MCM proteins are associated with RNA polymerase II holoenzyme.

MCMs are a family of proteins related to ATP-dependent helicases that bind to origin recognition complexes and are required for initiation of DNA replication. We report that antibodies against MCM2(BM28) specifically inhibited transcription by RNA polymerase II (Pol II) in microinjected Xenopus oocytes. Consistent with this observation, MCM2 and other MCMs copurified with Pol II and general transcription factors (GTFs) in high-molecular-weight holoenzyme complexes isolated from Xenopus oocytes and HeLa cells. Pol II and GTFs also copurified with MCMs isolated by anti-MCM3 immunoaffinity chromatography. MCMs were specifically displaced from the holoenzyme complex by antibody against the C-terminal domain (CTD) of Pol II. In addition, MCMs bound to a CTD affinity column, suggesting that their association with holoenzyme depends in part on this domain of Pol II. These results suggest a new function for MCM proteins as components of the Pol II transcriptional apparatus.

Transcriptional control: Tat cofactors and transcriptional elongation.

HIV-1 gene expression requires the transactivator Tat, which stimulates viral transcript elongation. Recent results show that two cellular cyclin-dependent kinases, which phosphorylate the carboxy-terminal domain of the RNA polymerase II large subunit, contact Tat and contribute to the control of transcriptional elongation.

Regulation of CDK7 substrate specificity by MAT1 and TFIIH.

The cyclin-dependent kinase (CDK)-activating kinase CAK has been proposed to function in the control of cell cycle progression, DNA repair and RNA polymerase II (pol II) transcription. Most CAK exists as complexes of three subunits: CDK7, cyclin H (CycH) and MAT1. This tripartite CAK occurs in a free form and in association with 'core' TFIIH, which functions in both pol II transcription and DNA repair. We investigated the substrate specificities of different forms of CAK. Addition of the MAT1 subunit to recombinant bipartite CDK7-CycH switched its substrate preference to favour the pol II large subunit C-terminal domain (CTD) over CDK2. We suggest that the MAT1 protein, previously shown to function as an assembly factor for CDK7-CycH, also acts to modulate CAK substrate specificity. The substrate specificities of natural TFIIH and free CAK were also compared. TFIIH had a strong preference for the CTD over CDK2 relative to free CAK. TFIIH, but not free CAK, could efficiently hyperphosphorylate the CTD. In the context of TFIIH, the kinase also acquired specificity for the general transcription factors TFIIE and TFIIF which were not recognized by free CAK. We conclude that the substrate preference of the CDK7-CycH kinase is governed by association with both MAT1 and 'core' TFIIH.

The C-terminal domain of RNA polymerase II couples mRNA processing to transcription.

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II.

We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5' capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5' ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.

TFIIH functions in regulating transcriptional elongation by RNA polymerase II in Xenopus oocytes.

We investigated the role of TFIIH in transcription by RNA polymerase II (pol II) in vivo by microinjection of antibodies against this factor into Xenopus oocytes. Five different antibodies directed against four subunits of TFIIH were tested for effects on transcription of coinjected human immunodeficiency virus type 2 and c-myc templates. Each of these antibodies severely reduced the efficiency of elongation through human immunodeficiency virus type 2 and c-myc terminator elements. In contrast, an anti-TFIIB antibody did not inhibit elongation. Anti-TFIIH antibodies also had a much smaller inhibitory effect on total transcription than did anti-TFIIB or anti-pol II large subunit. Three inhibitors of TFIIH kinase activity, H-7, H-8, and dichlororibofuranosylbenzimidazole (DRB), inhibited elongation similarly to anti-TFIIH antibodies. These results strongly suggest a role for TFIIH in the stimulation of transcriptional elongation in vivo.

The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase.

Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.

Transcriptional elongation by RNA polymerase II is stimulated by transactivators.

We report that a variety of transactivators stimulate elongation by RNA polymerase II. Activated transcription complexes have high processivity and are able to read through pausing and termination sites efficiently. In contrast, nonactivated and "squelched" transcription mostly arrests prematurely. Activators differ in the extent to which they stimulate processivity; for example, GAL4-VP16 and GAL4-E1a are more effective than GAL4-AH. The stimulation of elongation can be as important as the stimulation of initiation in activating expression of a reporter gene. We suggest that setting the competence of polymerase II to elongate is an integral part of the initiation step that is controlled by activators cooperating with the general transcription factors.

An immunocytochemical study of the proliferating cell nuclear matrix antigen p125/6.5 during rat spermatogenesis.

In previous studies of proliferating mammalian cells a p125/6.5 nuclear matrix antigen displaying a marked increase in mitotic cells has been identified. This antigen was investigated by immunocytochemistry of cryosections of testes at different stages of postnatal development: newborn, 20 days after birth and sexually mature rats. In Sertoli cells, the distribution of the p125/6.5 antigen parallels [3H]thymidine incorporation: present in newborn and absent in sexually mature testes. The p125/6.5 antigen is present also in some prespermatogonia of the newborn rat testis, which do not incorporate [3H]thymidine. At later stages of development, the p125/6.5 antigen is present also in first meiotic prophase spermatocytes displaying an extrachromosomal nucleoplasmic distribution, while absent in spermatids and spermatozoa. These results show that the p125/6.5 antigen increases not only during mitosis, but also during meiosis. They suggest further that this antigen is characteristic of both proliferating cells and cells (prespermatogonia) committed to proliferation.

The use of a monoclonal antibody against a proliferating cell nuclear matrix antigen in the study of solid human tumors.

A monoclonal antibody (MAb) identifies the nuclear antigen p125/6.5 associated with the nuclear matrix and present in proliferating human cells. By direct and indirect immunofluorescence, the presence and distribution of the antigen p125/6.5 in cryostat sections from primary and metastatic solid human tumors has been investigated. The antigen is present in nuclei of malignant and benign tumor cells, but is not detected in adjacent normal tissues. The antigen displays a speckled nucleoplasmic distribution, while nucleoli are negative. The intensity of the immunofluorescence reaction is markedly higher in the nuclei of some individual or grouped tumor cells.