PhD Catherine Feart
email: catherine.feart-couret@u-bordeaux.fr

Special Issue "VSI: Aging

Keywords: frailty, aging, nutrition, Mediterranean diet, vitamins

Purpose of the review

Nutrition, as part of lifestyle and modifiable environmental factors, constitutes an interesting approach for the prevention of geriatric syndromes. The objective of this review was to examine the most recent evidence on the association between nutrition, from dietary patterns to specific nutrients, and frailty, before the onset of disability, among elderly individuals.

Recent findings

Based on available epidemiological studies, three meta-analyses published in 2018 have outlined a protective effect of greater adherence to a Mediterranean-type diet (MeDi) on the risk for frailty, with up to a 60% reduction in risk. Several studies focusing on particular food groups, macronutrients and micronutrients have also been published and have highlighted that a protein intake of 1 g/kg in body weight per day should be fulfilled (except for patients suffering from kidney or hepatic dysfunction) and that vitamin deficiencies should be avoided. Available interventional studies of nutritional supplements and/or physical activity programs have mainly been limited to disabled participants to date.

Summary

Research efforts should target both developing a better understanding of the mechanisms underlying frailty and improving detection tools and the effectiveness of intervention studies, alongside efforts to address the specific needs of older people. For instance, ensuring an adequate nutritional status, by fighting the age-related increased prevalence of undernutrition or sarcopenic obesity, should be privileged.

INTRODUCTION

According to the World Health Organization, aging is a continuous process which results from interactions between genetic and environmental factors that alter the structure and function of the organism. Advancing age is accompanied by common geriatric syndromes that can coexist, such as falls and fractures, thinness, isolation, incontinence, sensory disorders, disability, and frailty [START_REF] Tabue-Teguo | Prevalence and Co-Occurrence of Geriatric Syndromes in People Aged 75 Years and Older in France: Results From the Bordeaux Three-city Study[END_REF].

For the last two decades, frailty has become a particularly attractive concept because of its place in the continuum from robustness to age-related disability [START_REF] Tabue-Teguo | Frailty in elderly: A brief review[END_REF]. Frailty involves uncompensated adaptation to changes in the environment over time, and the frailty status is characterized by a depletion in the functional reserves of physiological systems. The French Society of Geriatrics and Gerontology defines frailty as "a clinical syndrome expressing a multisystemic reduction of physiological capacities limiting the adaptation to stress" [START_REF] Rolland | Frailty in older population: a brief position paper from the French society of geriatrics and gerontology[END_REF]. The frail older adult is therefore unable to respond appropriately to situations of stress, from the cellular level to the societal level. The frail older adult thus has an unfavorable prognosis, i.e., is more at risk of adverse health events, disability, loss of autonomy and death [START_REF] Tabue-Teguo | Frailty in elderly: A brief review[END_REF][START_REF] Gill | Trajectories of disability in the last year of life[END_REF][START_REF] Crow | Mortality Risk Along the Frailty Spectrum: Data from the National Health and Nutrition Examination Survey 1999 to 2004[END_REF][START_REF] Harmand | Comparing the predictive value of three definitions of frailty: Results from the Three-City study[END_REF]. However, from a public health point of view, this transitional state is particularly interesting, since it is potentially reversible and is accessible to preventive measures [START_REF] Clegg | Frailty in elderly people[END_REF][START_REF] Gill | Transitions between frailty states among community-living older persons[END_REF][START_REF] Santos-Eggimann | Screening for frailty: older populations and older individuals[END_REF].

Today, the concept of frailty seems commonly accepted by most medical practitioners in addition to geriatricians, while its translation into clinical practice seems to still be limited due to the absence of a standardized operational definition. Similarly, the underlying pathophysiological processes that lead to frailty are still debated [START_REF] Harmand | Comparing the predictive value of three definitions of frailty: Results from the Three-City study[END_REF][START_REF] Rodriguez-Manas | Searching for an operational definition of frailty: a Delphi method based consensus statement: the frailty operative definition-consensus conference project[END_REF][START_REF] Aguayo | Comparative analysis of the association between 35 frailty scores and cardiovascular events, cancer, and total mortality in an elderly general population in England: An observational study[END_REF]. In the current scientific literature, two ways of thinking coexist; the first defines frailty as the accumulation of deficits, with a multidomain and cumulative approach to pathologies and dependencies [START_REF] Mitnitski | Frailty, fitness and late-life mortality in relation to chronological and biological age[END_REF][START_REF] Rockwood | A global clinical measure of fitness and frailty in elderly people[END_REF], while the second mainly considers frailty as an impairment of physical function resulting from a pathological process underlying various clinical manifestations [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF].

According to this latter hypothesis, pathologies are excluded from the definition, whereas the loss of lean mass with undernutrition, decreased muscle strength, and decreased metabolic and physical activity are components of a self-sustaining circle and are also fueled by adverse external environmental factors [START_REF] Fried | Frailty in older adults: evidence for a phenotype[END_REF]. This phenotype, as proposed by Fried et al., seems to be the most commonly used definition in the scientific literature to date. A third stream of thought has also emerged over several years and favors a multidimensional approach. Social, cognitive and psychosocial components, as well as factors associated with decreasing the elderly's physiological reserves, including nutritional factors, define different scores of frailty [START_REF] Aguayo | Comparative analysis of the association between 35 frailty scores and cardiovascular events, cancer, and total mortality in an elderly general population in England: An observational study[END_REF][START_REF] Avila-Funes | Cognitive impairment improves the predictive validity of the phenotype of frailty for adverse health outcomes: the three-city study[END_REF][START_REF] Gobbens | Towards an integral conceptual model of frailty[END_REF].

Frailty is therefore part of a multifactorial dynamic process in which the nutritional status appears as a key element in the assessment of frailty. Moreover, the main hypothesis of the present article is that nutrition should also be considered as a modifiable environmental factor which potentially could be associated with the prevention of the frailty status. The purpose of this article is to create a nonexhaustive inventory of the relationship between nutrition and frailty in the elderly, summarizing the work of observational studies up to the intervention trials already established.

TEXT OF THE REVIEW

Prevalence of frailty

The French "Haute Autorité de Santé" suggests identifying frailty in people over 70, free from serious illness, by tools that are complementary to standardized geriatric evaluation. Among the most commonly used tools is the Short Physical Performance Battery (SPPB), which is the sum of the scores on three criteria: balance test, walking speed and chair lift test. The walking speed alone, at the 0.8 m / s threshold, is also a relevant criterion and a marker of health status [START_REF] Van Kan | Gait speed at usual pace as a predictor of adverse outcomes in community-dwelling older people an International Academy on Nutrition and Aging (IANA) Task Force[END_REF][START_REF] Studenski | Gait speed and survival in older adults[END_REF]. Other scales derived from the frailty phenotype items of Fried et al. have been proposed, such as the "SOF (Study of Osteoporotic Fracture) criteria for frailty" [START_REF] Ensrud | Comparison of 2 frailty indexes for prediction of falls, disability, fractures, and death in older women[END_REF], which combines chronic fatigue, chair lift and weight loss, or the "FRAIL scale", which combines fatigue, resistance, ambulation, diseases and weight loss [START_REF] Morley | A simple frailty questionnaire (FRAIL) predicts outcomes in middle aged African Americans[END_REF]. The common feature of these numerous scales is their ability to predict the higher risk for adverse events in frail individuals [START_REF] Aguayo | Comparative analysis of the association between 35 frailty scores and cardiovascular events, cancer, and total mortality in an elderly general population in England: An observational study[END_REF].

Because of the lack of a standardized definition, prevalence data for frailty in the elderly community vary widely. From 2012, Collard et al. reported a prevalence in the order of 4 to 60%. Overall, the weighted prevalence of frailty in their analysis was 10.7%, based on 21 studies involving nearly 61,500 participants aged 65 and over, with an increased prevalence with age and among women, compared to men [START_REF] Collard | Prevalence of frailty in communitydwelling older persons: a systematic review[END_REF].

