The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

Abstract

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3′ -end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes premRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

A day in the life of the spliceosome

“…This process is facilitated by the pol II CTD, which reportedly interacts directly with U1 snRNP 95, 96 although the functional role of this interaction is still under debate 97 . The interaction between the 5′ss and U1 snRNP in complex E is ATP independent and fairly weak; it is stabilized by other factors such as SR proteins 98, 99 and the cap-binding complex 100 . The 3′ss of the pre-mRNA is recognized by the U2 snRNP and associated factors, such as SF1 and U2 auxiliary factors (U2AF), which are also components of complex E.…”

The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion

“…Because formation of the m 7 G cap is essentially complete by the time nascent Pol II transcripts are 20–30 nt long (83, 84), and this is the sole feature recognized by the nuclear CBC, mRNP assembly likely initiates with CBC acquisition. CBC then promotes TREX complex acquisition in the cap-proximal region (85) and enhances cotranscriptional pre-mRNA splicing by recruiting several spliceosomal components (Figure 2a) (86, 87). Although CBC binding is not necessary for EJC deposition, it does promote stable association of several proteins that specifically bind spliced mRNA in vitro (e.g., ILF2, THRAP3) (Figure 2a) (39).…”

Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate

“…In the case of conventional mRNAs, the CBC stimulates the splicing of cap-proximal introns, the processing of RNA 3′ ends, and the formation of export-competent ribonucleoproteins (RNPs) (Cheng et al., 2006, Flaherty et al., 1997, Izaurralde et al., 1994). Stimulation of RNA splicing and export has been proposed to involve interactions of the CBC with the U4/U6.U5 tri-small nuclear RNP (snRNP) and ALYREF, respectively (Cheng et al., 2006, Pabis et al., 2013). In the case of non-adenylated histone mRNAs, the CBC promotes their 3′ end formation in a process involving interactions with the ARS2, NELF-E, and SLBP proteins (Gruber et al., 2012, Hallais et al., 2013, Narita et al., 2007).…”