Regulation of alternative splicing by the RNAi machinery – iSPLICE

The nuclear AGO complex contains specific subtypes of splicing factors, chromatin factors, and small RNAs. Precisely identifying these components and their positioning on the genome will provide much new data on the cross-talk between the splicing and the RNAi machineries.

Mass spectrometry analysis of the nuclear AGO complex has shown its enrichment in U5 snRNP components.

We are currently analyzing more thoroughly the population of small RNAs associated with the nuclear AGO complex.

Still to come.

Alternative splicing is a process by which the exons of a pre-mRNA produced by transcription of a gene, are reconnected in multiple ways. This process is an important step in the regulation of gene expression and has a major impact on the complexity of the proteome. Alternative splicing is, in part, regulated by enhancers and repressors that bind the pre-mRNA and control the recruitment of the spliceosome. In addition, much recent data suggest that chromatin structures inside the genes as well as the elongation rate of the RNA polymerase II (RNAPII) play an important role in the selection of the exons that ultimately will be present in the mature mRNA.
In this context, the group of CM has recently shown that HP1? and tri-methylation of histone H3 on lysine 9 (H3K9me3) can reduce RNAPII elongation and thus alternative splicing. These observations are in agreement with genome-wide studies carried out by the lab of JCA and showing a co-accumulation of HP1? and RNAPII inside the coding region of genes.
In parallel, the group of AHB has provided evidence that in mammalian cells, nuclear complexes containing Argonaute (AGO) proteins participate in the writing of H3K9me3 inside the coding region of genes via a mechanism related to the “RNAi transcriptional gene silencing” described in yeast. Unexpectedly, this group also finds that the nuclear AGO complexes contain a large number of splicing factors.
A collaborative work associating the three groups has now shown that the nuclear AGO complexes can function as regulators of alternative splicing.
In the present project, we intend to:
- Further document nuclear mechanisms allowing AGO complexes to regulate alternative splicing.
- Determine the nature and the origin of the non-coding RNAs involved.
- Explore the impact of the nuclear AGO complexes on RNAPII elongation.
These issues are important because the existence of a mechanism using RNAi transcriptional silencing to control gene expression in mammalian somatic cells is still at debate. By examining alternative splicing rather than transcriptional initiation, we expect to get a new perspective on that conundrum.
We anticipate that our data will:
- Bring new insight on RNAi transcriptional gene silencing in mammals
- Allow identifying novel pathways for the regulation of alternative splicing.
- Bring the proof of concept that active synthetic small RNAs able to correct splicing defects associated with human diseases can be efficiently predicted.