arguments, some elements of Friedman's critique can bring up elements for a further reflection. The paper will then explore some tropes that this episode in the debate over "cultural hybridity" leads to. As Friedman sets his critique of hybridity in opposition to what he considers "true" cosmopolitanism to be, we will show how his understanding can be considered as flawed and how hybridity can in turn be considered as being less but meaning more than cosmopolitanism. The paper does not have the pretension to provide a comprehensive study of hybridity theories or of the debates around it, and as such is only the starting point of a wider reflection on contemporary social modes of exclusion and inclusion.

Varieties of Hybridity and the Discourse of Critique

As much as hybridity theory aims for the deconstruction of essentialist categories, it has been criticised on grounds that it can only make sense "on the assumption of purity" (Nederveen Pieterse 2001:221; see also [START_REF] Young | Colonial Desire: Hybridity in Theory, Culture and Race[END_REF]. Nederveen Pieterse who has more extensively analysed the debate over hybridity, distinguishes two different varieties. One of those, "new hybridity", is a process that can be observed (Mandarin pop, e.g.). The other variety, "existing or old hybridity", is a discourse and a perspective, which creates a "hybridity consciousness". Additionally, they connect in the experience of the "new" phenomona ("new hybridity") and through the self-conscious perspective taken on performing and experiencing the processes ("exisiting or old hybridity").

"Hybridization as a process is old as history, but the pace of mixing accelerates and its scope widens in the wake of major structural changes, such as new technologies that enable new phases of intercultural contact. [...] If practices of mixing are as old as the hills, the thematization mixing as a discourse and perspective is fairly new." (ibid. 222) This twofold project, of describing hybrid narratives and "thematising" the experience and the self-conscious perspective, is at the core of Bhabha's works on cultural hybridity. In his theorisation, it precisely refers to "the creation of new transcultural forms within the contact zone produced by colonisation." (Ashcroft et al. 2003:118) These new forms, Bhabha argues, come together as a counter-discourse to the discursive dominance of the hegemonic structures and institutions of colonisation. The main narratives it opposes are what Bhabha considers to be essentialist national narratives of culture and belonging. The significance of these counternarratives is their "negotiation" of space where hegemonic discourses homogenise culture and society. This negotiation as a constant endeavour "that seeks to authorize cultural hybridities that emerge in moments of historical transformation." (Bhabha 1994:2) The coerciveness of hegemonic narratives can nevertheless be overcome. The "Third Place of Enunciation" in which cultural hybridity comes into constant formation is a place of movement, of "fluidity", which opposes the traditional fixity of national narratives. Referring to artist Renee Green's metaphor of the art gallery as a stairwell Bhabha develops the metaphor for describing the cultural negotiation which takes place in this space of "différance"1 :

" The stairwell as liminal space, in-between the designations of identity, becomes the process of symbolic interaction, the connective tissue that constructs the difference between upper and lower, black and white. The hither and thither of the stairwell, the temporal movement and passage that it allows, prevents identities at either end of it from settling into primordial polarities. This interstitial passage between fixed identifications opens up the possibility of a cultural hybridity that entertains difference without an assumed or imposed hierarchy. " (1994:5) Cultural hybrid expressions ,which have emerged from colonisation, are marginal cultural narratives. The space they need to be expressed cannot be imagined in the binary categories traditionally associated with essentialist discourses: " [...] the very idea of a pure, 'ethnically cleansed' national identity can only be achieved through the death, literal and figurative, of the complex interweavings of history, and the culturally contingent borderlines of modern nationhood." (1994:7)

At the same time, Bhabha sees the discourse of these narratives (as well as his) as the discourse of critique that can break down these essentialist barriers and make sense of the historical complexity (opposed to the historicity of the hegemonic narratives of the nation). As such, he proposes a further, political perspective for hybridity to take on:

"The language of critique is effective not because it keeps forever separate the terms of the master and the slave, the mercantilist and the Marxist, but to the extent to which it overcomes the given grounds of opposition and opens up a space of translation: a place of hybridity, figuratively speaking, where the construction of a political object that is new, neither the one nor the other, properly alienates our political expectations, and changes, as it must, the very forms of our recognition of the moment of politics." (Bhabha 1994: 37) From this very brief overview of Bhabha's contribution to the postcolonial theory of hybridity, two dilemmas already emerge. The first is related to the already mentioned problematic "assumptions of purity". The ambivalence of Bhabha's account on the inclusive and exclusive properties of hybridity is confusing. On the one hand, liminal space is supposed to prevent "identities at either end of it from settling into primordial polarities", suggesting that the polarities are in fact the boundaries of liminal space (and not spaces themselves), and as such, are included within the former. On the other hand, it is an "interstitial passage between fixed identifications", between fixed "imaginaries", which supposes (just like the concept of "Third Space") that they are indeed spaces. This lack of clarity, although not necessarily contradictory, needs to be clarified. We shall focus on it further below.

The second dilemma, which is one of the point of criticism mentioned before, concerns the sources Bhabha uses to describe and conceptualise narrative processes of cultural hybridisation. In The Location of Culture (1994), he draws on a very large array of literary, artistic and theoretical texts. The interweaving of all these texts create a complex and hermetic whole which can indeed give the impression to voice an elite condition (albeit marginal) rather than the commonality and daily experiences of displacement. The lack of clarity, as mentioned above, helps to produce this impression. But Bhabha describe his endeavour in different terms, and reverses the argument:

"There is a damaging and self-defeating assumption that theory is necessarily the elite language of the socially and culturally privileged. It is said that the place of the academic critic is inevitably within the Eurocentric archives of an imperialist or neo-colonial West." (1994:19) It is sometimes seen as ironic coming from one of the leading postcolonial theorists that has throughout his career been subject to charges of elitism and the like [START_REF] Graves | An Overview[END_REF]. Friedman is no exception to the rule. We shall take the charge of elitism as the starting point of his critique.

Essentialist Hybridity and Essentialist Critique

Jonathan Friedman seems to hold a deeply rooted grudge against postcolonial theory of cultural hybridity as well as towards its theorists. In relation to Bhabha's approach, we shall review [START_REF] Friedman | Global Crisis, the Struggle for Cultural Identity and Intellectual Porkbarelling: Cosmopolitans versus Locals, Ethnics and Nationals in an Era of Dehegemonisation[END_REF] main arguments. As Nederveen Pieterse's analysis shows, Friedman's arguments against hybridity can be considered "[...] as representative of a wider view." (Nederveen Pieterse 2001:224) Contrary to Bauman's metaphor of "liquidity", Friedman gives a dramatic and alarming account of the world literally falling into pieces, moving away from the neat modernist classifications. Asserting that "the general fragmentation process of the world system" is under way, he considers the concurrent "theorisation of creolisation, métissage, mestisaje, and hybridity" [START_REF] Gilroy | The Black Atlantic: Modernity and Double Consciousness[END_REF]:2, quoted in Friedman 1997:75) to be: "[...] the intellectual cosmopolitan reaction to that process, one that contains a highly ambivalent posture with regard to to the ethnification process itself and the desire for something broader, more global, truly cosmopolitan and above it all. This is the hyphenated reality of the postmodern cosmopolitan, a reality that is defined not by the modern, the abstract, but by the plurality of knowledges, of cultures and of their continuous fusion." (Friedman 1997:75) If the theorisation of hybridity is the "self-identification" of postmodern cosmopolitans, Friedman argues that "it has little to do with everyday problems of identity in the streets, even as it is part of the same world." (1997:74) This is the recurrent elitist argument against theorists like Bhabha or Paul Gilroy. Friedman rhetorically asks: "[...] For whom, one might ask, is such cultural transmigration a reality? In the works of the post-colonial border-crossers, it is always the poet, the artist, the intellectual, who sustains this displacement and objectifies it in the printed word. But who reads the poetry [...]?"(1997:79)

One would like to ask whether Friedman does not have a narrow view on the question. But, we shall not linger discussing it more here (for a more thorough analysis see Nederveen [START_REF] Pieterse | Hybridity, So What? The Anti-Hybridity Backlash and the Riddles of Recognition[END_REF]. What explains it is his own ideological perspective, the criticism of which has been left out from previous reviews of the debate. Through elements than run throughout his critique, Friedman also presents himself as a proponent of cosmopolitanism, but not of the same "age", or of the same "sort". It is significant in the sense it establishes a framework from which Friedman expresses the more "direct" criticisms.

The new condition of the world, the one Bauman describes as "liquid", is defined in Friedman's "neomedievalism" assertions as a sort of chaotic dark age (which "is one of the accounts for current political conditions", Nederveen Pieterse 2001:238) In this light, Friedman considers that the "model" for postmodern cosmopolitanism "is not the macro-nation but the medieval Church, the great encompasser. Ecumenical pluralism is the complementary counterpart of fragmented ethnic identities." (Friedman 1997:75) What transpires here is an opposition between modernity, or the modernist project Friedman associates with it, and what he describes as the postmodern condition of the world ("ethnification") and postmodern cosmopolitanism. Friedman suggests that this condition has taken the world over, "forsaking modernity" (1997:72). This "abandonment of the ideal of a strong social project and assimilation to that project" for the sake of multiculturalism, "is the expression of a broad shift in the 'identity space' of declining Western modernity." It is in these dark ages that a new cosmopolitanism is thriving. Friedman considers the cosmopolitan project that dominates this new age to be very different from the one that used to dominated in the modern world. It is a "[c]osmopolitanism without modernism" and yet "not without modernity as such, but without the rationalist, abstract and developmentalist project of modernism. " (1997:76) By describing what is lacking, Friedman gives a gist of what the "old" cosmopolitanism was. Indeed:

"The cosmopolitan of old was a modernist who identified above and beyond ethnicity and particular cultures. He was a progressive intellectual, a believer in rationality who understood cultural specificities as expressions of universal attributes. The new cosmopolitans are ecumenical collectors of culture. They represent nothing more than a gathering of differences, often in their own self-identifications." (1997:83)

The harsh criticism of the last sentence may divert attention from what the remainder of the quote suggests: Friedman appears to take the position of a nostalgic herald of the cosmopolitanism "of old". By taking side with a suggested idealist modernist approach, his critique appears to formulate a struggle for hegemony against "hybridization [which] is a political and normative discourse. " (1999:242) The binary opposition constructed by Friedman is in fact representative of the essentialist categories opposed by postcolonial theory. This provoking set of mind is also contained in the title of one of his articles: "Global Crisis, the Struggle for Cultural Identity and Intellectual Porkbarelling: Cosmopolitans versus Locals, Ethnics and Nationals in an Era of Dehegemonisation" (1997).