Nutritional status of frail elderly people

According to the definition of Fried et al., unintentional weight loss is a susceptibility criterion for frailty. Therefore, undernutrition is frequent among frail populations [START_REF] Bollwein | Nutritional status according to the mini nutritional assessment (MNA(R)) and frailty in community dwelling older persons: a close relationship[END_REF][START_REF] Lorenzo-Lopez | Nutritional determinants of frailty in older adults: A systematic review[END_REF]. Although undernutrition and frailty are two distinct entities, according to the studies, up to 90% of malnourished elderly people are also more frequently frail [START_REF] Bollwein | Nutritional status according to the mini nutritional assessment (MNA(R)) and frailty in community dwelling older persons: a close relationship[END_REF][START_REF] Verlaan | High Prevalence of Physical Frailty Among Community-Dwelling Malnourished Older Adults-A Systematic Review and Meta-Analysis[END_REF]. Moreover, the recent concept of sarcopenic obesity should be introduce in this field [START_REF] Batsis | Sarcopenic obesity in older adults: aetiology, epidemiology and treatment strategies[END_REF]. Indeed, although apparently in satisfying health due to overweight, elderly individuals are also likely to lose muscle mass and strength, a phenomenon called sarcopenia [START_REF] Bahat | Sarcopenia: revised European consensus on definition and diagnosis[END_REF].

Few studies have been interested in the association between sarcopenic obesity and frailty. One crosssectional analysis of the National Health and Nutrition Survey (NHANES) database reported that frail older adults exhibited higher adiposity (expressed as % of body fat) and greater average waist circumference than non-frail participants [START_REF] Crow | Association of Obesity and Frailty in Older Adults: NHANES 1999-2004[END_REF]. There is to date a single longitudinal analysis on the CHAMP participants (100% Australian men, n=1685, aged 77 years on average), which reported that low muscle mass and sarcopenic obesity were associated with a significantly doubled risk for frailty over a 5-y period [START_REF] Hirani | Longitudinal associations between body composition, sarcopenic obesity and outcomes of frailty, disability, institutionalisation and mortality in community-dwelling older men: The Concord Health and Ageing in Men Project[END_REF]. Overall, these studies confirmed that an altered nutritional status, as evaluated by both undernutrition and over-nourishment, should be considered as helpful criteria for the identification of frail individuals. This hypothesis was already suggested by Buch et al. who reported that central obesity or diabetes can provide useful marker of frailty among obese young elderly [START_REF] Buch | Severe central obesity or diabetes can replace weight loss in the detection of frailty in obese younger elderly -a preliminary study[END_REF].

Relationship between energy, macronutrients and frailty

Much of the literature concerns the contributions of total energy and macronutrients, especially proteins [START_REF] Lorenzo-Lopez | Nutritional determinants of frailty in older adults: A systematic review[END_REF][START_REF] Bonnefoy | Frailty and nutrition: searching for evidence[END_REF][START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF]. It is noted in the majority of cross-sectional studies that frail participants have the lowest energy intakes [START_REF] Lorenzo-Lopez | Nutritional determinants of frailty in older adults: A systematic review[END_REF][START_REF] Bonnefoy | Frailty and nutrition: searching for evidence[END_REF][START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF]; the threshold of 21 kcal / kg / d or less was significantly associated with a higher prevalence of frailty. Similarly, protein intake is inversely associated with frailty, except in one study where the distribution of daily protein intake, rather than the quantity itself, could explain this result [START_REF] Bollwein | Distribution but not amount of protein intake is associated with frailty: a cross-sectional investigation in the region of Nurnberg[END_REF]. Animal or vegetable protein sources are not essential in the relationship between protein intake and frailty [START_REF] Kobayashi | Three-generation Study of Women on D, Health Study G: High protein intake is associated with low prevalence of frailty among old Japanese women: a multicenter cross-sectional study[END_REF]. This finding is particularly true in our cohort of seniors who responded to a thorough dietary survey (cohort 3-City) [START_REF] Feart | Energy, macronutrient and fatty acid intake of French elderly community dwellers and association with socio-demographic characteristics: data from the Bordeaux sample of the Three-City Study[END_REF]: a protein intake of nearly 1 g / kg / day was associated with a lower prevalence of frailty (odds ratio = 0.41, 95% confidence interval 0.19-0.89), and this was independent of total energy intake (set at 30 kcal / kg / d, alone not associated with frailty) [START_REF] Rahi | Higher Protein but Not Energy Intake Is Associated With a Lower Prevalence of Frailty Among Community-Dwelling Older Adults in the French Three-City Cohort[END_REF]. Longitudinal studies confirm these results, showing that participants with the lowest protein intake are the most at risk of becoming frail over time [START_REF] Artaza-Artabe | The relationship between nutrition and frailty: Effects of protein intake, nutritional supplementation, vitamin D and exercise on muscle metabolism in the elderly. A systematic review[END_REF][START_REF] Beasley | Protein intake and incident frailty in the Women's Health Initiative observational study[END_REF][START_REF] Sandoval-Insausti | Macronutrients Intake and Incident Frailty in Older Adults: A Prospective Cohort Study[END_REF].

Relationship between micronutrients and frailty

To date, several studies have focused on micronutrients in relation to frailty, with a particular interest in vitamin D. Older people often suffer from hypovitaminosis D; in 2000, in our 3-City cohort, we observed a nearly 60% level of moderate impairment (25 (OH) D = 25-50 nmol / L) and a 24% level of deficit (25 (OH) D = 12.5 -25 nmol / L2) [START_REF] Feart | Associations of lower vitamin D concentrations with cognitive decline and long-term risk of dementia and Alzheimer's disease in older adults[END_REF]. Elderly frail participants also exhibited low circulating levels of vitamin D, which are themselves significantly associated with an increased risk of frailty among the robust [START_REF] Artaza-Artabe | The relationship between nutrition and frailty: Effects of protein intake, nutritional supplementation, vitamin D and exercise on muscle metabolism in the elderly. A systematic review[END_REF]. Two metabolic pathways are involved, namely, phosphocalcic metabolism, since vitamin D plays an essential role in bone mineralization, and muscle strength, since vitamin D binds its receptor in skeletal muscle cells to induce protein synthesis [START_REF] Lorenzo-Lopez | Nutritional determinants of frailty in older adults: A systematic review[END_REF][START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF][START_REF] Artaza-Artabe | The relationship between nutrition and frailty: Effects of protein intake, nutritional supplementation, vitamin D and exercise on muscle metabolism in the elderly. A systematic review[END_REF].

For other micronutrients, data from the Italian InCHIANTI cohort are among the most interesting: frail participants consume significantly less vitamin D, E, C and folate, regardless of energy consumption, than nonfrail participants [START_REF] Bartali | Low nutrient intake is an essential component of frailty in older persons[END_REF]. Rates of vitamin E levels were also associated with an increased risk of frailty over time [START_REF] Semba | Low serum micronutrient concentrations predict frailty among older women living in the community[END_REF][START_REF] Ble | Lower plasma vitamin E levels are associated with the frailty syndrome: the InCHIANTI study[END_REF]. These results were partly confirmed by other studies: the lowest levels of consumption or circulating biomarkers of carotenoids, such as β-carotene, lutein and zeaxanthin, vitamin A, vitamin E, and vitamin B6, were associated with a higher prevalence and an increased risk of frailty [START_REF] Lorenzo-Lopez | Nutritional determinants of frailty in older adults: A systematic review[END_REF][START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF]. Semba et al. suggested that the higher the number of nutritional deficits is, the higher the risk of frailty [START_REF] Semba | Low serum micronutrient concentrations predict frailty among older women living in the community[END_REF]. In the Spanish Seniors-ENRICA cohort, also interested in the associations between micronutrient intakes and the frailty risk, an analysis of more than 1,600 aged individuals reported that poor intake of several vitamins (B6, C, E and folates), and non-adherence to the recommended dietary allowances for thiamine, niacin and vitamin B6 were all independently associated with the frailty risk over 3.5y [START_REF] Balboa-Castillo | Low vitamin intake is associated with risk of frailty in older adults[END_REF]. As with food consumption, we recently developed an original approach that consisted of building a pattern of circulating biomarkers of micronutrients. As part of the European project FRAILOMIC (study case-control design) [START_REF] Erusalimsky | In Search of 'Omics'-Based Biomarkers to Predict Risk of Frailty and Its Consequences in Older Individuals: The FRAILOMIC Initiative[END_REF], we pooled 4 cohorts (including InCHIANTI and 3-City) for which blood tests of αand β-carotene, lycopene, cryptoxanthin, lutein and zeaxanthin, retinol, αand γ-tocopherol and 25 (OH) D3 were assessed from 1,324 participants. We identified 3 different nutrient patterns and observed that frail participants had significantly lower vitamin E and A levels (component 2), but there was no association between the patterns characterized by either the carotenoid levels (component 1) or by the vitamin D levels (component 3) and frailty [START_REF] Pilleron | Patterns of circulating fat-soluble vitamins and carotenoids and risk of frailty in four European cohorts of older adults[END_REF]. Even more surprisingly, no nutrient pattern was associated with the risk of frailty in this sample, suggesting that these patterns were only status markers and were not predictive of the risk of frailty [START_REF] Pilleron | Patterns of circulating fat-soluble vitamins and carotenoids and risk of frailty in four European cohorts of older adults[END_REF]. These results raise the question of the reverse causality bias that may have been observed in previous studies: the underlying frailty may have led to changes in eating behavior rather than the reverse. Nevertheless, to our knowledge, this original and unique study should be reproduced before forming a definitive conclusion about this relationship.

