The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

Abstract

A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3 part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3 splice site recognition. Our results suggest an essential role of WW/FF domaincontaining factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.Gene expression in eukaryotic cells involves several steps, including transcription, mRNA processing, and export. Synthesis of mRNA by RNA polymerase II (Pol II) is coordinated with subsequent RNA processing events such as capping, splicing, and cleavage and polyadenylation (4,12,20,23,37). The largest subunit of RNA Pol II recruits mRNA processing activities during transcriptional elongation via its heptapeptide repeat-containing C-terminal domain (CTD) (4, 23). Truncation of the Pol II CTD results in reduced splicing in vivo (34). Under some circumstances, in vitro splicing of pre-mRNA can be stimulated by phosphorylated Pol II or CTD (22, 52). Several transcriptional activators or coactivators associate physically with small nuclear ribonucleoproteins (snRNPs) or serine/arginine-rich splicing factors (SR proteins) and thereby may modulate pre-mRNA splicing (18,29,35). Moreover, promoter structure affects alternative splice site selection during pre-mRNA splicing (11), and further analyses reveal that transcriptional activators differentially modulate the rate of transcriptional elongation, which in turn determines the outcome of two competing alternative splicing reactions (15,26,38). Nevertheless, several lines of evidence support the concept of a physical and functional coupling between transcription and splicing (12,20).Since the RNA Pol II CTD is implicated in promoting premRNA splicing, proteins that associate with the CTD may mediate this function. A two-hybrid screen previously revealed a set of RS domain-containing proteins via their interaction with the CTD (51). Amon...

Coupling pre-mRNA processing to transcription on the RNA factory assembly line

“…73,75,76 Moreover, early reports have shown that the WW and FF domain-containing TCERG1 interacts with the phosphorylated CTD and the splicing factors that associate with the 3' end of the intron and that TCERG1 can activate splicing via its WW and FF domains. 77,78 More recent evidence indicates that TCERG1 regulates alternative splicing via modulating pol II transcription rate 48 (see above) and, thus, supports previous findings. Therefore, yeast WW-containing proteins may promote early spliceosome formation, whereas mammalian analogs have an additional function in alternative splicing.…”

Spatial Distribution of Di- and Tri-methyl Lysine 36 of Histone H3 at Active Genes

“…Consistent with the second possibility, the transcribing RNA polymerase (to which Set2p binds) has been shown to be involved in the early phase of RNA processing (18). Furthermore, the yeast Set2 protein contains a WW motif, a motif recently shown to be essential in proteins involved in pre-mRNA splicing, which is a process that occurs in vivo in concert with transcription (19). Whatever the detailed explanations, the mapping studies reported here suggest that methylated K4/H3 and methylated K36/H3 have distinct roles at active genes.…”

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)

“…This abnormal interaction can be considered to be a partial contribution of the polyQ-expanded Htt to the pathogenesis of HD. PolyQ-expanded Htt Causes Post-transcriptional Dysfunction of HYPA-HYPA is one of the constitutive components of splicesome U1 complex that can enhance the efficiency of RNA splicing in mammalian cells (7,8). Because HYPA could be sequestered by polyQ-expanded Htt, investigating the biological consequences of the abnormal interaction and redistribution is critical to understanding the pathogenesis of HD caused by mutant Htt.…”

“…HYPA, a human homolog of mouse forming-binding protein 11 (FBP11), is a component of mammalian mRNA splicing U1 complex (7) and is able to enhance the splicing efficiency in mammalian cells (8). Actually, the HYPA level was also found to be significantly reduced in the stratum of mouse HD model (6).…”