The following point of his critique lies in what he calls a simple "descriptive statement" (1997:82), namely that hybridity bases its possibility of identification on essentialist notions of identity and culture. In turn, it is hybridity theory that is considered essentialist. Commenting on what he calls the "programme Bhabha envisaged", he considers it establishes: "[...] a world in which the homogenising tendency of all identification are eliminated not via modernist anti-cultural identity, but by a postmodernist total fusion of all cultures into a new heterogeneous homogeneity of the 'third space', which, if it is a space, must have boundaries of its own, and thus be based on oppositions to its own others. [...] It is precisely in the metaphor of border-crossing that the notion of homogeneous identity is carried and reinforced, since it is a prerequisite of such transgression. " (1997:79) In relation to the lack of clarity and ambiguities found in Bhabha, and regardless of the elitist argument, it is hard not to give credit to Friedman's criticism. It shows how the possible interpreting of liminal space as exclusive of essentialist notions (like those of homogeneous identities), can be considered to consequently reinforce their perceived fixity. A fixity opposed to the movement, to the "différance" contained in the liminal space.

And yet, "différance" hints to a different interpretation. Friedman is again right to point a finger at the fact that "all cultures have always been the product of import and a mix of elements" adding with a sense of superiority that this "was a commonplace for early cultural anthropology". As a consequence, post-colonial theorists suffer from "a confusion of perspectives" (1997:80). But this is a point where Friedman's argument is flawed. Reversely, his suggestive defence of modernist cosmopolitanism shows that his take on the work of post-colonial theorists, including Bhabha's, is also based a misconception. It is in terms of representations and imaginaries that liminal space and hybridity as well as essentialist, homogeneous notions of culture and identity can make sense. In consequence, his criticism of "postmodern cosmopolitanism" as an elite discourse may well be justified. But his own discourse, albeit self-identified to be "above and beyond ethnicity" is also, as we will see, hegemonic and particularistic.

From One Cosmopolitanism to Another

The brief summary in the first part of this paper of Bhabha's theory of hybridity suggested how its interpretation is not necessarily clear. The lack of clarity in theorising the complexity and the "varieties" of hybridity is particularly evident the lack of coherence between the various aspects of the theory. This opens space for simplified interpretations on which a certain type of criticism can thrive. As it is reviewed in the second part, Friedman bases his critique on such interpretation. His aim appears in fact to be the deconstruction of the theory. He points at what he sees to be the limitations of the theory, its essentialist foundations, and that hybridisation is an anthropoligical evidence. This somehow closes the scholarly discussion. He concludes though that the theory is in fact a political endeavour representative of the postmodern condition of the world: the discourse of a new cosmopolitan elite looking for an overarching identity. In other words, looking to establish its discursive hegemony. Notwithstanding Bhabha's ambiguities, the political project (the "hybrid perspective") is indeed clearly stated. Similarly, other theorists do in fact argue in favour of what is pervieved as a political resistance of cultural representations or a "radical imaginary" hybridity can produce. So it seems that Friedman is right, and that there is a self-conscious element of the theorisation of hybridity. But one could argue that this, as well as hybridisation, is self-evident. Discursive formations are precisely about knowledge and power (although again, Friedman seems to discard the Foucauldian approach, 1997:72).

More precisely, the reason why pointing at the political aspect is a criticism in Friedman's eyes lies in the position he takes. His rather idealistic and naïve belief in the modernist cosmopolitan project, shows that despite his acute critical look on hybridity, his is not self-critical. He equates his position to a wider anthropological approach, taking Claude Lévi-Strauss as the main reference for the "critique of the chimera of a new multicultural world" (1997:81).

But had he taken Lévi-Strauss' word for granted, he would maybe have formulated a more critical standpoint. He uncritically shows how Lévi-Strauss' support for a possible "larger cooperation" of "the world's cultures" has shifted towards the impossibility to transcend "ethnocentrism": "[...] racism is not the same as ethnocentrism, and [...] the latter is an inescapable tendency that has little to do with learning to think correctly. No intellectual process can eliminate this phenomenon, simply because it has nothing to do with falsity of fallacy. " (1997:77) This leads to the conclusion that "strong cultural identities are the source of cultural creativity, and that there is nothing wrong with this as long as it does not lead to racism." Still referring to Lévi-Strauss, Friedman writes: "[h]e goes even further, suggesting that if cultures exchanged all their elements with one another on a continuous basis, there would no longer be any differences, and thus no mutual attraction." (ibid.)

The exclusion of relations of power and dominance in the abstract cultural exchanges he mentions show the oversimplification of his perspective. This is a crucial point as the political endeavour of postcolonialist theory can precisely be summed up in the deconstruction of power relations that have lead to hybridisation -and often not the least of them: the coloniser/colonised relation. This in short means that they are historical relations and that their localised context can hardly produce a systematic theory, even though certain patterns can reappear in different contexts. This why Bhabha for instance highlights a necessary interrelation between theory and practice (1994:19). But it might be here useful to turn to critical theories not generally associated with postcolonialism. In relation to ethnocentrism and racism, Friedman (through Lévi-Strauss) claims that they are not the same. Friedman dismisses their complex correlation maybe too quickly. Although ethnocentrism is indeed not racism as such, it can in fact lead to it. Philosopher Cornelius Castoriadis offers a more profound understanding of the relationship between racism and other forms of particularism and exclusion. In his article "Reflections on Racism" (1990), Castoriadis theorises racism as the "most acute tip" of exclusionary phenomena (40). Here these phenomena can be summarised as those that reject the Other, more or less violently, more or less figuratively. He further suggests how the tendency of rejecting the Other, like ethnocentrism, is perceived as "natural" given the constitution of identity throuh binary oppositions. But Castoriadis is also concerned with social representations, with imaginary formations and their institutionalisation. He thus draws a very subtle but significant distinction. He argues that the perceived "natural" tendency is in fact the "extreme probability" for social institutions and imaginaries to take ( 41), but that a different formation, a "radical imaginary" is possible, although historically highly improbable. Of course, this consideration does not close the discussion, it only shows the continuum and complexity of social modes of exclusion.

Focusing then back on Friedman, we have seen how he suggests a rationale for modernist cosmopolitanism:

"The cosmopolitan of old was a modernist who identified above and beyond ethnicity and particular cultures. He was a progressive intellectual, a believer in rationality who understood cultural specificities as expressions of universal attributes. " (1997:83) It is representative of the type of Western ethnocentrist discourses that carries essentialist notions. It also raises the issue of loyalty as it is to be found in the political or "ethical cosmopolitanism" defended by Jürgen Habermas to move beyond particularistic solidarities (1998).

Apart from the nostalgic note on the "faith" in rationality, the corollary attribute of the modern cosmopolitan is the knowledge about "universal attributes", or more simply, his universal identity.

It is again here useful to turn to another scholar, sociologist Craig Calhoun, who has produced a critical work on belonging and "modern" cosmopolitanism. Calhoun shows how cosmopolitanism renders "culture an object of external consumption rather than internal meaning", which relates to Friedman's and Habermas's "abstract" loyalty. And he concludes:

"No one lives outside particularistic solidarities. Some cosmopolitan theorists may believe they do, but this is an illusion made possible by positions of relative privilege and the dominant place of some cultural orientations in the world at large. The illusion is not a simple mistake, but a misrecognition tied to what Pierre Bourdieu called the "illusio" of all social games, the commitment to their structure that shapes the engagement of every player and makes possible effective play. In other words, cosmopolitans do not simply fail to see the cultural particularity and social supports of their cosmopolitanism, but cannot fully and accurately recognize these without introducing a tension between themselves and their social world. And here I would include myself and probably all of us. Whether we theorize cosmopolitanism or not, we are embedded in social fields and practical projects in which we have little choice but to make use of some of the notions basic to cosmopolitanism and thereby reproduce it." (2007:25-26) 4. From "Third Space" to "Second Nation"

One could wonder what we are left with. If on one hand hybridity theories are simply elite discursive formations of a certain sort of cosmopolitanism fighting over a hegemony with other cosmopolitanisms, and on the other, none of them is able to transcend essentialist categories, are we left with bitter cynicism? However, Calhoun does not refrain from formulating a political perspective in continuum to his critique: "Cosmopolitanism by itself may not be enough; a soft cosmopolitanism that doesn't challenge capitalism or Western hegemony may be an ideological diversion; but some form of cosmopolitanism is needed." (2002) He suggests that: " [...] we should want to transform it, not least because as usually constructed, especially in its most individualistic forms, it systematically inhibits attention to the range of solidarities on which people depend, and to the special role of such solidarities in the struggles of the less privileged and those displaced or challenged by capitalist globalization. " (2007:26) Postcolonial theory and related studies appear as one domain where the idea to provide a space for the expression and the formulation of marginal stories of solidarities is fundamental. But as Friedman critique of Bhabha's show, and in the same vein of Calhoun's insight, it has to carefully reflected and studied.

A certain number of studies show how what may be interpreted as new imaginings, as the promotion of hybrid identities, can in turn become exclusionary practices. In the case of such a narrative reaching the comfort of discursive hegemony, however new it is, it appears to falls back into the traditional set piece of the Self and the Other. Viranjini Munasinghe shows for instance in his case study on Indo-Trinidadians in Trinidad, how the Trinidadian national narrative celebrates hybridity and impurity, and as such seems to form a counter-narrative to Western national discourse. However "[l]ike all nationalist narratives [...] it remains a narrative that excludes, in this case those people who were thought to embody purity because they never mixed in the first place." (2002:685) Like Friedman's "postmodern cosmopolitanism", this suggests that hybridity as a theme, formally and consciously addressed, can become an essentialist category in itself. Social imaginaries are maybe necessarily exclusive at some point. But it becomes rather contradictory and even tragic when this exclusion is justified on grounds of an essential hybridity.

