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RNAmedTGS Report Summary

Project ID: 616015
Funded under: FP7-IDEAS-ERC
Country: France

Mid-Term Report Summary - RNAMEDTGS (RNA-mediated Transcriptional Gene Silencing in Humans)

While there is ample evidence from model systems that small RNAs are the lynchpin of powerful mechanisms that silence transcription, the demonstration of transcriptional gene silencing (TGS) mediated by a small RNA in humans is lacking. We had shown that the HIV-1 LTR is controlled by premature termination of transcription and TGS mediated by a small RNA derived from the stem-loop structure, TAR, that forms the 5’ end of HIV-1 transcripts. This pathway depends on Microprocessor, Xrn2, Setx and Rrp6. These findings provided the first evidence showing that small RNA-mediated TGS acts in human cells, and they also uncovered a new mechanism of gene regulation involving premature termination of transcription by RNAPII. The goals of this project are to identify the mechanism underlying small RNA-mediated TGS and determine whether small RNA-mediated TGS operates at human genes, as well as to determine the contribution of TGS coupled to premature termination in the global control of transcription. We are using a battery of biochemical and molecular approaches to dissect the molecular mechanism of RNA-mediated TGS, together with complementary genome-wide approaches to assess the global importance of small RNA-mediated TGS and premature termination of transcription in the control of human gene expression.

So far, we have succeeded in identifying the factors associated with the HIV LTR using the unbiased technique PICh (proteomics of isolated chromatin fragments). This analysis confirmed the presence of TGS factors such as Mtr4, and termination factors Xrn2 associated with the gene in its transcriptionally repressed state. The analysis furthermore revealed the unanticipitated recruitment of numerous factors in the DNA damage response that are specifically associated with the locus during active transcription. The implication of such factors in the process of transcription is currently under investigation.

We have successfully identified the interactome of factors whose homologues in models including S. pombe, C. elegans and D. melanogaster are critical for RNA-mediated transcriptional gene silencing. We used Crispr-Cas9 genome editing technology to tag the endogenous factors, which overcame a major technical challenge posed by the large size of these factors that precluded the use of standard lentiviral based techniques. This approach has led to the identification of a putative MTREC complex in human cells. The characterization of this complex is underway, and its genome wide association and impact on transcription are also under investigation. Using a similar strategy, we have identified the interactome of Senataxin, a helicase that resolves RNA:DNA hybrids and which is implicated at the cross-roads between transcription termination, DNA replication and DNA damage repair. Accordingly, its interactome revealed factors that could help to unravel the role of SETX in these diverse processes.

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