Recently, omega-3 fatty acids have gained interest regarding the preservation of muscle mass. We already observed that a higher circulating long-chain omega-3 fatty acid status (high in EPA and DHA) was associated with lower odds of exhibiting low gait speed among 3-City participants [START_REF] Frison | Plasma fatty acid biomarkers are associated with gait speed in community-dwelling older adults: The Three-City-Bordeaux study[END_REF].

Another longitudinal study confirmed that a higher intake of omega-3 fatty acids contributed to a lower risk of frailty development [START_REF] Garcia-Esquinas | Major dietary patterns and risk of frailty in older adults: a prospective cohort study[END_REF]. To our knowledge, no additional studies have assessed this association in a different context.

Relationship between food and frailty

With regard to food consumption and its relationship to frailty, studies are rare. Fruits and vegetables represent the food group that has attracted the most interest, including in our cohort. In collaboration with Spanish colleagues, we have studied the relationship between fruit and vegetable consumption and the risk of frailty in three European cohorts [START_REF] Garcia-Esquinas | Consumption of fruit and vegetables and risk of frailty: a dose-response analysis of 3 prospective cohorts of community-dwelling older adults[END_REF]. Among a sample of 2,926 people aged 65 and over, followed for up to 2.5 years, the initial intake of at least 3 fruits per day was significantly associated with a 52% decrease in the risk of developing frailty, while a consumption of 2 servings of vegetables a day led to a 44% decrease in the risk of frailty. Combined, the daily consumption of 5 or more servings of fruits and vegetables was significantly associated with a nearly 70% reduction in the risk of frailty among elderly people in the community. These results suggested a beneficial effect of fruit and vegetable consumption on the short-term risk of frailty, even among people who were almost 70 years old on average [START_REF] Garcia-Esquinas | Consumption of fruit and vegetables and risk of frailty: a dose-response analysis of 3 prospective cohorts of community-dwelling older adults[END_REF]. Regarding other food groups, a recent Spanish study has also underlined a significant association between sugar consumption and a higher risk for frailty, but this finding was limited to sugars from processed foods (i.e., added sugars) and not naturally occurring sugar in food [START_REF] Laclaustra | Prospective association between added sugars and frailty in older adults[END_REF]. Overall, it is important to emphasize that participants with a high consumption of plant products are also people who, in general, have a healthier diet and lower added sugar intake, which is now encouraging researchers to take into account for all food intake within the context of dietary patterns.

Relationship between dietary patterns and frailty

There are several methods of developing dietary patterns: either we have a priori assumptions about the beneficial or deleterious effects of food groups on health, which allow us to build scores for each participant whether or not they conform with these hypotheses (i.e., Mediterranean diet, Diet Quality Index), or we apply multidimensional statistical techniques to observational data, thus allowing the identification of dietary patterns specific to the studied sample (i.e., prudent diet, Western diet). An advantage of these approaches by pattern is the ability to capture the potential interactions between microconstituents, whether they are synergistic, additive or antagonistic [START_REF] Hu | Dietary pattern analysis: a new direction in nutritional epidemiology[END_REF].

These methodologies have been developed in the field of frailty. Thus, the Mediterranean diet would also have beneficial effects applicable to frailty, in addition to the well-known benefits on cardiovascular health and longevity [START_REF] Sofi | Mediterranean diet and health status: an updated meta-analysis and a proposal for a literature-based adherence score[END_REF][START_REF] Psaltopoulou | Mediterranean diet and stroke, cognitive impairment, depression: A meta-analysis[END_REF]. Several studies, including some recently published studies, confirmed that greater adherence to a diet of Mediterranean-type food was associated with a lower odds/risk of frailty over time [START_REF] Bollwein | Dietary quality is related to frailty in community-dwelling older adults[END_REF][START_REF] Talegawkar | A higher adherence to a Mediterranean-style diet is inversely associated with the development of frailty in community-dwelling elderly men and women[END_REF][START_REF] Guallar-Castillon | Mediterranean diet and risk of frailty in community-dwelling older adults[END_REF][START_REF] Milaneschi | Mediterranean diet and mobility decline in older persons[END_REF][START_REF] Veronese | Adherence to a Mediterranean diet is associated with lower incidence of frailty: A longitudinal cohort study[END_REF][START_REF] Ntanasi | Adherence to Mediterranean Diet and Frailty[END_REF]. Using data from the 3-City cohort, our analysis focused specifically on a representative sample of people aged 75 years and older, for whom a significant 68% decreased risk of developing frailty in the next 2 years was observed, suggesting that, even at advanced ages, this eating behavior could be beneficial [START_REF] Rahi | High adherence to a Mediterranean diet and lower risk of frailty among French older adults communitydwellers: Results from the Three-City-Bordeaux Study[END_REF]. We had already shown that following a Mediterranean-type diet was beneficial in terms of the risk of disability in the 3-City cohort; these last projects emphasized the importance of this dietary pattern in the phase that precedes this state of disability and suggested a long-term effect of the eating habits on health [START_REF] Feart | Adherence to a Mediterranean diet and onset of disability in older persons[END_REF]. Results from these studies on the Mediterranean diet and the risk of frailty were recently included in a meta-analysis, where participants who had the highest scores -with the strongest adherence to this dietary patternhad a significantly decreased risk of frailty by 56%, signaling a major strength of association [START_REF] Kojima | Adherence to Mediterranean Diet Reduces Incident Frailty Risk: Systematic Review and Meta-Analysis[END_REF].

More recently, a report observed that the benefit of a higher adherence to a Mediterranean diet on preventing frailty was also obvious on at-risk older women from the Nurses' Health Study suffering from type-2 diabetes [START_REF] Lopez-Garcia | Mediterranean diet and risk of frailty syndrome among women with type 2 diabetes[END_REF]. In addition to eating a Mediterranean diet to fight frailty, adopting a Mediterranean lifestyle would be a deterrent to frailty [START_REF] Voelker | The mediterranean diet's fight against frailty[END_REF][START_REF] Bach-Faig | Mediterranean diet pyramid today. Science and cultural updates[END_REF]. Moreover, as chronic inflammation may play a role in frailty, an a priori dietary pattern marker of foods and nutrients intakes associated with inflammation has been built in the Seniors-ENRICA cohort. The authors observed that the highest adherence to the "dietary inflammatory index" was associated with a higher risk for frailty (and slow gait speed) among 1948 participants followed for up to 4 years [START_REF] Laclaustra | The inflammatory potential of diet is related to incident frailty and slow walking in older adults[END_REF]. To our knowledge, this is the single study interested in the inflammatory part of the usual diet in the field.

Using the alternative approach of dietary pattern creation, namely, techniques without a priori hypotheses, we observed that the elderly men of the cohort 3-City who were characterized by a high consumption of pasta and women who were characterized by a higher consumption of biscuits and snacking, had a 2-fold greater risk of frailty after 12 years of follow-up [START_REF] Pilleron | Dietary Patterns and 12-Year Risk of Frailty: Results From the Three-City Bordeaux Study[END_REF]. By a similar approach, "cautious" or "traditional" profiles identified among Spanish or Dutch participants were also significantly associated with a lower risk of frailty 3.5 to 4 years later [START_REF] Garcia-Esquinas | Major dietary patterns and risk of frailty in older adults: a prospective cohort study[END_REF][START_REF] De Haas | Dietary patterns and changes in frailty status: the Rotterdam study[END_REF]. Since these dietary patterns are derived from observational data, they are not strictly comparable in terms of foods that comprise them, and the accumulation of knowledge in other elderly populations is required before drawing definitive conclusions. As a proof, an a posteriori inflammatory dietary pattern identified among the Spanish older individuals enrolled in the Seniors-ENRICA study failed to be associated with the frailty risk, while the a priori pattern was, in part due to the lack of reproducibility of such a posteriori patterns to other populations than the original ones [START_REF] Laclaustra | The inflammatory potential of diet is related to incident frailty and slow walking in older adults[END_REF].

