A Human RNA Polymerase II-containing Complex Associated with Factors Necessary for Spliceosome Assembly

Abstract

Transcription and splicing are coordinated processes in mammalian cells. We have used affinity chromatography with immobilized transcription elongation factor SII to purify a protein complex that contains core RNA polymerase II (RNA Pol II), the general transcription initiation factors, and several splicing factors, including the U1, U2, and U4 small nuclear RNPs, the U2AF 65 , and serine/arginine-rich proteins. The splicing factors and the transcription machinery co-purify through a gel filtration column and co-immunoprecipitate in experiments using an anti-U2AF 65 antibody, indicating that they are part of a unique complex. Although the RNA Pol II-containing complex does not possess splicing activity, it can complement small nuclear RNP-inactivated extracts and can promote the formation of a pre-spliceosome complex. Because interactions between components of the splicing and transcription machineries occur in the context of a complex containing a hypophosphorylated RNA Pol II capable of initiating transcription, our results suggest that the coupling between transcription and splicing begins before transcription initiation.The splicing of pre-mRNA in mammalian cells is the nuclear process during which introns are removed from primary transcripts synthesized by RNA polymerase II (RNA Pol II).1 Splicing is achieved by the spliceosome, a large macromolecular complex, composed of small nuclear ribonucleoprotein (snRNP) particles and additional proteins including members of the serine/arginine-rich (SR) protein family (reviewed in Refs. 1 and 2). Cytological studies have revealed that splicing can occur in a co-transcriptional manner (see Refs. 3 and 4 for examples) and that factors necessary for splicing are recruited to sites of transcription (5-7). A growing body of data indicates that coupling between transcription and a variety of RNA maturation events may be achieved through the C-terminal domain (CTD) of the RPB1 subunit of RNA Pol II (reviewed in Refs. 8 -11). The CTD is a seven-amino acid motif tandemly repeated 52 times in humans and 26 -27 times in yeast. It is highly conserved among eukaryotic organisms and is subject to reversible phosphorylation during the transcription cycle (reviewed in Refs. 12 and 13). Two isoforms of RNA Pol II exist in vivo, namely RNA Pol IIA and IIO. RNA Pol IIA possesses a hypophosphorylated CTD and preferentially enters the preinitiation complex, whereas RNA Pol IIO harbors an extensively phosphorylated CTD and is found in the elongation complex (13,14). Biochemical studies indicate that RNA Pol II physically interacts with components involved in capping (15-18), polyadenylation (19), and splicing (20 -24). Whereas a phosphorylated CTD is essential for the interaction with capping enzymes, the polyadenylation factor CPSF is brought to the transcription complex through an interaction with TFIID and transferred to the phosphorylated CTD after transcription initiation (19,25). The phosphorylated RNA Pol IIO isoform has been found to associate with splicing factors and has been i...

Transcriptional Activators Control Splicing and 3′-End Cleavage Levels

“…Association of these factors with the pol II CTD during transcription elongation would then facilitate the assembly of splicing and 3Ј-end processing complexes on nascent transcripts. Consistent with this model, several reports have described interactions between pre-mRNA processing factors and transcriptional activators (40), coactivators (41)(42)(43), and the holoenzyme (22,23). Thus, it is possible that increased splicing and cleavage levels promoted by transcriptional activators is the consequence of increased recruitment of pre-mRNA processing components to promoters.…”

“…The 3Ј-end cleavage polyadenylation stimulatory factor (CPSF) interacts with the general transcription factor TFIID during transcription initiation (21), as well as with the CTD (15), indicating that it is recruited to transcription initiation complexes prior to its role in cleavage. A number of splicing or splicing-related factors have recently been detected in association with holoenzyme complexes involved in transcription initiation, further suggesting that specific pre-mRNA processing components may be recruited to active genes at the stage of transcription initiation (22,23). Recent work has also shown that the type of promoter or activator used to drive transcription can influence the level of inclusion of alternative exons in transfected pre-mRNA reporters (24 -27), suggesting that events occurring as early as promoter recognition may play an important role in the regulation of splicing.…”

Functional coupling of RNAP II transcription to spliceosome assembly

“…As a consequence, the RNAP II transcripts are efficiently directed into the spliceosome assembly pathway rather than into the inhibitory H complex. A number of spliceosomal proteins that associate with RNAP II or the CTD in vitro have been identified, including Prp40, SCAFs, PSF, p54, SR proteins and all of the spliceosomal small nuclear RNPs (snRNPs) (Mortillaro et al 1996;Yuryev et al 1996;Corden and Patturajan 1997;Kim et al 1997;Patturajan et al 1998;Morris and Greenleaf 2000;Tian 2001;Emili et al 2002;Robert et al 2002;Kameoka et al 2004;Phatnani and Greenleaf 2004), but a function for these proteins in coupling remains to be demonstrated.…”

Proteomics of RNA polymerase II holoenzymes during P19 cardiomyogenesis

“…Their gentle technique allowed the recovery of a mixture of transcriptionally active RNAPII holoenzymes (Mr 2.4 MDa) containing all the general transcription factors from HeLa cells [12]. The protein composition of the different TFIIS-purified holoenzymes from HeLa cells was shown to consist of subunits from the spliceosome [19] and to vary according to cell cycle [22]. The changes in protein composition of TFIIS-purified holoenzymes during cell differentiation have not been addressed.…”

“…For example, RNAPII forms different holoenzymes capable of receiving and integrating cellular signals or modifing chromatin structure [13]. The diversity of complexes that interact with RNAPII span a vast spectrum of functions in transcription, genomic maintenance and splicing [14][15][16][17][18][19].…”