I would like to further the second interpretation of hybridity as an inclusive process rather than as an type of identities or cultures. Calhoun suggests that we should not dismiss the value of particularisms. It is, like Bhabha suggests, on the margins of the nation that hybrid perspectives can promote inclusive imaginary significations (1990:291-319). As we also read in The Location of Culture, it is "in moments of historical transformation" that "cultural hybridities [...] emerge" (994:2). In other words, it is during critical moments of struggles for recognition, and not necessarily for hegemony, that new imaginings of can emerge. On the same thread, recognition can mean inclusion, but of course, does not necessarily mean their institutionalisation. These issues would demand a wider reflection on the establishing of structural and institutional social and political formations which goes beyond the scope of the present paper.

In light the situational aspect and its importance in the emergence of hybridity, it would be useful to look a bit closer at such a situation not so far from "us". In 2005, France saw a wave of socalled "suburban riots" during which, teenagers living in housing estates took the streets. Images of French cities burning and falling into chaos were thoroughly aired throughout media networks. Politicians and alike also took advantage of the situation, and risk-driven and nationalist discourses were easily justified. What interests me here though are not the "crisis of the banlieues", but the period that followed it.

The spectacle and experience of the crisis fostered an already latent uneasiness towards French new minorities and immigrants (particulary as representative of the peoples who inhabit the housing estates). In 2007, the "failure of the French integration system" was part of president Nicolas Sarkozy's nationalist rhetoric during his campaign. On the other hand, a certain number of press articles, radio shows, television documentaries, and books on the "banlieues" and their "infamous" inhabitants have emerged [START_REF] Horobin | Figuring the banlieues: contemporary political discourse in France h, Modern Languages and Critical Theory MA thesis[END_REF].

But this crisis has also produced a mobilisation to counter this dominant narrative. Many rap or hip hop artists for instance who have for the past two decades been the heralds of the inhabitants of the "banlieues", have recently reaffirmed this status. I would like to mention just one, Kery James, whose latest video clip, "Banlieusards" (the term derives from "banlieues" and refers to its inhabitants) was extensively screened on French television networks and the internet earlier this year. At least since 1996, he is regarded as a spokesman for the "banlieues" along with other members of his rap formation of the 1990's Ideal J. They became famous with their first album Original MC's sur une mission with notably a song entitled "Ghetto français" ("French Ghetto"). They obviously refer to the "banlieues" (although born in Guadeloupe, James was raised in a housing estate in the south-east of Paris, in Orly).

In the video clip of "Banlieusards", we can see a picture frame being passed on from James to many famous and professionally successful people who come from the "banlieues". The frame is passed on throughout the video clip, taking snapshots of each of the participants. Under each frame appears a caption with the name and profession of the person in the frame. In the end of the clip, we can see all the participants singing the final verse with James.

The lyrics do not focus on cultural or ethnic themes (there is a brief mention about the varied skin colour of the "banlieusards" and about colonisation). But the main theme that runs throughout the song is about the marginal social and economic situation of the peoples of the "banlieues". The people, "we", are opposed here to the state, "them" -and not to the mainstream community: "[...]nous dans les ghettos, eux à L'ENA/ Nous derrière les barreaux, eux au sénat/ Ils défendent leurs intérêts, éludent nos problèmes"2 . Here James does not address "them", but the "banlieusards". In a nutshell, his message holds in the rhetorical question: "Mais [...] qu'a-t-on fait pour nous même ?"3 The motto "on n'est pas condamné à l'échec"4 is repeated throughout the song and also appears on the T-shirt James wears in the video clip.

What is important is that the theme of ethnicity and cultural particularism is turned upside down. James explicitly expresses the idea of an inclusive French society:

" Le 2, ce sera pour ceux qui rêvent d'une France unifiée/ Parce qu'à ce jour y'a deux France, qui peut le nier ?Et moi je serai de la 2eme France, celle de l'insécurité/ Des terroristes potentiels, des assistes/C'est c'qu'ils attendent de nous, mais j'ai d'autres projets qu'ils retiennent ça."5

Leaving aside the rest of the lyrics which would demand a much more extensive analysis, I would like to extract some of the significations from the elements already presented. James, who would certainly fit the "elitist" box in Friedman's critique, makes a very significant distinction between the people and the state, arguing for a unity of the people without the state, without a revolutionary project of hegemonic institutionalisation. This sort of shift in the significations from the master narrative of the nation-state and nationalism (e.g. "one people, one state"), is exactly what one would expect to find in the expression that emerges from a critical moment. Thematically though, there is no hybrid theme. What we find for instance are themes related to pluralism (the images form the video clip also bring this out) and to a lesser degree of a working class consciousness. It not a call for anarchy, but a message driven by a very Marxist assumption that the ruling class wants them to play a certain role (the scapegoats?) and is driven by its own interest.

What is interesting in terms of hybrid imaginary representations, is that the process is inclusive of what would traditionally (or in the dominant discourse) be considered the Self and the Other. Both, the "banlieusards" and the rest of French society are part of the project of unity James expresses.

Conclusion: Rap and Différance

The potential for hip-hop movements to "transcend the divisions that are ever more openly fostered by the French state" was already identified in 1997 by Steve Canon (1997:163, quoted in Horobin 2007). The interweaving of cultural, political and social significations that can be emerge from these productions present what should remain at the centre of hybridity theory: the transcending of exclusionary imaginaries. All other elements in a study are historically and geographically localised. It should then be clarified that it is about providing a space for marginal historis (for analysis and expression) rather than leaving it for a deification of "new" forms -even though they might as well be new.

Hybridity is an theoretical tool that is helpful in describing and analysing marginal phenomena, non-hegemonic productions of meaning, not necessarily in terms of identity or culture, but in terms of social and singular imaginary significations. As such, they are indeed rarely unrelated to political significations, and it is not wrong to oppose these to fixed, traditional expressions of culture and identity. What Friedman points out in a simplistic and biased way, is the importance of class relations in the context of cultural expressions and significations. This may lack in Bhabha's account, inasmuch as one does not consider national and traditional hegemonic cultures related to one particular class.

If we take Friedman anthropological statement for granted, namely that all cultures are the result of a process of hybridisation, logically, the spaces of the "one" and of the "other" are the original "third space of enunciation" for those that identify with one. This raises of course problems about the historical development of cultures. It suggests that cultures mix, and eventually reach a certain maturity which finally equals a form of purity, the third space becomes the first one. Similarly, when an identified "hybrid" culture is reified and institutionalised (fixed), however novel it may appear, it eventually becomes a represented closed space of enunciation. It consequently loses its radical power. This is why, as Bhabha writes it in the Location of Culture, it is significant to stress the historical "locality" and not the linear historicity of these phenomena. The historicity can bring validation to expressions perceived as long-running historically (perceived tradition, meaning legitimation) whereas "new" forms will be discarded as simply invented, momentary creations of marginal, up-rooted, unstable or ill-identified people.

Hybridity is then not only a question of race, of métissage, but also of gender, class and maybe above all of imaginary significations that are constantly subjected to "différance". Bhabha is maybe wrong in defining the "place of hybridity" as something "new". Theorising hybridity as " neither the one nor the other" is maybe theoretically very attractive, a revolutionary tabula rasa, but it does not have the performative potential he claims it has (1994:37).

For "différance" to be performed, I would rather suggest a localised and yet unlimited potential of inclusion. If spaces close, the struggle becomes indeed a struggle for hegemony and not for recognition. Binary categories can not be transcended if they are discarded. Literally, Calhoun says we should seek to "transform" them as none is above particularistic and hence essentialist modes of thinking. "Nothing is lost, nothing is created, everything is transformed" goes Greek philosopher Anaxagoras' famous sentence. However, in the world of human relations and expressions, some things get certainly lost. But if "différance" is the mode through which social meanings change, then indeed, no signification is created out of nothing, and all can and will be somehow transformed.

For Derrida, "différance" is how "concepts are inscribed in a chain or a system within which it refers to the other, to other concepts, by means of the systematic play of difference". In Jacques[START_REF] Derrida | Margins of Philosophy[END_REF] (trans. Alan Bass), Margins of Philosophy, Chicago, University of Chicago Press, p.11 (see also pp.3-27).

The ENA stands for the National School of Administration, famously the one institution of higher education in which the majority of French politicians are educated. "We, in the ghettos, they, in the ENA/ We, behind bars, they, in the Senate/ They defend their interests, elude our problems."

"But what have we done for ourselves?"

"We are not doomed to failure"

"The 2 will be for those who dream of a unified France/ Because today there are two France(s), who can deny it?/ And I will be of the second France, that of insecurity/ Of potential terrorists, of social security/ This is what they expect from us, but I have other projects, let them hold to that."


Thomas Koed Doktor

Lisbeth Dahl Schroeder

Anne Vested

Johan Palmfeldt

Henriette Skovgaard Andersen

Niels Gregersen

Brage S Andresen
email: bragea@bmb.sdu.dk

Lisbeth Dahl

Brage Storstein Andresen

Storstein Brage

Andresen

inhibited by binding of hnRNP A1 to a common ESS motif that spans the 3' splice site

Keywords: A1, exon skipping, splicing, exon splicing silencer, SMN1, SMN2, hnRNP A1, exon skipping, splicing, exonic splicing silencer, ESS

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Spinal Muscular Atrophy (SMA) type I (OMIM #253300), type II (OMIM #253550) and type III (OMIM #253400) are neurodegenerative disorders characterized by progressive loss of lower motor neurons (Crawford and Pardo, 1996). SMA is caused by decreased levels of survival of motor neuron (SMN) protein usually caused by deletion of the functional SMN1 gene (Lefebvre, et al., 1995). A secondary gene, SMN2, provides a low level of SMN protein, which is sufficient for survival, but not adequate for proper development and continued survival of the lower motor neurons (Lefebvre, et al., 1997;Monani, et al., 1999). Interindividual variation in the copy numbers of the SMN2 gene account for most of the phenotypic differences in the clinical subtypes of SMA (Feldkotter, et al., 2002;Lefebvre, et al., 1997;Velasco, et al., 1996;Wirth, et al., 1999).

