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bioRxiv · 10.1101/177980

The RS Domain of Human CFIm68 Plays a Key Role in Selection Between Alternative Sites of Pre-mRNA Cleavage and Polyadenylation

Hardy, J. G.·Tellier, M.·Murphy, S.·Norbury, C. J.
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Abstract

Many eukaryotic protein-coding genes give rise to alternative mRNA isoforms with identical protein-coding capacities but which differ in the extents of their 3{acute} untranslated regions (3{acute}UTRs), due to the usage of alternative sites of pre-mRNA cleavage and polyadenylation. By governing the presence of regulatory 3{acute}UTR sequences, this type of alternative polyadenylation (APA) can significantly influence the stability, localisation and translation efficiency of mRNA. Though a variety of molecular mechanisms for APA have been proposed, previous studies have identified a pivotal role for the multi-subunit cleavage factor I (CFIm) in this process in mammals. Here we show that, in line with previous reports, depletion of the CFIm 68 kDa subunit (CFIm68) by CRISPR/Cas9-mediated gene disruption in HEK293 cells leads to a shift towards the use of promoter-proximal poly(A) sites. Using these cells as the basis for a complementation assay, we show that CFIm68 lacking its arginine/serine-rich (RS) domain retains the ability to form a nuclear complex with other CFIm subunits, but selectively lacks the capacity to restore polyadenylation at promoter-distal sites. In addition, nanoparticle-mediated analysis indicates that the RS domain is extensively phosphorylated in vivo. Overall, these results suggest that the CFIm68 RS domain makes a key regulatory contribution to APA.

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Hardy, J. G., Tellier, M., Murphy, S., Norbury, C. J.. 2017-08-18. The RS Domain of Human CFIm68 Plays a Key Role in Selection Between Alternative Sites of Pre-mRNA Cleavage and Polyadenylation. https://doi.org/10.1101/177980

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