Nutritional interventions to prevent or delay frailty

Intervention studies provide the best causal evidence in support of associations suggested by observational studies. In the case of nutritional exposure, it is particularly difficult to set up pragmatic trials to modify dietary behavior, but not impossible [START_REF] Estruch | Primary prevention of cardiovascular disease with a Mediterranean diet[END_REF], and complementary approaches are often preferred. The prevention of frailty at the level of the elderly community includes conventional health prevention messages such as the promotion of physical activity, a healthy diet, the cessation of tobacco, an active social life, weight maintenance, and the control of vascular and metabolic risk factors, such as dyslipidemia, diabetes, and blood pressure [START_REF] Sternberg | The identification of frailty: a systematic literature review[END_REF]. Multidomain interventions, including nutrition and physical activity, seem more relevant than the isolated nutritional approach, although nutrition alone can be considered as a multilevel approach through its potential benefits on several biological, clinical and social systems [START_REF] Dedeyne | Effects of multi-domain interventions in (pre)frail elderly on frailty, functional, and cognitive status: a systematic review[END_REF].

Overall, frailty is still a field of research where nutritional interventions are rare: participants are more often already frail and benefit from interventions to slow down the process and the onset of disability [START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF][START_REF] Cesari | The Multidomain Intervention to preveNt disability in ElDers (MINDED) project: rationale and study design of a pilot study[END_REF][START_REF] Puts | Interventions to prevent or reduce the level of frailty in community-dwelling older adults: a scoping review of the literature and international policies[END_REF]. Here, we described two main trials, but recent reviews will complete this noncomprehensive list [START_REF] Yannakoulia | Frailty and nutrition: From epidemiological and clinical evidence to potential mechanisms[END_REF][START_REF] Kelaiditi | Is nutrition important to postpone frailty? Current opinion in clinical nutrition and metabolic care[END_REF][START_REF] Kelaiditi | Frailty: role of nutrition and exercise[END_REF][START_REF] Hernandez Morante | Dietary Factors Associated with Frailty in Old Adults: A Review of Nutritional Interventions to Prevent Frailty Development[END_REF]. From 2013, a first trial was set up with robust women aged 65 and over who received 1-2 g of long-chain omega 3 fatty acids (EPA and DHA) daily for 6 months (n = 85), while the control group received 1.8 g of oleic acid in the form of olive oil (n = 41). The results showed no effect on the prevalence of frailty as a whole after 6 months, although a slight improvement in walking speed was observed in the intervention group [START_REF] Hutchins-Wiese | The impact of supplemental n-3 long chain polyunsaturated fatty acids and dietary antioxidants on physical performance in postmenopausal women[END_REF]. Another trial conducted in Singapore with 151 prefrail and frail women included nutritional supplementation, cognitive training, physical training or a combination of these treatments vs a nonintervention group, for 12 months [START_REF] Ng | Nutritional, Physical, Cognitive, and Combination Interventions and Frailty Reversal Among Older Adults: A Randomized Controlled Trial[END_REF]. At the end of the intervention, the frailty status was reduced in all groups, including the control group, and was significantly lower in the intervention group than in the control group. This trial has therefore demonstrated the effectiveness of these interventions on the reversibility of frailty [START_REF] Ng | Nutritional, Physical, Cognitive, and Combination Interventions and Frailty Reversal Among Older Adults: A Randomized Controlled Trial[END_REF].

While still rare today, the first available RCTs are valuable since they contribute to the improvement of future study plans of nutritional interventions on frailty, whether or not they are coupled with other interventions, and add to the definition of the best strategies for caring for frail people. Thus, hopes are now resting on SPRINTT-type trials, where sarcopenia, defined by a reduction in both mass and muscle strength, is considered the physiological substratum of frailty, while being a separate entity according to the ESPEN (European Society for Clinical Nutrition and Metabolism) [START_REF] Bahat | Sarcopenia: revised European consensus on definition and diagnosis[END_REF][START_REF] Cederholm | ESPEN guidelines on definitions and terminology of clinical nutrition[END_REF][START_REF] Landi | The "Sarcopenia and Physical fRailty IN older people: multi-componenT Treatment strategies" (SPRINTT) randomized controlled trial: design and methods[END_REF].

SPRINTT means "Sarcopenia and physical frailty in older people: multicomponent treatment strategies": for 36 months, 1,500 frail and sarcopenic elderly people will follow a multidomain program including structured physical activity programs, dietary counseling and intervention, and information and communication technology intervention vs a healthy aging education program in the control group. The goal is to demonstrate the effectiveness of this intervention on the prevention of disability, evaluated by the inability to walk 400 m in 15 min, without sitting, and without help. The results should help not only in informing better care for frail elderly people at high risk of disability but also in better defining preventive actions.

CONCLUSION

The growing interest in frailty in the scientific community lies in its impact on disability in our aging society. The identification of the frail individual depends primarily on the context, hence our current difficulties in making detection tools operational in a consensual manner. In terms of both diagnosis and prognosis, diet appears to be a major determinant in terms of frailty and its harmful consequences.

Altered nutritional status and inadequate protein, energy or micronutrient intake are associated with an increased risk of frailty, while all studies converge to suggest that adopting a Mediterranean-style dietary pattern rich in fruit and vegetable sources of antioxidants would be an effective way to combat the emergence of frailty. However, no scientific evidence is available to date. Research must continue to help better identify frail elderly people by providing validated tools and by testing the effectiveness of prevention interventions. As we move forward in this direction, we will be able to help make dietary recommendations to prevent frailty and its consequences and to meet the specific needs of the aging population.


Halidou Leïla

Diallo

Florence Tatin

David Florian

Anne-Claire Godet

Audrey Zamora

Anne-Catherine Prats

Barbara Garmy-Susini

Eric Lacazette
email: eric.lacazette@inserm.fr

How are circRNAs translated by non-canonical initiation mechanisms? by

Keywords: circRNA, translation, IRES, N 6 -methyladenosine

Circular RNAs (circRNAs) are covalently closed RNA loops produced by a very large number of expressed eukaryotic genes. Initially considered as splicing background and/or splicing side products, recent studies have shown that they are evolutionary conserved and abundant in cells. Yet, their functions remain largely unknown. Because of their circular shape, they were initially categorized as non-coding RNAs. However, recent studies based on mass spectrometry analysis indicate that some cytoplasmic circRNAs are effectively translated into detectable peptides. This raises the interesting question of which mechanisms regulate the translation initiation of those circular transcripts, i.e. unable to recruit the small ribosome subunit through the 5' cap. A possible mechanism for alternative translation initiation is the presence of an IRES (Internal Ribosome Entry Site) that allows direct recruitment of initiation factors and ribosomes on the RNA independently from the cap. This is the case for several circRNAs that exhibit IRESs upstream from the start codon. Yet, another process seems to be involved in initiating the translation of circRNAs: the presence of N 6 -methyladenosine (m 6 A) residues. These m 6 A can promote cap-independent translation and have been shown to be enriched in circRNAs. Interestingly, these two alternative translation initiation processes are generally activated under cellular stress to allow expression of specific stress response genes. These discoveries therefore link circRNA translation to cellular response to stress conditions, raising new enquiries about the regulation of circRNA expression under stress conditions and their functions. This review provides a state of the art on this emerging area.

