The Nuclear Matrix Protein Matrin 3 is a Regulator of Alternative Splicing

MIGUEL COELHO; NICOLAS BELLORA; JERNEJ ULE; CHRISTOPHER SMITH

Davos

Congreso; 18th Annual Meeting of the RNA Society; 2013

International RNA Society

Polypyrimidine Tract Binding protein (PTB) regulates an extensive range of alternative splicing events (ASE), andis composed of four RRM domains. In an MS2-tethering assay the second RRM and the following linker are sufficientto promote exon skipping. Short linear peptide motifs of the form [S/G][I/L]LGxfP, known as PTB RRM Interactingmotifs (PRI), which are present in multiple copies in the PTB-coregulator Raver1, bind to the ?dorsal? surface ofRRM2. A key Tyr247 residue in RRM2 is critical for this interaction. Seeking to better understand the mechanism ofsplicing regulation by PTB, we used proteomics to identify proteins that bind to the PTB minimal repressor domainand are sensitive to mutation of Tyr247. The two strongest interactors were Raver1 and the nuclear matrix proteinMatrin 3. The interaction with Matrin3 was mediated by a conserved GILGPPP motif, which is both necessaryand sufficient for interaction with PTB. Matrin3 is composed of two DNA binding zinc-finger domains as well astwo tandem RRMs. It is known to interact with RNA processing and transcription factors, and a mutation in thisprotein is associated with a type of distal myopathy, although its precise molecular function is unclear. We tested theconsequences of Matrin3 knockdown in HeLa cells using splice-sensitive microarrays. Multiple ASEs were stronglyaffected, suggesting the activity of Matrin3 both as a splicing repressor and activator. There was a significant overlapbetween ASEs regulated by Matrin3 and PTB, but strikingly the majority of Matrin3 events were not co-regulated byPTB. Matrin3 targeted events showed a significant enrichment for chromatin proteins. Structure-function analysesindicated that the ZF domains are dispensable but Matrin3 requires its RRMs for splicing activity suggesting it bindsdirectly to RNA. A number of 5-mer motifs are significantly enriched around Matrin3 repressed exons and adjacent tothe downstream constitutive exon, including pyrimidine-rich motifs similar to optimal PTB sites. These motifs werealso enriched among Matrin3 target exons that are not regulated by PTB. Strikingly, we found that Matrin3 activitywas abolished by mutations of its GILGPPP motif for both PTB co-regulated and PTB-independent events, suggestingthat this motif can mediate interactions with other splicing regulators. Our data indicate that Matrin 3 is not onlyfunctional as a nuclear matrix component but also as an active splicing regulator in the nucleoplasm.