Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

Abstract

, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.Expression of protein-encoding genes is a multistep process beginning with transcription by RNA polymerase II (RNAPII). During transcription, the nascent pre-mRNA undergoes several processing steps, including capping, splicing, and polyadenylation. Distinct cellular machines carry out each of the steps in gene expression, but growing evidence indicates that there are coupled interactions between these machineries and it is now believed that most mRNA processing reactions occur cotranscriptionally (23, 51, 54). The C-terminal repeat domain (CTD) of RNAPII has a principal role in coupling eukaryotic transcription and pre-mRNA processing. The CTD of RNAPII is composed of 52 (in mammals) or 26 (in yeast [Saccharomyces cerevisiae]) tandem repeats of the consensus heptapeptide YS PTSPS, and this domain is essential for viability in yeast (11). An RNAPII with an unphosphorylated CTD assembles into preinitiation complexes at the promoters, whereas the transition between initiation and elongation is accompanied by multiple phosphorylation events of the CTD. These events are catalyzed by several protein kinases, including cyclin-dependent kinase 7 (CDK7), which is part of the general transcription factor IIH (TFIIH) (14, 35), and CDK9, which is the kinase component of positive transcription elongation factor b (P-TEFb) (37,38). Phosphorylation of the CTD on Ser5 correlates with the presence of RNAPII at the promoter, while phosphorylation on Ser2 correlates with RNAPII at the coding regions of genes and thus is considered a mark of elongating polymerases (30). As first proposed by Greenleaf (19), the CTD of RNAPII has been shown to interact with capping, splicing, and polyadenylation factors (8,9,25,39,40), thus acting as a platform upon which mRNA processing factors bind. It is most likely that the phosphorylation events occurring at...

Structure of FBP11 WW1-PL Ligand Complex Reveals the Mechanism of Proline-rich Ligand Recognition by Group II/III WW Domains

“…In addition, Prp40, the yeast ortholog of FBP11, binds the branch-point bridging protein, U5 small nuclear ribonucleoprotein-associated protein (29,30), U1 small nuclear ribonucleoprotein, and C-terminal domain of RNA polymerase II of RNA polymerase II (31,32). RNA polymerase II is the main coordinator for pre-mRNA processing events, and CA150, another homologue of FBP11, assembles the splicing complex (33). Recent study has shown that MeCP2 regulates excitatory neurotransmission by repressing transcription (34).…”

Transcription Elongation Regulator 1 Is a Co-integrator of the Cell Fate Determination Factor Dachshund Homolog 1

“…The DRE element was assessed in each orientation by cloning the sequence into KpnI/BglII sites of pGL3 control or pGL3 basic (Promega). Expression vectors encoding TCERG1 were described previously (16). pCMV-FOXM1 expression vector and FOXA luciferase reporter vector were provided by Dr. R. Costa (17).…”

Functional integration of transcriptional and RNA processing machineries

“…In particular, the transcriptional elongation factor CA150 is able to bridge the splicing factor SF1 via its WW domain and phosphorylated CTD through its FF domain [35,36]; and interestingly, the interaction between CA150 and several splicing factors seems to subject to regulation by arginine methylation [37]. The transcriptional elongation factor TAT-SF1 forms a stable complex with the spliceosome [38] and its homologue in yeast (Cus2) has been shown to function as a critical proofreading enzyme during early spliceosome assembly [39,40], suggesting that TAT-SF1/Cus2 may be a dual function protein in transcription and mRNA splicing.…”