DS0401 -

Cross-talk between Splicing and RNA Silencing during gene expression – SPLISIL

Submission summary

RNA quality control (RQC) mechanisms eliminate aberrant RNAs, ensuring that only functional mRNAs are effectively translated into proteins. Defects in RQC or saturation by an excess of aberrant RNAs activate siRNA-mediated post-transcriptional silencing (PTGS), which cause the elimination of both aberrant and functional RNAs in a sequence-specific manner. Defective splicing is a major source of aberrant RNAs, resulting either in the introduction of premature stop codons leading to non-sense mediated decay (NMD), or removal of the correct stop codon leading to non-stop decay (NSD). Unexpectedly, a novel genetic screen for Arabidopsis PTGS-deficient identified five genes encoding proteins homologous to components of the splicing machinery in yeast, suggesting that the splicing machinery interplays with the PTGS/RQC equilibrium. Furthermore, emerging evidence suggest that long non-coding RNAs, potentially equivalent to aberrant RNAs, can regulate alternative splicing during plant development. In this project, classical genetics and new bioinformatics methodologies combined with high throughput RNA sequencing will allow us to explore the transcriptome to unprecedented depth and analyze the impact of RQC/PTGS/SPLICING on the transcriptome to establish mechanistic links between RQC/PTGS/SPLICING components and mRNAs, long non-coding RNAs and small RNAs.

The SPLISIL project combines RNA sequencing, cell biology and genetic approaches to dissect the molecular mechanisms involved in the regulation of gene expression by the RQC, PTGS and SPLICING machineries. The basis for the specificity of splicing factors towards certain subset of RNAs and their link with the RQC/PTGS degradation processes will be unveiled. The impact of mutations in several splicing factors on PTGS and siRNA production will serve to establish mechanisms of the cross-talk between PTGS and SPLICING. Sub-cellular localization of these proteins will be performed to address potential interactions between PTGS, RQC and SPLICING components. Tagged proteins will also be used to identify RNA substrates regulated through these pathways. Then, combining mutations in these pathways will allow determining how splicing factors interplay with RQC and PTGS. The integration of the complete transcriptomes (mRNAs, long ncRNAs, and siRNAs) in these different mutants will shed light on the coordinated regulation of splicing, RQC and PTGS during gene expression and will identify non-coding RNAs targeted by these mechanisms, which can serve to modulate these processes in vivo. The simultaneous presence of aberrant RNAs (such as antisense RNAs or other non-canonical RNAs) with triggering capacities of gene silencing and the dissection of their role in splicing regulation will open the way to identify novel mechanisms for gene regulation as well as novel tools for controlling splicing in vivo. The fundamental and applied data that we expect to obtain will increase our knowledge on global regulatory mechanisms controlling splicing and gene silencing, and may have a direct impact, through novel patents, on the development of new tools to control these important steps of eukaryotic gene expression.


Project coordination

Hervé VAUCHERET (Institut Jean Pierre Bourgin)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IPS2 Institute of Plant Sciences Paris Saclay
INRA Institut Jean Pierre Bourgin

Help of the ANR 433,043 euros
Beginning and duration of the scientific project: - 36 Months

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