Elucidating the roles of PARP1 and RBBP6 in the regulation of pre-mRNA 3' end processing

摘要

The mature 3' ends of most mRNAs are created by a two-step reaction that involves an endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream product. The 3' processing machinery is composed of four multisubunit complexes, which, together with a few other proteins, constitute the core components required for cleavage and polyadenylation. A proteomic analysis led to the identification of approximately 80 proteins that associate with the human pre-mRNA 3' processing complex, including new core 3' factors and other proteins that might mediate crosstalk between 3' processing and other nuclear pathways. This thesis focuses on two of the newly identified proteins, which we found particularly intriguing: PARP1 and RBBP6. PARP1 is an enzyme that, when activated, catalyzes the polymerization of ADP-ribose units from donor NAD molecules to acceptor proteins, a reaction known as PARylation. This post-translational modification has been shown to modulate critical events such as DNA damage response and transcription. We found that PARP1 binds PAP, the enzyme responsible for polyadenylating the 3' ends of mRNAs, and modifies it by PARylation. In vivo PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. Finally, we show that the observed inhibition reflects decreased PAP association with 3' end of genes. These results identify PARP1 as a regulator of polyadenylation during thermal stress and show for the first time that PARylation can control gene expression by modulating processing of mRNA. The second project involves RBBP6, a large multidomain protein that is known to interact with p53 and Rb. The N-terminal part of the human RBBP6 includes a DWNN domain, which is particularly interesting because it adopts a ubiquitin-like fold and, in addition to forming part of the full-length RBBP6 protein, is also expressed as a small protein (RBBP6 isoform3) which has been shown to be downregulated in several human cancers. We found that RBBP6 is essential for the cleavage activity of the 3' processing complex and that an N-terminal derivative of RBBP6 (RBBP6-N), containing only the DWNN, Zinc and Ring domains, is enough to rescue cleavage activity. The RBBP6 and RBBP6 isoform3 can compete with each other in binding to Cstf64 (an interaction mediated by the DWNN domain). In addition, overexpression of isoform3 inhibits cleavage raising intriguing possibilities of modulation of 3' processing by fine-tuning the levels of the two RBBP6 isoforms. To better characterize the function of RBBP6 globally, we also performed genome-wide analysis, both by microarray and deep sequencing. Following RBBP6 knockdown we observed a general lengthening of 3' UTRs accompanied by an overall downregulation in gene expression, especially of RNAs with AU-rich 3'UTRs. We show that this is the result of a defect in their 3' cleavage and subsequent degradation by the exosome. All together our results point to a role for RBBP6 as a new core 3' processing factor able to regulate the expression of AU-rich mRNAs.