Introduction

Circular RNAs (circRNAs) are endogenous to eukaryotic cells. Discovered more than 25 years ago, circRNAs have for long been considered as irrelevant artefacts of RNA processing [START_REF] Nigro | Scrambled exons[END_REF][START_REF] Cocquerelle | Mis-splicing yields circular RNA molecules[END_REF]. However, recent studies have uncovered their peculiar properties and large scale involvement in physiological and pathological gene expression [START_REF] Huang | The emerging role of circular RNAs in transcriptome regulation[END_REF][START_REF] Bolha | Circular rnas: biogenesis, function, and a role as possible cancer biomarkers[END_REF][START_REF] Dragomir | Circular RNAs in Cancer -Lessons Learned From microRNAs[END_REF][START_REF] Xu | A comprehensive review of circRNA: from purification and identification to disease marker potential[END_REF]. Indeed, data arising from RNA-seq projects demonstrated the significant amount of circRNAs in eukaryotic cell and the evolutionary conservation of this new category of RNAs, clearly stating that they are not merely splicing background. The first article describing a massive presence of circular RNAs in eukaryotic cells was from Salzman et al. [START_REF] Salzman | Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[END_REF]. In that study, the authors aimed to identify cancer-specific exon scrambling events, corresponding to non-colinear exon junctions. For this, they selected and analyzed non-colinear sequencing reads, evidence of "scrambled" exons and potential circRNAs in RNA-seq data. In this article they identified a total of 2,748 scrambled isoforms, or circRNAs, in HeLa and H9 embryonic cells. At the same time, a study conducted by Memczak et al. extended the exploration to different species and led to the identification of approximately 2,000 circRNA in humans, 1,900 in mice and 700 in nematodes and showed that circRNAs are often expressed with specific expression patterns depending on tissues or developmental stages [START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF]. Subsequently, Jeck et al. modified the approach by adding a 3' exoribonuclease (RNase R) treatment prior to sequencing, thus enriching the population of circular RNAs in the samples. This enhancement led to the identification of a significantly higher number of circular RNAs, and the results suggested that 14.4% of actively transcribed genes produced circular RNAs in human fibroblasts [START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF]. It is also the development of specific bioinformatics tools, based on split mapping of RNA-seq reads, that allowed the efficient prediction of circular RNAs from RNA-seq data [START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF][START_REF] Westholm | Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation[END_REF][START_REF] Zhang | Diverse alternative back-splicing and alternative splicing landscape of circular RNAs[END_REF][START_REF] Gao | CIRI: an efficient and unbiased algorithm for de novo circular RNA identification[END_REF][START_REF] Wang | MapSplice: accurate mapping of RNA-seq reads for splice junction discovery[END_REF].

A relatively large number of actively transcribed genes generate circular RNAs in mammalian cells. They mostly originate from coding genes by a spliceosome-mediated alternative splicing of the pre-messenger RNA called backsplicing [START_REF] Huang | The emerging role of circular RNAs in transcriptome regulation[END_REF][START_REF] Salzman | Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[END_REF][START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF][START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF][START_REF] Eger | Circular RNA Splicing[END_REF]. Using the spliceosome to link a splice acceptor (3' splice site) to a downstream splice donor (5' splice site) on the same exon or further, circRNA biogenesis can compete with the maturation of its linear counterpart [START_REF] Salzman | Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[END_REF][START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF][START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF][START_REF] Ashwal-Fluss | circRNA biogenesis competes with pre-mRNA splicing[END_REF][START_REF] Starke | Exon circularization requires canonical splice signals[END_REF]. This non-colinear splicing forms a backspliced junction specific to each circular RNA species and absent from the canonical linear RNAs. This process is promoted by the presence of complementary inverted repeats and RNA binding proteins (RBPs) binding motifs in introns flanking the two splice sites (Figure 1). The pairing of complementary sequences or formation of RBP dimers brings the circularized exons close together [START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF][START_REF] Eger | Circular RNA Splicing[END_REF][START_REF] Liang | Short intronic repeat sequences facilitate circular RNA production[END_REF][START_REF] Ivanov | Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals[END_REF][START_REF] Conn | The RNA binding protein quaking regulates formation of circRNAs[END_REF]. In primates, Alu repeats are the most abundant form of repeated sequences and have thus been associated with circRNA biogenesis in human cells. Formation of these double stranded RNA structures can also imply trans-acting RBPs that will modulate backsplicing either positively, by stabilizing the structure as is the case with NF90/NF110, or negatively, by destabilising the base-pairing like the editing enzyme adenosine deaminase that acts on RNA (ADAR1) [START_REF] Li | Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection[END_REF][START_REF] Rybak-Wolf | Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed[END_REF].

The covalently closed circular structure of circRNAs renders them resistant to 3' or 5' exoribonucleases. From this characteristic derives their high stability with a half-life reaching up to 10 times that of linear RNAs (median half-life 2.5 times longer on 60 analyzed circRNAs) [START_REF] Enuka | Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor[END_REF]. In eukaryotes, the length of circRNAs is variable but tends to average between 200-800 nucleotides (nt) with a range of 100 to 4000 nt in length [START_REF] Memczak | Identification and characterization of circular rnas as a new class of putative biomarkers in human blood[END_REF]. One estimate that in humans, circular RNA may account for 1% as many molecules as poly (A) RNA [START_REF] Salzman | Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[END_REF].

On the functional level, circRNAs are tuned to retain tissue and developmental stage specificity and can be sensitive to cellular stresses [START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF][START_REF] Salzman | Cell-type specific features of circular RNA expression[END_REF]. They could also be involved in pathological processes such as cancer [START_REF] Yang | Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis[END_REF][START_REF] Zhang | A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[END_REF]. The circular transcripts have well-conserved sequences among eukaryotes. Based on the type of sequences they contain, they are divided into exonic (EcRNA), intronic (CiRNA) which have a different biogenesis mechanism and exon-intron (EIcRNA) circRNAs. Like in other RNAs, these differences in structure and sequence define their sub-cellular localisation and function [START_REF] Li | Exon-intron circular RNAs regulate transcription in the nucleus[END_REF][START_REF] Hansen | Natural RNA circles function as efficient microRNA sponges[END_REF] (Figure 1). Some intronic and exon-intron circRNAs are reported to be sequestered in the nucleus [START_REF] Li | Exon-intron circular RNAs regulate transcription in the nucleus[END_REF][START_REF] Conn | A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation[END_REF], while exonic circRNAs are mostly exported to the cytosol via a length-dependent mechanism involving drosophila's Hel25E dead-box helicase and its homologues in human cells [START_REF] Huang | A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs[END_REF]. Circular RNAs are predominantly exonic and thus predominantly cytosolic [START_REF] Salzman | Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types[END_REF][START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF].

In the nucleus, circRNAs participate in gene expression regulation at different levels. First, circRNA biogenesis is largely post-transcriptional and use the canonical splicing apparatus, thus competing with the maturation of its linear counterparts [START_REF] Ashwal-Fluss | circRNA biogenesis competes with pre-mRNA splicing[END_REF][START_REF] Starke | Exon circularization requires canonical splice signals[END_REF][START_REF] Zhang | The biogenesis of nascent circular rnas[END_REF][START_REF] Vicens | Biogenesis of circular rnas[END_REF]. Second, exemplified by circPAIP2 in human cells, intronic and exon-intron circRNAs have been described as transcriptional regulators of their parental genes by interacting with RNA polymerase II, snRNPs and hnRNPs [START_REF] Li | Exon-intron circular RNAs regulate transcription in the nucleus[END_REF][START_REF] Conn | A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation[END_REF][START_REF] Kramer | Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins[END_REF] (Figure 1-1).

In the cytoplasm there is an accumulation of EcRNAs with two types of functional outcomes described so far. Firstly, because they share parts of the mature linear RNA sequence, exonic circRNAs act as competing endogenous RNAs, sequestering microRNAs (miRs) and RBPs away from their cytosolic targets (Figure 1-2). For example, one of the first and most studied circRNAs, circCDR1as/ciRS-7 in brain tissue harbours 63 conserved binding sites for miR7, acting as an effective miR7 sponge. Over-expression of the human circCDR1as/ciRS-7 revealed a negative impact on brain development in zebrafish [START_REF] Memczak | Circular RNAs are a large class of animal RNAs with regulatory potency[END_REF][START_REF] Hansen | Natural RNA circles function as efficient microRNA sponges[END_REF]. CircRNA interaction with RBPs is illustrated by the cases of circMbl and circFoxo3. The drosophila muscleblind gene (Mbl) produces a circular RNA (circMbl) when the Mbl protein, involved in circRNA biogenesis, accumulates. CircMbl then binds and retains the excessive Mbl protein [START_REF] Ebbesen | Circular RNAs: Identification, biogenesis and function[END_REF]. In mammalian cells the tumour suppressor gene Foxo3 generates a circRNA (circFoxo3) that regulates cell cycle progression by binding to cell division protein kinase 2 (CDK2) and cyclin-dependent kinase inhibitor 1 (p21) [START_REF] Du | Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2[END_REF]. Hence, circRNAs participate in miR and RBP transportation, storage and availability creating yet new ways of post-transcriptional regulation.