SMN2 differs from SMN1 primarily by a silent c.840C>T transition (corresponding to position +6 in exon 7), which disrupts an exonic splicing enhancer (ESE) within exon 7. The result is predominant skipping of exon 7 (Cartegni, et al., 2002;Cartegni, et al., 2006;Lorson and Androphy, 2000;Monani, et al., 1999) and production of an unstable protein with decreased activity (Burnett, et al., 2009;Butchbach, et al., 2007;Cho and Dreyfuss;Le, et al., 2005). It has also been suggested that the c.840C>T transition instead creates an hnRNP A1 binding exonic splicing silencer (ESS), which causes exon 7 skipping in SMN2 (Kashima and Manley, 2003;Kashima, et al., 2007a). Additionally, several other hnRNP A1 binding elements in intron 6 and intron 7 of SMN2 have been reported to affect splicing (Hua, et al., 2010;Hua, et al., 2007;Hua, et al., 2008;Kashima, et al., 2007b), and inactivation of one or several of these elements increases inclusion of SMN2 exon 7.

We initially noted that there is a strong similarity between the region surrounding the 6 th nucleotide of SMN1 and SMN2 exon 7 and a region located in exon 5 in the MCAD gene (ACADM). In this region MCAD exon 5 harbors a SF2/ASF binding ESE and exon 5 is (Nielsen, et al., 2007). A synonymous single nucleotide polymorphism (SNP) (c.351A>C) located upstream of the ESE in MCAD exon 5 confers immunity towards a mutation (c.362C>T) that disrupts the SF2/ASF binding ESE causing exon skipping and ultimately MCAD deficiency. We have demonstrated that the c.351C variant in MCAD disrupts a CAGGGG ESS element, which functions by binding of hnRNP A1 (Nielsen, et al., 2007). In the MCAD gene, the function of the SF2/ASF binding ESE is to suppress binding of hnRNP A1 to the ESS. When the ESS function is disrupted, the ESE is no longer required for exon inclusion. Interestingly, a sequence very similar to the ESS element in MCAD is present in SMN1 and SMN2 exon 7 at the exact same position within the region of high similarity (Fig 1). The potential ESS sequence element in SMN1 and SMN2 is located across the 3'ss of exon 7 and includes nucleotides previously associated with an extended splicing inhibitory context, which has been suggested to be mediated by formation of a terminal stem-loop structure that inhibited access to the 3'ss (Singh, et al., 2004).

Based on our observations from the MCAD gene, we hypothesized that instead of an inhibitory stem loop structure, the sequence spanning the 3' splice site of exon 7 in SMN1 and SMN2 may function as an ESS and that binding to the ESS is counteracted by the functional ESE at position +6 in SMN1, whereas loss of the ESE in SMN2 shifts the balance, allowing increased binding of hnRNP A1 to the ESS, thereby blocking the 3'ss and splicing of exon 7.

The principal protein recognizing the 3'ss is the U2AF 35 protein, the smaller subunit in the U2AF complex (Merendino, et al., 1999;Wu, et al., 1999). It resembles an SR protein by having an RRM motif and an RS-domain but also interacts specifically with the larger subunit of the U2AF complex, U2AF 65 , which binds to the polypyrimidine tract (Wu and Maniatis, 1993;Zamore, et al., 1992;Zhang, et al., 1992). The U2AF complex is thought to be recruited by SR proteins and together with the branch site binding protein SF1 it recruits the U2 snRNP to the branch site to initiate spliceosome assembly (Brow, 2002;Graveley, et al., 2001;Kent, et al., 2005). Prevention of binding of the U2AF complex to the 3'ss inhibits formation of the spliceosome and usage of the 3'ss (Domsic, et al., 2003;Masuda, et al., 2008;Warf, et al., 2009), and ESS motifs in the immediate vicinity of the 3'ss may consequently cause skipping of the exon. Although some splice sites are AG-independent due to a strong polypyrimidine tract and able to undergo splicing in the absence of U2AF 35 , direct blocking of a 3'ss by binding of splicing inhibitory factors to an overlapping ESS would still be expected to inhibit splicing. Furthermore, the polypyrimidine tract of the 3'ss of SMN1/2 exon 7 has been shown to be functionally suboptimal as substitutions of purines for pyrimidines within the PPT have been shown to increase inclusion of SMN2 exon 7 (Lorson and Androphy, 2000).

In the present study we show that the hypothesized ESS element functions as an ESS in a heterologous context. It binds hnRNP A1 in vitro and mutations that disrupt the ESS element partially restores splicing of SMN2 minigenes in transfection assays. We show by siRNA mediated knock-down that hnRNP A1 can regulate splicing of SMN2 transcripts and that this effect is decreased in an SMN2 minigene with a disrupted ESS element. Thus, the present study identifies a new important splicing regulatory element spanning the 3'ss of SMN exon 7 and shows that it is part of the fine balance between positive and negative regulatory elements, which are decisive for SMN exon 7 splicing. Additionally, this is an example of how hnRNP A1 may function as a general inhibitor of splicing by binding to the 3'ss of canonical exons. By binding directly to a 3'ss, hnRNP A1 may inhibit exon inclusion either by directly blocking the 3'ss or by propagating along the exon or along the PPT to prevent spliceosomal recognition of and recruitment to the exon.

GGCCTTTAAGCTCAGCAAGTACTTACTTGATATTTTGAAGAAATGAGGCCAGTTA

) with HiFi Phusion DNA polymerase (Finnzymes, Espoo, Finland).

The PCR product contains 230 bp of the 3' part of intron 6, exon 7, intron 7 and the first 111 bp of exon 8 from SMN1 and SMN2. This resulted in an 881 bp fragment, containing 839 bp of the gene, additional restriction sites and an artificial exon 8 5'ss site, which was cloned into the pCR-Blunt II-Topo vector (Invitrogen Co., Carlsbad, CA). After sequence analysis the generated plasmids were digested with XhoI and BamHI and ligated into an MCAD minigene (Nielsen, et al., 2007) with an optimized exon 5 3'ss and a BamHI site in intron 5

(for schematic, see Fig 5A). All plasmids were verified by DNA sequencing on the ABI Prism 3100-Avant (Applied Biosystems, Foster City, CA). Variant SMN1/2 minigenes were generated by site-directed mutagenesis using the megaprimer method previously described (Sarkar and Sommer, 1990) with the SMN2wt minigene as template.

pSXN13 minigene. The pSXN13 minigene consists of an artificial small (<40bp) exon positioned between β-actin exon 1 and exon 2 as well as a downstream exon 18 from cTNT.

On both sides of the artificial exon is β-actin intron 1 in order to preserve the natural context of the two neighboring exons (Coulter, et al., 1997;Zatkova, et al., 2004). To generate pSXN13 constructs we used sense and antisense oligonucleotides with desired sequences, which were mixed 1:1, phosphorylated and ligated to the BamHI and SalI sites in the (Zatkova, et al., 2004). The sequences of the generated plasmids were verified by DNA sequencing.

Transient transfection of Chang cells and splicing analysis

Chang cells were seeded in 6 well plates and left to grow until reaching app. 25% confluence.

Cells were then transfected with 0.4 µg expression plasmid, either SMN constructs or pSXN13 constructs, using FuGENE6 transfection reagent (Roche, Mannheim, Germany). All transfections were performed at least 3 times. 48 hours after transfection cells were washed in 1xPBS-EDTA, cells lysed by adding 900 µL TRIzol reagent (Invitrogen) and incubating on ice for 10 min and RNA extracted according to manufactory instructions (Invitrogen).

Purified total RNA was used as template in first strand cDNA synthesis using the Advantage AUUUUUCCUUACAGGACUUUA. Pull-down assay and immunodetection of purified proteins were carried out as previously described (Nielsen, et al., 2007). For immunodetection we used antibodies from Santa Cruz Biotechnologies against hnRNP A1

(sc-10029) or hnRNP H (sc-10042).

Tandem mass spectrometry (MS/MS) protein analysis

Protein eluate from RNA pull down (ca 15 µL) was loaded on a 12% SDS-PAGE gel and run

for 60 min at 220V, 4°C. The gel was stained with Coomassie Blue and individual sample lanes divided into 3 to 5 samples. After destaining the proteins were in-gel trypsin digested, peptides purified on C18 PepClean spin columns (Pierce Biotechnology Inc., Rockford, IL, USA) and vacuum-dried. The peptides were then subjected to reverse phase nano liquid chromatography (nLC) and gradually eluted to the MS, as previously described (Hansen, et al., 2008). Individual proteins were identified (p<0.001) by Mascot (Matrix Science Inc., Boston, MA, USA) and the Mascot score was used as a rough measure of protein abundance in the sample (Kikuchi, et al., 2004). For a more detailed quantification of protein abundance, the corresponding peptides were quantified from the nLC chromatogram (Hansen, et al., 2008). 2 or more peptides per protein were used (in the case of hnRNP A1 4 peptides were used) and each peptide was identified with better than p<0.01. The amount of individual peptides across the samples were normalized relative to the SMN2 3'ss wt sample and taken as a relative measure of protein abundance. In this way, several independent measurements of individual protein abundances were obtained through the peptides produced by trypsin digestion. The whole procedure was repeated twice and data pooled accordingly between experiments. Sequences reported to bind the U2AF complex (Wu, et al., 1999) were aligned and the pictogram program (http://genes.mit.edu/pictogram.html) used to calculate information content bit scores for each position as well as base frequencies and a graphical depiction of the motif based on the frequencies. Using the bit scores, positions -3 to +2 relative to the splice site were selected as the U2AF 35 defining motif. We assumed that the high bit scores observed at position -12 to -5 were due to U2AF 65 binding since Wu and co-workers purified the oligonucleotides using the entire U2AF complex. Using the default background frequencies (0.25 for each base), a position weight matrix (PWM) was constructed (Fig 3)

Generation of U2AF 35 Position Weight Matrix

using the method previously described (Cartegni, et al., 2003).

