Alternative splicing plays a major role in the adaptation of cardiac function exemplified by the isoform switch of titin, which adjusts ventricular filling. We previously identified a rat strain deficient in titin splicing. Using genetic mapping, we found a loss-of-function mutation in RBM20 as the underlying cause for the pathological titin isoform expression. Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy. We showed that the phenotype of Rbm20 deficient rats resembles the human pathology. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20 dependent regulation of alternative splicing. Additionally to titin we identified a set of 30 genes with conserved regulation between human and rat. This network is enriched for genes previously linked to cardiomyopathy, ion-homeostasis, and sarcomere biology. Our studies emphasize the importance of posttranscriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

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RNA-binding motif protein 20 (RBM20) is a cardiac splice regulator that adapts cardiac filling via its diverse substrates—including the sarcomeric protein titin. The molecular basis and regulation of RBM20-dependent exon exclusion are largely unknown. In tissue culture experiments, we show that the combination of RNA recognition motif (RRM) and C-terminus is necessary and sufficient for RBM20 activity, indicating an important function of the ZnF2 domain in splicing repression. Using splice reporter and in vitro binding assays targeting titin exons 241–243, we identified a minimal genomic segment that is necessary for RBM20-mediated splicing repression of the alternative exon. Here, RBM20 binds the cluster containing most RBM20 binding motifs through its RRM domain and represses the upstream and downstream introns. For subsequent exon exclusion, specific regions upstream, downstream and within the alternative exon 242 are required. Regulation of exon exclusion involves PTB4 as a novel titin splice regulator, which counteracts RBM20 repressor activity in HEK293 cells. Together, these mechanistic insights into the regulation and action of RBM20 and PTB4 provide a basis for the future development of RBM20 modulators that adapt titin elasticity in cardiac disease.

The essential splicing factor ASF/SF2 activates or represses splicing depending on where on the pre-mRNA it binds. We have shown previously that ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing by binding to an intronic repressor element. Here we used MS2-ASF/SF2 fusion proteins to show that the second RNA binding domain (RBD2) is both necessary and sufficient for the splicing repressor function of ASF/SF2. Furthermore, we show that the completely conserved SWQDLKD motif in ASF/SF2-RBD2 is essential for splicing repression. Importantly, this heptapeptide motif is unlikely to be directly involved in RNA binding given its position within the predicted structure of RBD2. The activity of the ASF/SF2-RBD2 domain in splicing was position-dependent. Thus, tethering RBD2 to the IIIa intron resulted in splicing repression, whereas RBD2 binding at the second exon had no effect on IIIa splicing. The splicing repressor activity of RBD2 was not unique to the IIIa pre-mRNA, as binding of RBD2 at an intronic position in the rabbit ␤-globin pre-mRNA also resulted in splicing inhibition. Taken together, our results suggest that ASF/ SF2 encode distinct domains responsible for its function as a splicing enhancer or splicing repressor protein.The human ASF/SF2 protein is a member of the evolutionary conserved SR family of splicing factors (1). SR proteins are essential splicing factors required for constitutive splicing and, furthermore, play a regulatory role in alternative RNA splicing (reviewed in Refs. 2-4). SR proteins partake at multiple steps during spliceosome assembly. For example, they help to commit a pre-mRNA for splicing by stabilizing U1 snRNP 1 and U2AF binding to the 5Ј and 3Ј splice sites, respectively (5-8). Also, they facilitate U4/U6-U5 snRNP recruitment to the spliceosome and are believed to bridge between splicing factors binding to the ends of the intron during spliceosome formation (9 -11).SR proteins contain one or two N-terminal RNP-type RNA binding domains (RBDs) and a highly charged variable-length C-terminal domain rich in arginine-serine dipeptide repeats (the RS domain), hence the name SR proteins (reviewed in Ref.3). ASF/SF2 contain two RBDs, and both are required for high affinity RNA binding (12,13). SR proteins are modular in structure with the RBDs making sequence-specific contact with the RNA (14, 15) and the RS domain mediating protein-protein interactions with multiple general splicing factors (3,6,8,16).Previous studies (3) have shown that SR proteins function as enhancer proteins, stimulating both constitutive and alternative splicing. SR proteins bind to splicing enhancer elements, which typically are located downstream of the affected intron (reviewed in Ref. 2). SR protein binding to enhancer elements helps to recruit U2AF65 to a nearby weak 3Ј splice site (8,17,18). Thus, the RS domain of ASF/SF2 appears to be required for U2AF recruitment to weak polypyrimidine tracts, whereas the RS domain is dispensable for ASF/SF2 activation of strong 3Ј splice sites that bind U2AF efficiently (19...

We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2) preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein (EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar accumulation signal within the Mu sequence. In addition, we noted that both preMu-EGFP and Mu-EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in adenovirus-infected cells. Surprisingly, we observed that cells in which preMu-EGFP (but not Mu-EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late adenovirus genes. Further investigation suggested this might be due to a failure to express pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein, DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu responsible for these observations. Thus our data suggest that preMu may play a role in modulating accumulation of proteins from the E2 region.