In addition, interesting recent studies have shown that a subset of exonic circRNAs is found associated with polysomes and most of them comprise the native start codon of their mRNA counterparts and exhibit favourable open reading frames [START_REF] Yang | Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis[END_REF][START_REF] Zhang | A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[END_REF][START_REF] Pamudurti | Translation of CircRNAs[END_REF][START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis[END_REF][START_REF] Yang | Extensive translation of circular RNAs driven by N6-methyladenosine[END_REF][START_REF] Wang | Efficient backsplicing produces translatable circular mRNAs[END_REF][START_REF] Zhang | A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[END_REF][START_REF] Begum | Novel tumour suppressive protein encoded by circular RNA, circ-SHPRH, in glioblastomas[END_REF][START_REF] Wilusz | Circular RNAs: Unexpected outputs of many protein-coding genes[END_REF][START_REF] Tatomer | An unchartered journey for ribosomes: circumnavigating circular rnas to produce proteins[END_REF]. This revealed an unexpected translation potential for circRNAs. Originally mislabelled as "noncoding", circRNAs can actually be translated into regulatory peptides (Figure 123). These observations nevertheless raise the question of translation initiation for this particular class of circular transcripts.

circRNA translation

The question of circRNAs translation in cells is highly relevant since they are mainly cytoplasmic and comprised of exonic sequences (EcRNAs), often carrying an initiator codon and sometimes the canonical AUG of the associated mRNA with potential open reading frames. In addition, Chen an Sarnow have proven, in early studies in 1995, that synthetic circular RNAs could recruit the 40S ribosomal subunit and initiate translation of detectable peptides in human cells through an internal entry site [START_REF] Chen | Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs[END_REF]. However, that study does not investigate the case of endogenous circRNA molecules. Therefore, several teams investigated whether endogenous circular RNAs were translated. The early results provided rather negative responses to that problematic. In a first study Jeck et al. selected three EcRNAs that had a translation start site (common with the corresponding mRNA) and found out that they were absent from the polysome fraction [START_REF] Jeck | Circular RNAs are abundant, conserved, and associated with ALU repeats[END_REF]. This indicated that these three circular RNAs were not bound to the ribosomes and therefore not translated. Another study, by Guo et al. systematically searched for evidence of circRNA translation by examining data from ribosome footprinting of non-poly(A) selected RNA-Seq [START_REF] Guo | Expanded identification and characterization of mammalian circular RNAs[END_REF]. They concluded that no evidence supported the possibility of circRNAs undergoing translation in cells. The only demonstration of EcRNAs translation by ribosome footprinting is the detection of reads spanning the backsplicing junctions. In this case, it is presumably this constraint combined with the lesser abundance of circRNAs, compared with corresponding linear RNAs, that could explain these results (i.e., no evidence of circRNA translation). In a later study, Schneider et al. investigated the association of circRNAs with proteins by sucrose gradient fractionation [START_REF] Schneider | CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs[END_REF]. Like the study by Jeck et al. they focused on a limited number of abundant circRNAs and failed to find any association with polysomes. Despite these negative responses, the investigations continued and, from 2017 to this date, seven distinct circular RNAs have been deemed translated in human cells and, for some, the function of the deriving peptide have been identified [START_REF] Yang | Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis[END_REF][START_REF] Zhang | A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[END_REF][START_REF] Pamudurti | Translation of CircRNAs[END_REF][START_REF] Zhang | A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[END_REF][START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be[END_REF][START_REF] Zheng | A novel protein encoded by a circular RNA circPPP1R12A promotes tumor pathogenesis and metastasis of colon cancer via Hippo-YAP signaling[END_REF][START_REF] Liang | Translation of the circular RNA circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway[END_REF]. In addition to that, a recent large scale study by Ragan et al., identified circRNAs with a great potential of translation [START_REF] Ragan | Insights into the biogenesis and potential functions of exonic circular RNA[END_REF]. By performing TrIP-seq (Transcript Isoform in Polysomes sequencing, [START_REF] Floor | Tunable protein synthesis by transcript isoforms in human cells[END_REF]) they found approximately 15% of all circRNA read counts in the ribosomal fraction, representing 177 potentially translated circRNA candidates in HEK293 cells [START_REF] Ragan | Insights into the biogenesis and potential functions of exonic circular RNA[END_REF]. Most of these candidates for translation are associated with the monosomal to tetrasomal fractions, whereas linear mRNAs are mostly associated with hepta and octasomal fractions. These results, considering the seven studies mentioned above, support the coding potential of many more endogenous circular RNAs and raises the question of the mechanisms by which their translation is initiated.

cap-independent translation initiation mechanisms

The circular structure of circRNAs necessarily implies a cap-independent and internal mechanism to initiate translation. Indeed, eukaryotic mRNAs have their 5' end modified by the presence of a m7G (7-methyl guanosine) cap which is recognized by the eIF4E translation initiation factor [START_REF] Gross | Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E[END_REF]. The eIF4E factor combines with eIF4G (a scaffold protein) and eIF4A (an helicase protein) to form the eIF4F complex [START_REF] Schütz | Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions[END_REF][START_REF] Marintchev | Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation[END_REF]. The interaction between eIF4E and the cap is modulated by the mTOR (mammalian Target Of Rapamycin) kinase pathway.

When the cells are submitted to a stress like hypoxia, nutrient starvation, oxidative stress, etc… the mTOR kinase is inactivated, leading to an overall inhibition of the cap-dependent translation due to hypophosphorylation of 4E-BPs (eIF4E-binding proteins). Hypophosphorylated 4E-BPs compete with eIF4G for eIF4E binding and thus prevent eIF4F assembly and the 43S ribosome recruitment at the 5'end of the transcripts [START_REF] Pause | Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function[END_REF]. This is one of the main pathways for translation initiation control in eukaryotes [START_REF] Sonenberg | Regulation of translation initiation in eukaryotes: mechanisms and biological targets[END_REF]. The other major mean to modulate translation initiation is through the eIF2α factor phosphorylation. A number of serine kinases phosphorylate eIF2α. Those kinases are activated by stresses such as amino acid deprivation (GCN2), ER stress (PERK), the presence of dsRNA (PKR), heme deficiency (HRI) or interferon [START_REF] Ryoo | Two distinct nodes of translational inhibition in the Integrated Stress Response[END_REF]. When eIF2α initiation factor is phosphorylated, there is a blockade of GDP to GTP exchange in the eIF2 complex, which in turn prevents the assembly of the ternary complex eIF2-GTP-tRNAi Met . Thus, during stress, the cap-dependent translation is largely inhibited [START_REF] Holcik | Translational control in stress and apoptosis[END_REF]. Nevertheless, a subset of messenger RNAs maintain their translation during stress by using cap-independent translation initiation mechanisms [START_REF] Sonenberg | New modes of translational control in development, behavior, and disease[END_REF][START_REF] Meyer | m6A-mediated translation regulation[END_REF].

One possibility in the cells, for cap-independent translation initiation, is the presence of an IRES (Internal Ribosome Entry site) structure in the 5'UTR of mRNAs. IRESs are sequences that form secondary structures on RNA and allow translation initiation in the total or partial absence of canonical translation initiation factors, replaced by ITAFs (IRES Tran-Acting Factors) (for review, see [START_REF] Godet | IRES Trans-Acting Factors, Key Actors of the Stress Response[END_REF]). Although eIF2α is, in principle, required for the initiation of cap-dependent and cap-independent translation, several reports have shown that IRESdependent translation can occur despite eIF2α phosphorylation [START_REF] Fernandez | Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2alpha[END_REF]. The first IRES elements have been discovered in viruses [START_REF] Jang | A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation[END_REF] but are also present in the 5'UTR of many cellular mRNAs like the immunoglobulin heavy-chain binding protein (BiP) mRNA, the fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) mRNAs [START_REF] Godet | IRES Trans-Acting Factors, Key Actors of the Stress Response[END_REF] [START_REF] Macejak | Internal initiation of translation mediated by the 5' leader of a cellular mRNA[END_REF]. On cellular mRNAs, IRESs are particularly functional under stressful conditions. They allow the maintenance or activation of translation for a pool of RNAs equipped with IRESs, providing the cell with important proteins for its survival. These genes include master regulators like transcription factors, growth factors, etc….

