Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA

Abstract

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the polymerase via the hyperphosphorylated CTD.

New insights into the biogenesis of nuclear RNA polymerases?This paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…Alternatively, the complex might have an activity that is closer in nature to the canonical prefoldin complex in that it could be involved in cytoskeletal assembly. Given that hyperphosphorylated RNAP II is known to migrate away from the DNA during mitosis (Parsons and Spencer 1997;Kim et al 1997), association with RNAPs might therefore be a trigger to promote a cell-cycle dependent microtubular rearrangement or more simply, it could be responsible for directly segregating the RNA polymerases from DNA during this timeframe. Whatever the case might be, reviewing the literature on the many subunits and interactors of this complex, as we have done here, helps defining its role in RNAP biogenesis and its putative coordination with other cellular processes.…”

Promoter-driven splicing regulation in fission yeast

“…The CTD is preferentially phosphorylated at Ser 5 when pol II is recruited at the promoter sites but becomes phosphorylated at Ser 2 when located at the coding region (Cho et al, 2001;O'Brien et al, 1994). This change of phosphorylation pattern might be relevant for the recruitment of splicing factors (Kim et al, 1997).…”

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

“…These early genomic efforts suggested that, similar to DNA binding proteins, functional groupings of genes are being bound by RNA binding proteins perhaps as post-transcriptional operons (Keene and Tenenbaum 2002). However, it is not known at which stage of RNA processing these operons are established.Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al 1996;Yuryev et al 1996;Kim et al 1997). The CTD is phosphorylated as transcription begins.…”

“…Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al 1996;Yuryev et al 1996;Kim et al 1997). The CTD is phosphorylated as transcription begins.…”