> Stages de M2 > Liste des stages proposés pour l’année 2017-2018 > Exploring the function of an RNA methyl transferase (RMT) involved in (...)

Exploring the function of an RNA methyl transferase (RMT) involved in non-coding RNA silencing pathways (mi, si &piRNA).

oposé par Clément CARRÉ, - Institut de Biologie Paris Seine - 9, Quai St Bernard, Boîte courrier 24 ; 75252 Paris Cedex 05

Projet de stage :

Three classes of small non-coding RNAs (sncRNA) emerged as a vast silencing framework in metazoan. They are involved in numerous biological processes including development, defense against pathogens and transposable element as well as chromatin dynamics. Precursors of these three types of small RNAs, namely miRNAs (microRNA), siRNAs (small interfering RNA) and piRNAs (piwi interacting RNA) are all transcribed by RNA polymerase II. After distinct biogenesis processes, si, pi and miRNAs are all loaded inside Argonaute proteins and guide them to target RNAs with sequence complementarity. The post-transcriptional repression that will take place using these ribonucleoprotein complexes (RNA-Induced Silencing Complexes, RISC) can operate through endonucleolytic cleavage or translational repression of the target RNA. Argonaute proteins loaded with si or piRNA are also involved in transcriptional repression by recruiting, at nascent RNAs, complexes that are able to modify DNA or histone proteins. These varieties of functional and mechanistic outputs explain why misregulation of RNA silencing pathways has been correlated to the emergence of cancer, neurodegenerative diseases and infertility troubles. In mice, fruit flies and humans, it has been shown that defects due to faulty methylation in tRNA (transfer RNA) biogenesis exhibit neurodegenerative diseases similar to those recently observed in misregulation of siRNA and piRNA pathways. The degradation of tRNA into tRF (tRNA Fragments) partly depends on their degree of methylation. When tRF are abnormally present in the cell, they titrate down RNA binding protein, including Dicer-2, which then produce less siRNA. Regarding these data, it is thus tempting to think that neurodegenerative pathologies observed in tRNA biogenesis defects are actually provoked by their ability to shut down the sncRNA pathways. The Master2 student project aims at characterizing the precise role of those tRFs observed in our RNA methylase mutant. Interestingly, this methylase is conserved in human and we also would like to shed light on its potential involvement during known disease development. This project will be conducted using two key activities, first disentangle the mechanism of action of abnormal tRFs observed in our methylase mutant along with the identification of those tRFs inside Argonautes proteins using Immunoprecipitation followed by Northern blot and/ or small RNA sequencing. Second, showing that the human homologues has conserved function on sncRNA pathways (using specific in vivo sensors) and interrogates the ‘tRFs status” of the patient cells carrying a mutation in this methylase. This Master2 project involves significant interdisciplinary contribution going from genetics and biochemistry principally to high throughput sequencing and bioinformatics analysis.

Techniques mises en œuvre par le stagiaire :
  Immunoprecipitation followed by Northern blot and/ or small RNA sequencing.
  Fly genetics
  Human patient cells (cell culture, Cell transfection).
  Methylation assay

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