Another mechanism described recently is the cap-independent translation initiation under stress through methylated adenosine residues in the form of N 6 -methyladenosines (m 6 A) in the 5' untranslated region (5'UTR) of some transcripts [START_REF] Meyer | 5' UTR m(6)A Promotes Cap-Independent Translation[END_REF]. 5'UTR m 6 A residues can directly recruit the eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex and initiate translation in the absence of the cap-binding factor eIF4E. 5'UTR m 6 A residues can also modulate translation initiation during integrated stress response by redirecting ribosomes on different ORFs. This is illustrated by the case of ATF4 mRNA, which is regulated by both the eIF2α signaling pathway and the presence of m 6 A residues, upon amino acid starvation [START_REF] Zhou | N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response[END_REF]. Indeed, ATF4 mRNA is m 6 A methylated in the uORF2 located in the 5'UTR. This methylation is decreased upon amino acid starvation in a ALKBH5 and FTO demethylase-dependent manner. In fact, ribosome retention increases on the uORF2 m 6 A residues, leading to a slower ribosome scanning, which in turn promotes translation initiation at non-canonical upstream initiation sites. Ultimately, there is a decrease of translation initiation on ATF4 main ORF. This concept has been enlarged to other transcripts by global profiling of m 6 A and translation initiation sites occupancy [START_REF] Zhou | N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response[END_REF]. Thus, both m 6 A and IRES dependent translation initiation are potential mechanisms for cytoplasmic circular RNAs translation.

To date, only seven translated circRNAs have been uncovered by Legnini, Pamudurti, Zhang, Yang, Zheng and Liang et al. in separate studies published during the last two years [START_REF] Yang | Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis[END_REF][START_REF] Zhang | A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[END_REF][START_REF] Pamudurti | Translation of CircRNAs[END_REF][START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis[END_REF][START_REF] Zhang | A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[END_REF][START_REF] Zheng | A novel protein encoded by a circular RNA circPPP1R12A promotes tumor pathogenesis and metastasis of colon cancer via Hippo-YAP signaling[END_REF][START_REF] Liang | Translation of the circular RNA circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway[END_REF]. In the following segments we will present each of these studies, focusing on the crucial question of which cap-independent translation initiation mechanisms are triggered by circular RNAs. Consequently, we exclude the study by Zheng et al. on circPPP1R12A for which the translation initiation mechanisms were not addressed [START_REF] Zheng | A novel protein encoded by a circular RNA circPPP1R12A promotes tumor pathogenesis and metastasis of colon cancer via Hippo-YAP signaling[END_REF].

IRES driven circRNA translation

IRES-driven translation of circZNF609

In the study by Legnini et al. the authors showed that circZNF609 is the backsplicing product of the ZNF609 gene exon 2, encoding a poorly characterized transcription factor belonging to the zinc-finger protein family [START_REF] Legnini | Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis[END_REF]. Interestingly, the Open Reading Frame (ORF) of circZNF609 starts at the same AUG codon as its linear counterparts but encounters a stop codon 3 nucleotides after the backspliced junction where a frameshift occurs. Supporting its in vivo translation, circZNF609 has been found in the heavy polysomes fraction after RNase R treatment. The authors then tested whether the sequence upstream from the initiation codon (i.e. 80 nt from the 3' end of exon 2 and across the backspliced junction fused to the UTR) displayed an IRES activity. This sequence cloned into a bicistronic vector, containing the Renilla luciferase under cap-dependent translation and the Firefly luciferase under the putative IRES-dependent translation, did not display any significant IRES activity. However, when they added small fragments of the endogenous intronic sequences in the construct, in order to mimic the backsplicing occurring to generate circZNF609, they obtained a stronger IRES activity than that of EMCV. Thus circZNF609 exhibits a splicing dependent IRES activity. This observation is particularly interesting because it suggests that factors necessary for IRES activity may be recruited through splicing. It is entirely conceivable, for example, that proteins of the hnRNP family involved in splicing could be recruited onto circRNA in the nucleus, afterwards acting as IRES Trans-Acting Factors (ITAFs) in the cytosol. This process has already been described for several hnRNPs acting as ITAFs for mRNAs [START_REF] Godet | IRES Trans-Acting Factors, Key Actors of the Stress Response[END_REF].

Additionally, protein production from circZNF609 is increased after a heat-shock stress, which is known to activate IRES activity [START_REF] Holcik | Translational control in stress and apoptosis[END_REF] and modulate alternative splicing [START_REF] Biamonti | Cellular stress and RNA splicing[END_REF]. These two processes may be a functional link between stress and circRNA translation initiation.

IRES-driven translation of circMbl

In the same issue of Molecular Cell, Pamudurti et al. presented their study in which they show that a subset of circRNAs are associated with translating ribosomes in vivo in Drosophila by ribosome footprinting [START_REF] Pamudurti | Translation of CircRNAs[END_REF]. They focused on a circRNA, circMbl, generated from the muscleblind locus. Like circZNF609, circMbl shares the same start codon as the linear muscleblind mRNA and the ORF stops a few nucleotides after passing the backspliced junction. Similarly, they have found an IRES activity arising from the UTR of the circRNA (cUTR) cloned into a bicistronic vector. In their experiments, they also measured Firefly and Renilla activities in the presence or absence of 4E-BP, mimicking the blockade or not of capdependent translation. Overexpression of 4E-BP resulted in an increase by more than 4 fold of the Firefly luciferase/Renilla luciferase luminescence ratio, sustaining cap-independent translation initiation through the circMbl cUTR in a context mimicking cellular stress.

Importantly, the circMbl-encoded protein was also detected by mass spectrometry.

IRES-driven translation of circSHPRH

In another study, Zhang et al. identified a new circRNA produced by the gene coding the E3ligase SNF2 histone linker PHD RING helicase (SHPRH), called circSHPRH [START_REF] Zhang | A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[END_REF]. In this case, the backsplicing of exons 26 to 29 forms circSHPRH and organises a large ORF of 146 amino acids. The particularity of circSHPRH compared to the two previous examples is that the ORF occupies the totality of the circRNA by using a start codon overlapping with two tandem repeat stop codons: UGAUGA. The authors have identified an IRES able to drive translation initiation of circSHPRH. However, this IRES is located downstream from the start codon in contrast to circZNF609 and circMbl. It is also interesting to note that the functional IRES is formed by the joint sequences of exons 26 and 29 and thus spans across the backspliced junction. Indeed, if a portion of the backspliced junction arising from either exon 26 or exon 29 is deleted, the IRES activity is dramatically reduced. This illustrates yet another example of splicing dependence for IRES mediated translation initiation of a circRNA, and points again at the possibility of ITAF recruitment through splicing. Significantly, circSHPRH produces a novel 146 amino acids protein that was detected by mass spectrometry and displayed a tumour suppressor activity by acting as a decoy, protecting the related fulllength SHPRH protein from degradation.

IRES-driven translation of circFBXW7

In a similar way, Yang et al. have characterized a circRNA produced by the tumour suppressor E3 ligase FBXW7 [START_REF] Yang | Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis[END_REF]. The circular form of FBXW7, circFBXW7, derives from the backsplicing of exon 3 to 4 and forms a 620 nt circular RNA. In the case of circFBXW7, the authors have found a 120 nt long IRES in the cUTR that is located upstream from the AUG and downstream from the backspliced junction. The partial deletion of this sequence induced a decrease in the IRES activity. Unfortunately, it seems that the authors did not test the IRES activity of a longer sequence stretching upstream to cover the backsplicing junction, which would have been interesting to compare with the previous occurrences of circRNA IRESs. Similarly to circSHPRH, the 185 amino acids protein encoded by the circFBXW7 (FBXW7-185aa) was detected by mass spectrometry and displayed a tumour suppressor activity in a glioblastoma model. The analysis of circFBXW7 expression in glioblastoma clinical samples confirmed its effective translation in vivo and the potential tumour suppressor activity of FBXW7-185aa, as the protein levels were reduced in the samples compared with their paired tumour-adjacent tissues.

