RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5' splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5' splice site, but not the engineered strong 5' splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5' splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5' splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5' splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.     

Overlapping splicing regulatory motifs—combinatorial effects on splicing

Regulation of splicing in eukaryotes occurs through the coordinated action of multiple splicing factors. Exons and introns contain numerous putative binding sites for splicing regulatory proteins. Regulation of splicing is presumably achieved by the combinatorial output of the binding of splicing factors to the corresponding binding sites. Although putative regulatory sites often overlap, no extensive study has examined whether overlapping regulatory sequences provide yet another dimension to splicing regulation. Here we analyzed experimentally-identified splicing regulatory sequences using a computational method based on the natural distribution of nucleotides and splicing regulatory sequences. We uncovered positive and negative interplay between overlapping regulatory sequences. Examination of these overlapping motifs revealed a unique spatial distribution, especially near splice donor sites of exons with weak splice donor sites. The positively selected overlapping splicing regulatory motifs were highly conserved among different species, implying functionality. Overall, these results suggest that overlap of two splicing regulatory binding sites is an evolutionary conserved widespread mechanism of splicing regulation. Finally, over-abundant motif overlaps were experimentally tested in a reporting minigene revealing that overlaps may facilitate a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it.     

Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.     

The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II

We showed previously that the WW domain of the prolyl isomerase, Ess1, can bind the phosphorylated carboxyl-terminal domain (phospho-CTD) of the largest subunit of RNA Polymerase II. Analysis of phospho-CTD binding by four other WW domain-containing Saccharomyces cerevisiae proteins indicates the splicing factor, Prp40, and the RNA polymerase II ubiquitin ligase, Rsp5, can also bind the phospho-CTD. The identification of Prp40 as a phospho-CTD binding protein represents the first demonstration of direct interaction between a documented splicing factor and the phospho-CTD. Domain dissection studies reveal that phospho-CTD binding occurs at multiple locations in Prp40, including sites in both the WW and FF domain regions. Because the conserved repeats of the CTD make it an ideal ligand for multi-site binding events, the implications of multi-site binding are discussed. Our data suggest a mechanism by which the phospho-CTD of elongating RNA polymerase II facilitates commitment complex formation by juxtaposing the 5' and 3' splice sites.     

The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.     

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.     

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

RBFOX1 and RBFOX2 are alternative splicing factors that are predominantly expressed in the brain and skeletal muscle. They specifically bind the RNA element UGCAUG, and regulate alternative splicing positively or negatively in a position-dependent manner. The molecular basis for the position dependence of these and other splicing factors on alternative splicing of their targets is not known. We explored the mechanisms of RBFOX splicing activation and repression using an MS2-tethering assay. We found that the Ala/Tyr/Gly-rich C-terminal domain is sufficient for exon activation when tethered to the downstream intron, whereas both the C-terminal domain and the central RRM are required for exon repression when tethered to the upstream intron. Using immunoprecipitation and mass spectrometry, we identified hnRNP H1, RALY, and TFG as proteins that specifically interact with the C-terminal domain of RBFOX1 and RBFOX2. RNA interference experiments showed that hnRNP H1 and TFG modulate the splicing activity of RBFOX1/2, whereas RALY had no effect. However, TFG is localized in the cytoplasm, and likely modulates alternative splicing indirectly.