In silico genomic analysis

For initial analyses of splice sites we used SpliceRack (http://katahdin.cshl.edu/SpliceRack/index.cgi?database=spliceNew) (Sheth, et al., 2006).

For further analysis we used publicly available hg19 data tracks from the UCSC browser (http://genome.usc.edu) to obtain the genomic location of constitutive and cassette exons (Karolchik, et al., 2004;Rhead, et al., 2010). Genomic features were compared using BEDTools, which was also used to extract sequences (Quinlan and Hall, 2010). We wrote custom Perl scripts to analyze and quantitate motif frequencies.

Mutation nomenclature

When nucleotide changes are preceded by a c. (for instance c.351A>C) they refer to the cDNA sequence numbering where +1 corresponds to the A of the ATG translational initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen).

The initiation codon is codon 1. In all other cases, nucleotide numbering reflects SMN1/2 exon 7 numbering, where +1 corresponds to the first base of the exon and -1 to the last base of the preceding intron.

RESULTS

The ESS motif spanning the 3'ss of SMN1 and SMN2 is functional in a heterologous context and can be abrogated by mutations that decrease the hnRNP A1 binding score.

We hypothesized that a CAGGGT motif that spans the 3'ss of SMN1/2 exon 7 comprises a hnRNP A1 binding site, like the CAGGGG motif in MCAD (Nielsen, et al., 2007, see Fig 1), since both motifs score high in an hnRNP A1 binding matrix and hnRNP A1 has been demonstrated to affect inclusion of SMN1 and SMN2 exon 7 (Hua, et al., 2010;Hua, et al., 2008;Kashima and Manley, 2003;Kashima, et al., 2007a;Kashima, et al., 2007b).

The putative ESS motif in SMN1 and SMN2 spans the 3'ss, thus only a limited number of positions in the motif can be mutated in the native context without risking that they also disrupt the 3'ss itself. Therefore we first used the pSXN13 splicing reporter minigene, which allows testing of the antagonistic interplay between an ESS and an ESE element (Nielsen, et al., 2007;Zatkova, et al., 2004) to test mutations aimed at disrupting the potential splicing silencer. In the pSXN13-SMN1wt construct, we observed predominant skipping, while the pSXN-SMN2wt construct showed complete skipping of the exon (Figure 2). The inhibitory effect of the ESS motif is abrogated by introducing a -2A>C change, that disrupts the hnRNP A1 consensus sequence. This is analogous to the c.351A>C change that abolishes hnRNP A1 mediated splicing inhibition in MCAD (Nielsen, et al., 2007). We generated a position weight matrix (PWM) for U2AF 35 based on earlier binding studies (Wu, et al., 1999) and used this to predict the effect on U2AF 35 binding of various mutations by assigning scores to different motifs. The U2AF 35 PWM (Fig 3) and hnRNP A1 PWM (Cartegni, et al., 2006) Although the reduction in hnRNP A1 binding was not as pronounced by the 2A3C mutation, there was still a significant decrease in the amount of hnRNP A1 bound to the oligonucleotide both in the MS/MS analysis and in the western blot analysis compared to wt.

Because hnRNP A1 seems to bind preferentially to YAG motifs (Burd and Dreyfuss, 1994), it is possible that because the seemingly indispensable YAG motif is still present in the 2A3C mutant, it is still able to bind hnRNP A1 with some affinity. Because both the MCAD and SMN ESS are G rich, we suspected that they may potentially bind hnRNP H (Caputi and Zahler, 2001;Schaub, et al., 2007). We observed strong binding of hnRNP H to the wild type motif and hnRNP H binding to the variants were decreased and showed an inverse bindingpattern compared to that observed for hnRNP A1 (Fig 4B and 3C). This is consistent with the (Caputi and Zahler, 2001;Dobrowolski, et al., 2010;Schaub, et al., 2007) and the -2C mutation does not disrupt the central GGG motif, whereas the 2A3C mutant disrupts the central GGG motif and thus dramatically decreased hnRNP H binding. MS/MS analysis revealed that not only hnRNP H, but also other members of the hnRNP H family of hnRNP proteins exhibited reduced binding to the -2C and 2A3C mutant sequences (data not shown).

Mutations disrupting the ESS increase inclusion of SMN2 exon 7.

In order to investigate an effect of the ESS disrupting mutations in a native context, we constructed SMN minigenes containing a partial intron 6, a complete exon 7 and intron 7 and Since both the 2A3C and the 2C3C mutations would disrupt formation of the potential splicing inhibitory terminal stem loop (Singh, et al., 2004) it was possible that the observed increase in inclusion seen in the 2A3C and 2C3C constructs was due to disruption of this to promote inclusion of exon 7. Using Human Splicing Finder (http://www.umd.be/HSF/) [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF], we examined the 2A3G mutation and found that it gave rise to several new potential ESS elements, which may explain why it failed to restore splicing of the SMN2 minigene.

hnRNP A1 knockdown decreases the effect of an ESS disrupting mutation

The identification of hnRNP A1 and hnRNP H as potential mediators of the inhibitory activity of the CAGGGT motif in SMN1 and SMN2 indicated that knocking down hnRNP A1

or hnRNP H by siRNA would result in an increase of inclusion of exon 7. We expected this effect to be less pronounced in the 2A3C constructs, since a reduction in binding affinity is analogous to a reduction in protein level. To test this hypothesis, we conducted siRNA experiments in HEK-293 cells in which we knocked down cellular levels of either hnRNP A1

or hnRNP H. We chose to use HEK-293 cells for these experiments as we have obtained more efficient siRNA mediated knock-down in HEK-293 cells as opposed to Chang cells. A1 knockdown is due to the fact that the 2A3C mutant motif still retains some capability for hnRNP A1 binding (as observed in the pull down experiments) and possibly also that other hnRNP A1 binding sites (Hua, et al., 2010;Hua, et al., 2007;Hua, et al., 2008;Kashima, et al., 2007b) First we used the SpliceRack web-tool (Sheth, et al., 2006) to investigate the relative abundance of GT-AG U2 splice sites similar to the 3'splice site of SMN exon 7 with the 6 nt long CAGGGT motif, with the MCAD CAGGGG ESS motif, the mutant CAGGAC (2A3C) motif and the mutant CAGGCC (2C3C) motif. Interestingly, the relative distribution of the 6 nt long motifs did not show clear differences, although CAGGGT and CAGGGG motifs were less abundant than the other motifs. However, when we looked at the 7 nt long motifs it was clear that the sequence corresponding to the SMN exon 7 is dramatically underrepresented.

The native CAGGGTT sequence was observed in 597, the MCAD ESS CAGGGGT in 411, whereas the positive CAGGCCT and CAGGACT were observed in 1032 and 857, splice sites respectively.

We proceeded to extract the sequence of 29150 cassette exons and 175814 constitutive exons plus 20 bp of intron sequence, as determined by the Alt Events track in the UCSC genome browser from the hg19 scaffold (Karolchik, et al., 2004;Rhead, et al., 2010). We defined constitutive exons as exons in the UCSC knownGene track, which did not overlap with any of the exons in the Alt Events track. When we searched for the different 3' splice site sequences in these, we found the SMN CAGGGTT motif in 92 cassette exons and in 559 constitutive exons whereas the mutant CAGGACT motif was found in 123 and 769 exons

DISCUSSION

All patients with SMA posses the partly functional SMN2 gene and could therefore benefit from therapies, which correct or improve splicing of SMN2 exon 7. Therefore it is very important that the underlying molecular defect mechanisms and the involved splicing regulatory elements, that suppress correct splicing of SMN2 exon 7, are identified and characterized. Moreover, because the affected splicing regulatory elements and the molecular pathological mechanisms involved are general and there is increasing evidence suggesting that the regulatory elements are organized in common architectures between human disease genes this knowledge is also important for a better understanding of other inherited diseases.

In this study we report the identification of an ESS element positioned across the 3'ss, which is important for regulation of splicing of SMN1 and SMN2 exon 7. We have previously identified a nearly identical ESS, which binds the splicing inhibitory protein hnRNP A1 and together with a flanking ESE determines splicing of MCAD exon 5 (Nielsen, et al., 2007).

Furthermore, a synonymous c.574C>A mutation in HADH2 exon 5 has been reported to cause X-linked mental retardation through exon skipping (Lenski, et al., 2007). The c.574C>A mutation creates a CAGGGT motif, identical to the ESS in SMN1/2. This indicates that the CAGGGT motif can also function as an ESS in a different gene and that creation of this ESS motif is sufficient to disrupt splicing. Based on nearly identical sequences between MCAD and SMN1/2 in a longer region harboring both the MCAD ESS and ESE and the region harboring both the critical c.840C>T change that distinguishes SMN2 from the functional SMN1 and the flanking splicing inhibitory region (Hua, et al., 2007;Singh, et al., 2004), we speculated whether SMN exon 7 splicing could be regulated in a way similar to MCAD exon 5, involving the potential ESS that spans the 3'ss of both SMN genes.

When we compared the effect of the (Zatkova, et al., 2004) we observed that the splicing inhibitory effect was indistinguishable and that the SMN ESS could be inactivated by mutations, which disrupts hnRNP A1 binding in pull down experiments. Moreover, functional testing of mutations, which were designed on the basis of the constructed binding matrices for U2AF35 and hnRNP A1, and testing of mutations substituting the SMN ESS with the MCAD ESS in SMN1

and SMN2 minigenes supports the notion that a hnRNP A1 binding ESS is located across the 3' splice site of SMN1 and SMN2. This, together with experiments, where the effect of siRNA mediated knock down of hnRNP A1 on the splicing of SMN1 and SMN2 minigenes with either the native ESS or a mutated ESS with a compromised hnRNP A1 binding motif, also supported the notion that SMN1/2 exon 7 splicing is inhibited by the presence of an hnRNP A1 binding ESS spanning the 3'ss.

