Dissect the molecular mechanisms by which nuclear Argonaute proteins regulate gene expression
RNAi as positive regulator of gene expression.
Even if the localization of nuclear Argonaute proteins has been observed on active euchormatic regions [1-5], a compelling mechanistic understanding of how nuclear Argonautes regulate these active regions of the genome is lacking.
We have recentely characterized the role of the Argonaute protein CSR-1 and its associated short RNAs in transcriptional regulation in C. elegans .
In C. elegans the specific class of short RNAs, endogenous siRNAs (endo-siRNAs), are mainly produced by the activities of RNA-dependent RNA polymerases (RdRPs) on target transcripts, which create an antisense copy of the mRNA target in form of thousands of single-stranded short RNAs. Interestingly, the Argonaute CSR-1 localizes to the nucleus and is exclusively loaded with endogenous siRNAs (endo-siRNAs), called CSR-1 22G-RNAs, which are antisense to more than 4,000 active germline protein-coding transcripts . In addition, inactivation of the CSR-1 pathway components leads to several germline defects and embryonic lethality [7-9]. Despite these observations, the role of CSR-1 in gene regulation was unclear. We adapted the Global Run-On sequencing (GRO-seq) method  for the use in C. elegans to investigate the function of CSR-1 22G-RNAs in controlling the generation of nascent transcripts genome-wide. Because of the sensitivity of this approach, we were able to show that CSR-1 globally promotes transcription of its target genes . Moreover, we demonstrated that CSR-1 directly interacts with RNA polymerase II (Pol II) through nascent RNAs in a siRNA-dependent manner .
These results contribute to the proposal that the CSR-1 pathway may have a positive role in gene expression and challenged the long-standing dogmatic view of RNAi being simply adapted to silence gene expression.
Yet, several fundamental questions still remain unanswered regarding the molecular mechanism by which the Argonaute CSR-1 regulates Pol II transcription and propagates epigenetic modifications. For instance, CSR-1, as many other Argonaute proteins, has been shown to have endonucleolytic activity in vitro . Yet, whether such activity is retained in vivo and whether it plays an important role in transcriptional regulation remains to be addressed. Moreover, we have shown that CSR-1 binds nascent transcripts in a siRNA-dependent manner . Thus, the binding of CSR-1 to nascent RNAs can directly regulate the recruitment of chromatin- and RNA-associated proteins that play an important role in transcription, chromatin organization, epigenetic modifications, and RNA metabolism.
In Addition, the precise map of the binding sites of CSR-1 on nascent transcripts has still to be determined. This might reveals additional information about the mechanism by which CSR-1 regulate gene expression.
Our aim is to obtain a deep molecular characterization of RNAi-mediated regulatory mechanism targeting euchromatic genes.
New questions and directions:
Is the endonucleolytic activity required for transcriptional regulation?
Where CSR-1 binds nascent RNAs?
Can the binding of CSR-1 to nascent RNAs recruits chromatin- and RNA-associated proteins?
How CSR-1-bound siRNAs are generated?
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