IRES-driven translation of circLINC-PINT

A second study by Zhang et al. reported last year the translation of a circRNA originating from the long non-coding RNA and p53-induced transcript LINC-PINT [START_REF] Zhang | A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[END_REF]. The circLINC-PINT is generated by the circularisation of exon 2. The authors identified circLINC-PINT as a potential coding circular RNA from the analysis of ribosome nascent chain-sequencing data. This 1084 nt circular RNA contains several small open reading frames. The study has proven one of them to produce a 87 amino acid peptide, called PINT87aa, that participates in the control of cell proliferation and tumorigenesis in cancer cells. The authors cloned the sequence of 478 nt between the backspliced junction and the start codon and found an IRES activity able to drive cap-independent translation initiation in a bicistronic vector. In this case too, the cloned region tested for IRES activity did not span the backspliced junction.

Interestingly, the most potent region that retained IRES activity, when they performed deletion experiments, was the region closest to the backspliced junction (i.e. the first 231 nt display a comparable IRES activity to the full length 478 nt).

IRES-driven translation of circβ-catenin

This year, Liang et al. have published the latest article to date reporting the translation of a circular RNA into a novel β-catenin isoform [START_REF] Liang | Translation of the circular RNA circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway[END_REF]. Circβ-catenin is formed by the backsplicing of exons 2 to 7 of the β-catenin gene, resulting in a 1129 nt long circular molecule. The authors identified an ORF spanning from the same start codon as the linear β-catenin mRNA (nucleotide 49 in exon 2) across the backspliced junction to a new stop codon (nucleotide 30 in exon 2). The study demonstrated that a 370 amino acids β-catenin isoform was produced by this ORF and detected by mass spectrometry, on a liver cancer cell line. Further experiments revealed that this protein promotes liver cancer cells growth, both in vitro and in vivo, through the activation of the Wnt pathway. By acting as a decoy for the canonic βcatenin, the circRNA derived isoform protects it from degradation and thus promotes its action. The authors identified a putative IRES sequence of 107 nt, upstream of the start codon, displaying a significant activity when cloned into a dual luciferase reporter system, strongly suggesting the presence of an IRES in this region.

Thus, like for circZNF609 and circSHPRH, the location of the stop codon a few nucleotides upstream the start codon, with respect to the size of the IRESs that are described, implies that the sequence containing the IRES overlaps the translated region. In addition, the possibility of a similar layout for the other four circRNAs is not ruled out, if not comforted in the case of circLINC-PINT. The reoccurrence of this configuration in such a small population of translated circRNAs is interesting to note as it could be a specific feature of circular RNA IRESs. Indeed, for cellular IRESs, only one example of an IRES in a translated region has been reported in the literature. It is the case of the CAT-1 mRNA, which possesses an IRES overlapping a small ORF upstream of the main start codon. In this case, the translation of the small reading frame induces a conformational change that activates the IRES [START_REF] Yaman | The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader[END_REF]. Could this apply to circRNAs ? Though, in the case of the CAT-1 mRNA, the activation of the IRES depends on the cap-dependent translation of an upstream ORF, whereas for circular RNAs, the IRES would need to be active enough to initiate a pioneering translation round and then be further activated. It will be interesting to carry out an in-depth mechanistic study of these IRESs overlapping translated regions in circular RNAs to answer these questions in the future.

m 6 A driven circRNA translation

Nevertheless, it seems that mRNAs without IRES activity can still be translated independently of the eIF4F complex. Several recent articles report that methylation in the form of N 6methyladenosine (m 6 A) in the 5'UTR of messenger RNAs facilitates the translation of some RNAs, in a context where translation initiation via eIF4F is blocked but in an IRESindependent pathway [START_REF] Coots | m6A Facilitates eIF4F-Independent mRNA Translation[END_REF]. Therefore, this mechanism seems to constitute a third way to initiate translation in eukaryotic cells and an alternative to the use of IRESs for capindependent translation initiation. The mechanism for m 6 A-initiated translation is not well known but involves the methyltransferases METTL3, METTL14 and Wilm's tumor associated protein [START_REF] Liu | A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation[END_REF][START_REF] Ping | Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase[END_REF]. This system is reversible since translation by m 6 As can be inhibited by the demethylases FTO (fat mass and obesity-associated protein) and ALKBH5 (alkylated DNA repair protein alkB homolog 5) [START_REF] Jia | N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO[END_REF][START_REF] Zheng | ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[END_REF]. Interestingly, the initiation of m 6 Adependent translation seems to be finely regulated under stress conditions and could constitute an additional mechanism to modulate the translation of a pool of specific transcripts under stress conditions in parallel with the pool of IRES-controlled mRNAs [START_REF] Zhou | N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response[END_REF]. Here again, this mechanism, which allows the initiation of translation in an eIF4F-independent way, can be applied to translation initiation for circular RNAs, as reported by some recent studies [START_REF] Yang | Extensive translation of circular RNAs driven by N6-methyladenosine[END_REF]. In the article by Yang et al., the authors show that m 6 A residues are enriched on all circRNA sequences and that a single m 6 A is sufficient to initiate translation. This circRNA m 6 Adependent translation relies on the eIF4G2 factor, and the YTHDF3 m 6 A reader. It is activated under stress conditions such as heat-shock or by the overexpression of METTL3/14 and is inhibited by the FTO demethylase. The authors performed an RNA-Seq analysis of m 6 A-targeted immunoprecipitated RNAs, treated with RNase R. They estimate that at least 13% of circular RNAs carry the m 6 A modification, but this is probably an underestimated value since the technical approach focuses only on the analysis of m 6 A motifs adjacent to backspliced junctions. This study identified 250 circRNAs associated with polysomes and some may be actively translated since this association is sensitive to puromycin treatment, although this fact alone does not constitute absolute proof of translation.

Translation of synthetic circular RNAs

Very recently, studies have been initiated to produce synthetic circulars RNAs in order to investigate the biological characteristics of circular RNAs and their biogenesis. For this purpose, authors have designed a plasmid expression system where backsplicing is facilitated by the hybridization of complementary sequences in the synthesized "pre-circRNA" [START_REF] Wesselhoeft | Engineering circular RNA for potent and stable translation in eukaryotic cells[END_REF].

With an IRES upstream of the reading frame, the circRNAs derived from these expression vectors can produce a given protein. Yang et al. adopted this strategy to design a vector where a GFP reading frame is assembled by backsplicing [START_REF] Yang | Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA[END_REF]. The construct can therefore be used to study the efficiency of backsplicing or IRES dependent translation initiation. Considering the stable properties of circRNAs, this type of vector is also a promising tool in biotechnology for a prolonged production of recombinant proteins [START_REF] Yang | Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA[END_REF]. The IRES-dependent GFP split construct has already been transposed into an adenoviral genome to produce recombinant proteins in vivo [START_REF] Meganck | Tissue-Dependent Expression and Translation of Circular RNAs with Recombinant AAV Vectors In Vivo[END_REF]. Finally, very recently, a team proposed an original variant where the translation is initiated by an "eukaryotic IRES-like site" i. e. an m 6 A motif. In this new vector, the stop codon of the reading frame has been removed and the presence of a T2A sequence allows the ribosome to continuously re-initiate translation. Therefore, the entire sequence is translated. The use of an m 6 A motif drastically reduces the size of the sequence allowing translation initiation [START_REF] Costello | Continuous translation of circularized mRNA improves recombinant protein titer[END_REF]. This system enables the stable and continuous production of a protein in a given cell. The combination of the stability of circular RNAs and their high potential for continuous translation could make them major tools for future use in biotherapies. Nevertheless, much work remains to be done to optimize these vectors, especially by a thorough understanding of the translation initiation mechanisms on circular RNAs.

Outlook

New perspectives are opening for the field of research on RNAs and particularly on circular

RNAs. There is no doubt that new protein coding circRNAs will be discovered in the near future. It will be essential to uncover and understand the various cellular functions of these novel proteins. These mechanisms are of utmost relevance in physiopathological contexts since circRNA derived proteins are very likely involved in stress responses, due to their capindependent and stress responsive translation initiation, whether IRES or m 6 A-dependent.

Their implications in cell differentiation and tumor cell development are proof enough of the potential of circRNA derived proteins in deciphering complex systems and finding new therapeutics. There is no doubt that a growing number of publications will enlighten us on the importance of circular RNAs in many cellular processes, including cell metabolism, which has been poorly studied so far.

Figure captions

Figure 1 :

 1 Figure 1: Circular RNAs biogenesis and functions.

Figure 2 :

 2 Figure 2: Circular RNA translation

Conflict of interest

The authors declare no conflict of interest