Several of the mutations tested in the SMN1 and SMN2 minigenes and/or the pSXN splicing reporter indicate that the splicing inhibitory activity located near the 3'ss of exon 7 is not caused by the potential to form an inhibitory stem loop structure as proposed by Singh and coworkers (Singh, et al., 2004). The -2C mutation is located outside the potential stem loop structure, but still improves splicing dramatically in the pSXN splicing reporter minigene, while the 2A3G disrupts the potential to form the stem loop structure, but does not alter the splicing pattern of the SMN1 and SMN2 minigenes. Moreover, both the +3T>G and insertion of G at +1 (which both create the CAGGGG ESS) disrupt the potential to form the stem loop structure, but still inhibit splicing at a level indistinguishable from the native SMN CAGGGT ESS.

The 2A3G mutation failed to restore inclusion of exon 7 in the SMN2 minigene despite displaying a similar decrease in hnRNP A1 score as the 2A3C and 2C3C mutations. Analysis by the Human Splicing Finder tool [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF] indicates that this could be due to the fact that the 2A3G mutation creates 2 new potential ESS motifs (cagGAGTT and that even though a potential ESE motif may occur across the 3'ss it is likely not functional or at least not sufficient alone for an increase in inclusion of exon 7 and that the best explanation of increased inclusion is the disruption of an ESS motif. The possibility of new or remaining functional ESS motifs in the case of the 2A3G mutant thus seems the most likely explanation for the splicing pattern of this construct.

Our in vitro pull down experiments with RNA oligonucleotides with the wild type and mutant SMN1/2 exon 7 3'ss sequence further supported that splicing inhibition is mediated by hnRNP A1 binding. The wild type 3'ss sequence bound hnRNP A1 strongly, whereas introduction of the -2C substitution abolished hnRNP A1 binding, consistent with results obtained with a RNA oligonucleotides with the MCAD ESS wild type and the -2C mutant sequence. The 2A3C mutation also decreased binding of hnRNP A1, but not as dramatic as the -2C mutation. This shows that the 2A3C mutant sequence retains some affinity for hnRNP A1 and it is consistent with our observation that splicing of the 2A3C mutant SMN2 The in vitro binding studies additionally revealed binding of hnRNP H to the 3'ss region of SMN2 exon 7, but to our surprise siRNA mediated knock-down decreased inclusion of SMN2

exon 7 in a model minigene. The endogenous transcripts expressed in the same cells were, however, unaffected by the decreased levels of hnRNP H.

These conflicting results might simply be due to minigene specific motifs, which enable hnRNP H to influence splicing of SMN2 exon 7 in the minigene specific context and since these motifs are not present in the endogenous transcript, hnRNP H is unable to regulate splicing of endogenous SMN2 exon 7.

While we cannot rule out that hnRNP H might, in some circumstances, also regulate splicing of endogenous SMN2 exon 7, further studies would be needed in order to clarify this.

It has recently been suggested that the c.840C>T transition in SMN2 exon 7 forms a new binding motif for the negative splicing regulator Sam68 and that SMN2 exon 7 splicing inhibition by Sam68 is mediated by recruitment of hnRNP A1 (Pedrotti, et al., 2010). Such a model would fit well with our finding that hnRNP A1 can bind directly to the 3'ss, competing with U2AF 35 for binding. Binding of Sam68 to the motif immediately downstream would thus improve hnRNP A1 binding to the 3' splice site of SMN2 thereby inhibiting exon 7 inclusion. This is also compatible with a model where the SF2/ASF binding ESE in SMN1, stimulates recruitment of U2AF to the 3'splice site directly through SR domain interactions 6 and intron 7 also play a fundamental role in mediating exon 7 skipping in SMN2 (Hua, et al., 2010;Hua, et al., 2008;Kashima, et al., 2007b). Binding to these sites could further facilitate hnRNP A1 binding at the 3' splice site through cooperative binding and/or by propagation of hnRNP A1 binding proteins along the RNA either in a 3'-5' direction or less efficiently in a 5'-3' direction (Okunola and Krainer, 2009). The proximal hnRNP A1 binding site in intron 7 (Hua, et al., 2008) could for instance initiate propagation of hnRNP A1 binding in a 3'-5' direction towards the 3'ss in exon 7. This process may be antagonized by SF2/ASF binding to the ESE in SMN1 and stimulated by Sam68 binding in SMN2. These proposed mechanisms would imply that U2AF 35 and the U2AF complex would have a higher affinity for the 3'ss of exon 7 of SMN1 than that of SMN2 and this has in fact recently been reported (Martins de Araujo, et al., 2009).

The observation that hnRNP A1 can inhibit splicing of SMN2 exon 7 by direct binding to the 3'ss sequence is in agreement with earlier studies. The similarity of the motifs binding hnRNP A1 and the 3'ss consensus sequence has previously been noted (Burd and Dreyfuss, 1994), but the possible role of hnRNP A1 acting as a splicing inhibitor by direct binding to the 3'ss has not been investigated in detail. Binding of hnRNP A1 to 3'ss was initially thought to stimulate recognition of the 3'splice site (Buvoli, et al., 1990;Swanson and Dreyfuss, 1988a;Swanson and Dreyfuss, 1988b). Subsequent studies of hnRNP A1 binding to the 3'end of an intron indicated, however, that the observed interaction was non-specific or at least that the function of hnRNP A1 is not dependent on specific interactions with the 3'ss , 1996). The oligonucleotides used in these studies did not contain any exonic nucleotides, but only the polypyrimidine tract and the AG dinucleotide, and are therefore not in conflict with our observation, since we show that hnRNP A1 binding is dependent on the exonic nucleotides, as illustrated by the 2A3C mutation. It is thus likely that hnRNP A1 can function as a repressor of exon recognition by specific binding to the 3'ss, when the first few nucleotides in the exon completes a binding motif and that this is dependent on contextual conditions, such as the strength of the polypyrimidine tract, presence of nearby antagonizing ESEs and hnRNP A1 binding sites. Increased amounts of hnRNP A1 has previously been reported to affect some alternatively spliced exons dependent on the strength of the polypyrimidine tract (Mayeda, et al., 1993). In agreement with this is the fact that although the polypyrimidine tract appears very conserved, which could even point towards an AG-independent intron, and the strength of SMN2 exon 7 3'ss as estimated by several different splice site prediction programs is quite high (NNsplice score is 0.99 (Reese, et al., 1997), MaxEntScan score is 10.92 (Yeo and Burge, 2004)), strengthening of the polypyrimidine tract alone induces complete inclusion of exon 7 in an SMN2 minigene (Lorson and Androphy, 2000). This indicates that while the 3'ss of SMN2 exon 7 looks like a strong splice site, it is functionally weak and the binding of U2AF 65 to the PPT is suboptimal and dependent on U2AF 35 binding to the AG dinucleotide. This model is supported by the results of a paper describing PUF60 as a regulator of SMN2 exon 7 splicing (Hastings, et al., 2007). The authors examined SMN2 exon 7 splicing when U2AF 65 , PUF60 or both U2AF 65 and PUF60 were knocked down by siRNA and reported that SMN2 exon 7 inclusion increased upon knockdown of these factors. As the authors suggest, this could be explained by less competition from SMN2 exon 8 while SMN2 exon 7 3'ss strength is less affected by depletion of U2AF 65 as the binding of U2AF 65 to SMN2 exon 7 is already compromised (Hastings, et al., 2007). Further, this very clearly demonstrates that splice site scores should to overall splice site strength. The presence of an hnRNP A1 binding ESS spanning the SMN1/2 3'ss would compromise recognition of the 3' splice site and represents a plausible explanation for this. We therefore speculated if the SMN1/2 3'ss represent a generally weak 3'ss sequence and decided to investigate globally for the presence of the SMN1/2 3'ss motif among 3'splice sites. Comparing only the occurrence of the 6 nt CAGGGT motif in 3'ss to the occurrence of the corresponding 2A3C and 2C3C mutant motifs only revealed minor differences in frequency, but when we included the flanking exonic nucleotide, differences in frequency became apparent, and when we compared the frequencies of these motifs in the 3'ss of constitutive and cassette exons, there were clear indications that 3'ss with either the SMN1/2 CAGGGTT motif or the MCAD CAGGGGT motif were less frequent (i.e. weaker splice sites) than those with the CAGGACT and CAGGCCT motifs. The fact that these differences only became apparent when we included the nucleotide flanking the core 6 nt motif could either indicate that the hnRNPA1 binding motif in fact constitutes 7 nt with a requirement for a T at position 7, or that binding of hnRNP A1 to these 3' ss is strongly dependent on the flanking sequence or alternatively that some other unrelated feature determining 3'ss recognition is influenced by the presence of a T nucleotide at this position (+4 in the exon). It will require further experiments to elucidate the precise mechanism underlying this observation.

In conclusion, we show that an important ESS overlaps the 3'ss of SMN exon 7 and provide evidence to suggest a combination of both the ESE loss model and the ESS gain model for the splicing of exon 7 in SMN1 and SMN2.

