Abstract. A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol 17 LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol II0 and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.NA polymerase II transcripts (pre-mRNAs) are cotranscriptionally spliced and packaged into ribonucleoprotein (RNP) t particles in diverse eukaryotic nuclei (Sass and Pederson, 1984; Beyer and Osheim, 1988; Fakan et ai

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR)

A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol hIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.with pre-mRNA processing that are related to the SR family.In the present study, a new anti-NM mAb, B3, is characterized that recognizes a 250-kDa NM protein concentrated in speckles. Similar to anti-NM mAbs which recognize SR proteins, B3 preferentially binds in vitro to a subset of splicing complexes containing exon sequences. Surprisingly, the B3 antigen corresponds to a hyperphosphorylated form of the large subunit of pol II (pol IIo). In addition to splicing complexes, pol IIo is associated with a subset of snRNP and SR protein splicing factors. The possible implications of these findings in relation to the regulation of RNA processing are discussed. MATERIALS AND METHODSIncreasing evidence suggests that transcription and processing of RNA polymerase II (pol II) transcripts are temporally and spatially linked. Visualization of chromatin spreads by electron microscopy has revealed that the majority of introns are removed cotranscriptionally from pre-mRNA (1, 2). These studies are supported by recent fluorescent in situ hybridization experiments, indicating that the synthesis and splicing of specific pol II transcripts are coincident at discrete foci (3-5).In several cases, transcript foci appear to be localized in association with specific nuclear domains that are highly enriched in splicing factors, referred to as "speckles" (3, 5-7).Although not mutually exclusive with evidence implicating speckle domains in splicing factor storage and/or assembly (8, 9), these transcript localization experiments indicate a possible direct role of speckle domains in the processing of pre-mRNAs (10, 11). Mammalian nuclei typically contain 20-50 speckle domains, which, in addition to the four spliceosomal small nuclear ribonucleoprotein particles (snRNPs; Ul, U2, U4/6, and U5), are also enriched for non-snRNP splicing factors and poly(A)+ RNA (8, 9, 11). Many of the non-snRNP splicing factors in speckles are related to the Ser-Arg (SR) family of proteins, all of which contain one or more domains rich in alternating serine and arginine residues (12). Besides splicing components, speckle structures also contain elevated concentrations of proteins involved in transcription and cellular transformation (13-15). Since these structure...

7 . Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.The largest subunit of RNA polymerase II (pol II) 1 contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-ProSer (1, 2). The CTD plays an essential (3-6) but as yet poorly understood role in mRNA synthesis with evidence indicating potential roles in initiation or promoter clearance (7-9), elongation (10 -15), and pre-mRNA processing (16 -20).Phosphorylation of the CTD is a key feature of CTD function. SDS gel electrophoresis separates the largest subunit into two species as follows: IIA contains a hypophosphorylated CTD and pol II0 is hyperphosphorylated on the CTD (21). Serine is the predominant in vivo phosphoacceptor with minor amounts of phosphothreonine and phosphotyrosine detected (22, 23). Although in vivo phosphorylation sites have not been mapped, in vitro studies have identified serines in both positions 2 and 5 (22, 24, 25) and tyrosine in position 1 (23) as potential phosphoryl acceptors. Mutation of these sites to unphosphorylatable alanine or phenylalanine residues in each yeast CTD repeat is lethal, suggesting a requirement for CTD phosphorylation in vivo (26).The preferential inclusion of pol IIA into preinitiation complexes (27-30) together with the observation that elongating pol II is phosphorylated on the CTD (31) led to the hypothesis that the CTD is reversibly phosphorylated with each transcription cycle (8). The unphosphorylated CTD has been shown to contact basal transcription factors TATA binding protein (32), TFIIE, and TFIIF (33), and these contacts, together with as yet undefined interactions with SRBs (34 -37), suggest that the CTD acts as a structural framework for the preinitiation complex (38). The pol II preinitiation complex also contains several protein kinases that are capable of phosphorylating the CTD (39 -45) suggesting that one role of this complex is to effect the conversion of pol IIA to pol II0 thereby releasing pol II from the initiation complex. Finally, CTD phosphatase is required to dephosphorylate pol II0 thus completing the CTD phosphorylation cycle (46, 47).Se...

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.