We suggest that the ESS motif which is present in the MCAD and the SMN genes, and which is created by a disease causing mutation in the HADH2 gene, is an example of a general splicing regulatory motif that binds hnRNP A1 and can inhibit splicing. In particular, we This antagonism is depicted in the figure and we hypothesize that a similar antagonism exists in the case of SMN1 exon 7 and that it is disrupted in the case of SMN2 exon 7. In addition, position of the putative stem loop structure is indicated (Singh, et al., 2004), as well as the sequence of the SMN1/2 polypyrimidine tract. Purines in the PPT are underlined. published (Cartegni, et al., 2006) is additionally included for comparison. binding ESS immediately upstream. This antagonism is depicted in the figure and we hypothesize that a similar antagonism exists in the case of SMN1 exon 7 and that it is disrupted in the case of SMN2 exon 7. In addition, position of the putative stem loop structure is indicated (Singh, et al., 2004) (Cartegni, et al., 2006) Human Mutation

  ESE is disrupted by mutations, resulting in loss of activity and MCAD deficiency (OMIM: 607008)

  were used to select the mutations of +2 and +3 in exon 7 to either AC or CC, which decreased the hnRNP A1 score while keeping the U2AF 35 score essentially unchanged. We tested these mutations in the pSXN13 minigene and both inhibited the function of the ESS with the 2A3C mutation being the most efficient of the two (Fig 2B). We used RNA affinity chromatography to investigate changes in hnRNP A1 binding upon mutation of the ESS motif and to identify additional proteins, which were able to bind to the 3'ss of SMN2 exon 7. Because the 2A3C mutation decreased the hnRNP A1 score the most and did not improve the U2AF 35 score relative to the wt 3'ss of SMN exon 7, we used this variant for RNA-Protein affinity purification experiments along with the wild type sequence and the -2C mutant sequence as a control. Biotin-coupled RNA oligonucleotide sequences are shown in Fig 4A. MS/MS identification of peptides revealed a large reduction in hnRNP A1 derived peptides in the -2C sample relative to the wt oligonucleotide (Fig 4B). Western blot analysis also demonstrated that the -2C mutation drastically decreased hnRNP A1 binding, indicating that there is a specificity of hnRNP A1 for the CAGGGT motif (Fig 4C). This was corroborated by the results with the MCAD ESS oligonucleotides (Fig 4D), which also showed an equally dramatic effect of the -2C mutation on hnRNP A1 binding.

  a partial exon 8. In this context the 2A3C mutation resulted in an increase of exon 7 inclusion from both the SMN1 and SMN2 minigenes (Fig 5B) indicating that there is an ESS at the intron-6/exon-7 junction, and that the ESS function can be disrupted by the 2A3C mutation. Notably the 2C3C constructs behaved almost identical to the 2A3C constructs (Fig 5B). In order to examine the effect of having an MCAD like ESS in SMN exon 7, we designed SMN1 and SMN2 constructs with a +3G mutation which converts the ESS to one identical to the MCAD ESS. The CAGGGT SMN ESS and the CAGGGG MCAD ESS behaved identically in the SMN minigene context (Fig 5B). Additionally, we designed constructs where the MCAD ESS motif was instead generated by insertion of an additional G at position +1, and these constructs exhibited a similar splicing pattern as the constructs with the SMN CAGGGT ESS or the MCAD ESS CAGGGG generated by converting the +3T to a G (Fig 5B). This corroborates the hypothesis of a shared splicing mechanism between MCAD exon 5 and SMN1 and SMN2 exon 7.

Formatted:

  English (U.S.) Formatted: Font: Italic Deleted: This might reflect the limitations of the score matrices. of hnRNP H did not increase inclusion of SMN2 exon 7, but instead seemed to result in an increase in skipping (Fig 6A). The effect of the 2A3C mutation was still evident (Fig 6A).Knockdown of hnRNP A1 resulted in an increase in inclusion of SMN2 wt exon 7 transcripts(Fig 6A) and also an increase in inclusion from the 2A3C mutant construct compared to the scrambled siRNA control, despite the fact that the hnRNP A1 PWM indicates a reduction in binding of hnRNP A1 to the 2A3C variant motif. It is likely that the positive effect of hnRNP

  improved by siRNA mediated knockdown of hnRNP A1, although to a much lesser extent than the SMN2 minigene with the wild type high score hnRNP A1 binding motif. The fact that the wild type motif can be completely as well as partially abrogated by mutations indicates that hnRNP A1 binds to this region both strongly and specifically as opposed to binding indiscriminately along the oligonucleotide. We therefore propose that splicing of SMN2 exon 7 is inhibited by the ability of the sequence across the 3' splice site to bind hnRNP A1.

  same time also antagonizes hnRNP A1 binding to the 3' splice site in SMN1. The c.8406C>T substitution in SMN2 would thus have a dual effect by disrupting SF2/ASF binding and at the same time create a Sam68 binding motif, which would both increase binding of hnRNP A1 to the 3' splice site of SMN2 exon 7 and decrease U2AF binding resulting in splicing inhibition. It has been shown that other hnRNP A1 binding sites in intron

  caution in determining weak and strong splice sites, as other factors contribute

Figure 1 .

 1 Figure 1. Similarities between SMN1 and SMN2 exon 7 and MCAD exon 5. The region comprising the ESS and ESE motifs in SMN1 and SMN2 exon 7 is compared to the corresponding region in MCAD exon 5, showing the extensive sequence similarity between the two exons. Both contain an SF2/ASF binding ESE as well as an hTra2-β1 binding ESE further downstream. In the context of MCAD exon 5, we have previously described the antagonistic functions of the ESE and the hnRNP A1 binding ESS immediately upstream.

Figure 2 .

 2 Figure 2. Mutational analysis of the putative ESS. A) Schematic overview of the pSXN13 minigene and constructs used in this study. Capitals indicate exonic bases. B) RT-PCR analysis of splicing of pSXN13 constructs in Chang cells. Inclusion or exclusion of the alternative exon is indicated on the left. hnRNP A1 and U2AF 35 motif scores are indicated below.

Figure 3 .

 3 Figure 3. Binding consensus of U2AF 35 . A) Top: A pictogram depiction of the base composition of the U2AF 35 binding consensus. Bases that are more prevalent than the background frequency (25%) are shaded. Bottom: The Position Weight Matrix (PWM) of the U2AF 35 binding consensus. The individual scores of the most frequent bases at any given position are indicated in bold. We have included the 6 th base for easy comparison with the hnRNP A1 motif in both the pictogram representation and the scoring matrix representation,

Figure 4 .

 4 Figure 4. Protein binding analysis. A) The RNA oligonucleotides used to bind proteins from a HeLa cell Nuclear Extract. X indicates the position of covalently bonded biotin. B) Peptide quantification of selected proteins. Individual peptides were identified by nLC-MS/MS analysis and quantitated by measuring the area of the corresponding peak in the nLC chromatogram. Peptides were individually normalized to the wt sample and relative protein quantities estimated by taking the average of the normalized peptide quantities. Error bars indicate standard error of mean. C) Western blots of protein eluates from the samples containing SMN2 sequences. NE indicate control lane with Nuclear Extract. D) Western blot of protein eluates from the samples containing MCAD sequences. NE indicate control lane with Nuclear Extract.

Figure 5 .

 5 Figure 5. Analysis of an SMN1/2 exon 7 spanning ESS by mutation. A) Schematic overview of the SMN1/2 minigene and constructs used in this study. Capitals indicate exonic bases. The minigene is a hybrid construct consisting of both MCAD and SMN sequence, but retains the splicing pattern of SMN1 and SMN2 wt genes. Endogenous SMN2 was used as template and the minigene retains the SMN2 specific intron variations. B) RT-PCR analysis of splicing of SMN1/2 minigenes in Chang cells. Inclusion or exclusion of exon 7 are indicated on the left. hnRNP A1 and U2AF 35 motif scores are indicated below.

Figure 6 .

 6 Figure 6. Analysis of hnRNP A1 and hnRNP H function by siRNA. A) RT-PCR analysis of splicing of SMN1/2 minigenes in HEK293 cells. hnRNP A1 and hnRNP H were knocked

Figure 1 .

 1 Figure 1. Similarities between SMN1 and SMN2 exon 7 and MCAD exon 5. The region comprising the ESS and ESE motifs in SMN1 and SMN2 exon 7 is compared to the corresponding region in MCAD exon 5, showing the extensive sequence similarity between the two exons. Both contain an SF2/ASF binding ESE as well as an hTra2-β1 binding ESE further downstream. In the context of MCAD exon 5, we have previously described the antagonistic functions of the ESE and the hnRNP A1binding ESS immediately upstream. This antagonism is depicted in the figure and we hypothesize that a similar antagonism exists in the case of SMN1 exon 7 and that it is disrupted in the case of SMN2 exon 7. In addition, position of the putative stem loop structure is indicated(Singh, et al., 2004), as well as the sequence of the SMN1/2 polypyrimidine tract. Purines in the PPT are underlined. 275x190mm (600 x 600 DPI)

Figure 2 .Figure 3 .

 23 Figure 1. Similarities between SMN1 and SMN2 exon 7 and MCAD exon 5. The region comprising the ESS and ESE motifs in SMN1 and SMN2 exon 7 is compared to the corresponding region in MCAD exon 5, showing the extensive sequence similarity between the two exons. Both contain an SF2/ASF binding ESE as well as an hTra2-β1 binding ESE further downstream. In the context of MCAD exon 5, we have previously described the antagonistic functions of the ESE and the hnRNP A1binding ESS immediately upstream. This antagonism is depicted in the figure and we hypothesize that a similar antagonism exists in the case of SMN1 exon 7 and that it is disrupted in the case of SMN2 exon 7. In addition, position of the putative stem loop structure is indicated(Singh, et al., 2004), as well as the sequence of the SMN1/2 polypyrimidine tract. Purines in the PPT are underlined. 275x190mm (600 x 600 DPI)

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 3. Binding consensus of U2AF 35 . A) Top: A pictogram depiction of the base composition of the U2AF 35 binding consensus. Bases that are more prevalent than the background frequency (25%) are shaded. Bottom: The Position Weight Matrix (PWM) of the U2AF 35 binding consensus. The individual scores of the most frequent bases at any given position are indicated in bold. We have included the 6 th base for easy comparison with the hnRNP A1 motif in both the pictogram representation and the scoring matrix representation, but since the information content in this position is very low it has not been included when calculating U2AF35 scores. B) The PWM and scoring matrix of hnRNP A1 previously published(Cartegni, et al., 2006) is additionally included for comparison. 275x190mm (600 x 600 DPI)

siRNA reverse siRNA transfection and transient transfection of HEK-293 cells

	CAGAGUAACUGGUGAAGCAUU,	GUUCGCAACUCAUGAAGAUUU	and
	UAACAUUGCCGGUGGACUUUU; Scrambled control: UAGCGACUAAACACAUCAA)
	was reverse transfected into the HEK-293 using Dharmafect1 siRNA transfection reagent
	(Dharmacon, Chicago, IL). After 24 hours the cells were transfected in triplicates with 0.09
	µg SMN plasmid using FuGENE6 transfection reagent (Roche). 48 hours after transfection
	cells were harvested. Of the triplicate transfections, 2 cell pellets were used for total RNA
	isolation by TRIzol (Invitrogen) and cDNA synthesis using Advantage MMLV RT-PCR kit
	(BD Biosciences Clontech). Splicing analysis was carried out by PCR amplification and
	agarose gel electrophoresis with EtBr staining. Splicing analysis of endogenous SMN1 and
	SMN2 transcripts were performed by PCR with primers located in exon 6 and exon 8 F (SMN.endo.ex6s: ATATGTCCAGATTCTCTTGATG and SMN.endo.ex8as: o r ACCATTTGAAACATTTTAAGAC) followed by restriction digest of the PCR product with
	of the MCAD gene (MCex4ex5s: CATTCCAGAGAACTGTGGAGGT) and a primer located in SMN1/2 exon 8 (SMNtestex8as: GTGGTGTCATTTAGTGCTGCTC). For the pSXN13 DdeI. The third cell pellet was used for protein analysis by adding 75 µL lysis buffer, P containing 1% Triton-X100 and 3 µL PMSF protease inhibitor, and by incubating at 37°C for e 60 min. The lysate was spun and the supernatant extracted. The protein concentration in the e r supernatant was estimated using Bradford reagent (Bio-Rad, Hercules, CA, USA) and
	constructs we used a primer located in β-actin exon 1 (pSXN12s2: AAGGTGAACGTGGATGAAGTTGGTGGTG) and a primer spanning the β-actin exon 2 -knockdown of target proteins was evaluated by western blotting, using affinity purified R hnRNP A1 goat polyclonal antibody (sc-10029), an affinity purified hnRNP H goat
	cTNT exon 18 splice junction (pSXN13as: CCCACGTGCAGCCTTTGACCTAGTA). PCR e polyclonal antibody (sc-10042) or an actin antibody (sc-1616). All antibodies were from
	products were separated and visualized on agarose gels containing ethidium bromide (EtBr) on an Epi II Darkroom UVP Transilluminator. e i v Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
	App. 200,000 HEK-293 cells were seeded in each well in a 24 well plate (TPP AG) and 100 w
	pmol of the relevant siRNA (hnRNP A1: CAGCUGAGGAAGCUCUUCAUU; hnRNP H: A
	pool of 4 different siRNA oligonucleotides was used: GAUCCACCACGAAAGCUUAUU,
			8

MMLV RT-PCR kit (BD Biosciences Clontech, Franklin Lakes, NJ) with an oligo (dT) 18 primer. App. 1/10 of the cDNA synthesis product corresponding to 100 ng RNA was used as template in each PCR reaction using Tempase DNA polymerase (Ampliqon Aps, Skovlunde, Denmark). For the SMN constructs we used a primer spanning the exon 4 -exon 5 junction

Protein pull down by biotin coupled RNA oligonucleotides We used biotinylated RNA oligonucleotides spanning the area containing the ESS motif in

	AUUUUUCCUUACCGGGUUUUA-biotin	and	SMN2	3'ss	2A3C:	5'-
		F o r
		P
		e
			e r
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				e
					v i e	Deleted:
	MCAD and SMN2: MCAD-351A (wt): 5'-AUGGAUGUACAGGGGUUCAG-biotin, MCAD-w
	351C:	5'-AUGGAUGUACCGGGGUUCAG-biotin,	SMN2	3'ss	wt:	5'-
	AUUUUUCCUUACAGGGUUUUA-biotin,	SMN2	3'ss		-2C:	5'-
							9

  , thereby improving access to the 3'ss by the spliceosome. This is, however, contradicted by the fact that the MCAD CAGGGG ESS motif disrupts formation of the stem loop, but still causes a degree of exon skipping comparable to the normal CAGGGT motif. To further test the potential role of an inhibitory stem loop, minigenes containing a 2A3G mutation were constructed. This mutation would also remove the terminal stem loop by eliminating base-pairing in the stem. If a terminal stem loop is responsible for the skipping of SMN2 exon 7 this mutation would increase inclusion to similar levels as that of the 2A3C mutation. However, exon 7 inclusion was not improved in the 2A3G constructs(Fig 5B) further supporting that disruption of a potential terminal stem loop structure is not the underlying mechanism behind the increased inclusion demonstrated by the 2A3C and 2C3C constructs. The 2A3G represents a curious example, however. Like the 2A3C construct it has a moderately high U2AF 35 score and a very low hnRNP A1 score, but unlike the 2A3C it fails

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	terminal stem loop
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ss spanning hnRNP A1 motif is prevalent in human exons and may reduce splice site strength

  still play a role in splicing inhibition. Comparing the splicing pattern of the wt SMN2 construct to the 2A3C construct when both are expressed in hnRNP A1 knocked down cells reveals that the 2A3C mutation still increases inclusion of exon 7. This might be caused SMN2 exon 7 (Fig 6B), indicating that although hnRNP H might bind the ESS in vitro, it is not functionally linked in vivo.Because the CAGG portion of the CAGGGT/G ESS motif is present in the majority of human 3' splice sites we decided to investigate if the subset of splice sites harboring this CAGGGT/G ESS motif represent weak splice sites because they may be suppressed by hnRNP A1 binding.

	inclusion of endogenous The 3'
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by additional hnRNP proteins which are still able to bind to the 3'ss of SMN2, for instance members of the hnRNP A/B family.

All SMN1 constructs were unaffected by the siRNA mediated knockdown of interacting proteins.

hnRNP A1 but not hnRNP H can regulate splicing of endogenous SMN2 exon 7.

In order to investigate the effects of hnRNP A1 and hnRNP H knockdown on endogenous SMN1 and SMN2 transcripts, we performed a PCR with primers located in exon 6 and exon 8 of SMN2 and SMN1. To discriminate between SMN1 and SMN2 derived transcripts, we digested the PCR products with DdeI, which cleaves SMN2 exon 8, but not SMN1 exon 8, allowing for discrimination between SMN1 and SMN2 according to size. This splicing analysis revealed that hnRNP A1, but not hnRNP H knock-down induces a shift in the

  SMN ESS with that of the MCAD ESS on the SMN1 ESE or the corresponding SMN2 sequence in the heterologous context of the pSXN splicing

	reporter minigene
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  it removes several other ESS elements. Another alternative, but not mutually exclusive, explanation could be that in contrast to the 2A3G mutation, the 2C3C and 2A3C mutations are predicted by the Human Splicing Finder tool to result in formation of new ESE motifs, which span or are very closely located to the 3'ss and that these ESEs

		Field Code Changed
	AGTTTA) although facilitate increased inclusion. Several problems arise with this hypothesis, however. First of
	all, while not impossible, it is hard to imagine how an SR protein might efficiently recruit
	U2AF 35 to a 3'ss, which it is already blocking. Secondly, both the 2C3C and 2A3C mutation
	increase inclusion of exon 7, but the 2C3C mutation only forms one additional ESE motif, an
	SC35 motif, which spans the 3'ss (gGCCTTTA) and both the 2A3C and 2A3G mutations
		Formatted: Font: Italic
	form a 9G8 motif spanning the 3'ss (cagGAC and cagGAG). Finally, SMN2 already has a F naturally occurring SF2/ASF ESE motif spanning the 3'ss (tacagGG), which is either not o r functional or not sufficient to compensate for the loss of the downstream ESE. This indicates
	P	Deleted: the potential
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	21

  'ss of exons, where this inhibitory ESS motif constitute part of the 3'ss. This also illustrates how the third and fourth nucleotide of an exon may contribute to modulate splice site strength. Importantly, this means that variations within this region, which have

	propose that hnRNP A1 may function as a general inhibitor of exon inclusion by direct
	binding to the 3previously been assumed to be neutral, may cause partial or complete skipping of an exon, or	Formatted: Font: Italic Formatted: Font: Italic Deleted: strength of the Deleted:
	confer disease susceptibility depending on cell type or environmental changes in splicing
		Deleted: that of
	factor concentrations.	Deleted: did not
		Deleted: significant
		Deleted: one more
		Deleted: comparing
		Deleted: the
	F o r F o r F o r F o r F o r F o r	Formatted: Font: Italic Formatted: Font: Italic Deleted: are stronger splice sites than either the SMN CAGGGTT motif or the MCAD CAGGGGT motif Deleted: clear
	P P P P P P e e e e e e	Deleted: following Deleted: , not only illustrates the limitations of the binding score matrices, but it Deleted: s
	e r e r e r e r e r e r	Deleted: that the flanking immediate context of hnRNP A1 motifs may be decisive for motif strength or
	R R R R R R
	e e e e e e
	v i e v i e v i e v i e v i e v i e
	w w w w w w	Deleted: Finally, w Deleted: found
		Formatted: Font: Italic
		Deleted: may be
	26

  RT-PCR analysis of endogenous SMN1 and SMN2 transcripts in HEK293 cells. RT-PCR was performed on the same cDNA used for RT-PCR analysis in panel A. SMN1 and SMN2 were discriminated by digesting the PCR products with DdeI. Band sizes corresponding to SMN1 and SMN2 transcripts with or without exon 7 are indicated on the left. Also indicated is the band corresponding to the 3' end of SMN2 exon 8 which is a result of the digestion with DdeI. C) Western blot analysis of protein knockdown in HEK293 cells. Antibodies against Actin, hnRNP A1 and hnRNP H were used for staining. Actin was used as loading control.

	down by siRNA while a scrambled siRNA duplex was used as control. Band sizes
	Page 30: [1] Deleted	Thomas Koed Doktor	10/28/2010 10:38:00 PM
	corresponding to transcripts either including or excluding exon 7 are indicated on the left. B)
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	F F o r F o r F o r F o r F o r o r P P P P P P e e e r e e r e e r e e r e e r e r R e v i e R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
		w
			33

John Wiley & Sons, Inc.Human Mutation

Acknowledgments

This work was supported by grants from The Danish Medical Research Council (FSS grant no. 271-07-342 to BSA), The Riisfort Foundation (BSA) and The Augustinus Foundation (BSA). Tandem Mass Spectrometry analysis was supported by a grant from the John and Birthe Meyer Foundation to NG. We are grateful to Jane Serup Pedersen and Lone Sundahl for expert technical assistance. Deleted: and Page 26 of 43 John Wiley & Sons, Inc. Human Mutation

pesx/ (PESX) ¶