Nuclear c-Abl is a COOH-terminal repeated domain (CTD)-tyrosine (CTD)-tyrosine kinase-specific for the mammalian RNA polymerase II: possible role in transcription elongation.
(49/4175)
The c-Abl tyrosine kinase has been shown to interact with the COOH-terminal repeated domain (CTD) of mammalian RNA polymerase II and can phosphorylate the tyrosine residues in the CTD. Interestingly, the Drosophila or the yeast CTD were not efficiently phosphorylated by the mammalian c-Abl. This species-specificity was found to be determined by the extreme COOH-terminal CTD sequences that are not conserved through evolution. In vitro, COOH-terminal-truncated CTD could neither bind to, nor be phosphorylated by, c-Abl. In vivo, coexpression of a full length CTD prevents c-Abl from inducing the tyrosine phosphorylation of endogenous RNA polymerase II, and such inhibitory effect was not observed with the coexpression of COOH-terminal-truncated CTD. Serine/threonine phosphorylation of the CTD has been linked to the regulation of transcription elongation. Transcription from the human immunodeficiency virus type 1 (HIV-1) promoter requires CTD-phosphorylation, which is stimulated by the viral Tat protein through the recruitment of cellular Ser/Thr CTD kinases. In transient cotransfection experiments, the c-Abl kinase was found to activate the HIV promoter in the absence of Tat. The activation of the HIV promoter required the nuclear localization of c-Abl and could be correlated with increased tyrosine phosphorylation of RNA polymerase II. These observations suggest that tyrosine phosphorylation of the CTD may be functionally equivalent to its serine/threonine phosphorylation in stimulating transcription elongation. (+info)
The HIV-1-encoded Tat protein controls transcription elongation by increasing processivity of RNA polymerase II (Pol II). Here, we have identified a Tat stimulatory activity (Tat-SF) as a novel RNA Pol II-containing complex. Remarkably, Tat-SF contains the previously identified Tat cofactors Tat-SF1, P-TEFb and hSPT5/Tat-CT1, in addition to RNA Pol II and other unidentified polypeptides, but none of the SRB/MED proteins or other factors found associated with the previously described RNA Pol II holoenzyme complex. Tat-SF supports basal, Sp1-activated and Tat-activated transcription in a reconstituted system, and a Tat-SF-derived fraction lacking RNA Pol II can complement non-responsive RNA Pol II complexes for Tat-enhanced HIV-1 transcription, indicating that Tat-SF contains factors that are critical for Tat function. Both Tat-SF and RNA Pol II holoenzyme are present in HeLa nuclear extracts and each can be recruited to the HIV-1 promoter. Our results indicate that Tat-SF is a Tat cofactor-containing RNA Pol II complex whose recruitment to the promoter provides elongation factors important for Tat-enhanced HIV-1 transcription following TAR RNA synthesis. (+info)
A hyperphosphorylated form of RNA polymerase II is the major interphase antigen of the phosphoprotein antibody MPM-2 and interacts with the peptidyl-prolyl isomerase Pin1.
(51/4175)
The monoclonal antibody MPM-2 recognizes a subset of M phase phosphoproteins in a phosphorylation-dependent manner. It is believed that phosphorylation at MPM-2 antigenic sites could regulate mitotic events since most of the MPM-2 antigens identified to date have M phase functions. In addition, many of these proteins are substrates of the mitotic regulator Pin1, a peptidyl-prolyl isomerase which is present throughout the cell cycle and which is thought to alter its mitotic targets by changing their conformation. In interphase cells, most MPM-2 reactivity is confined to nuclear speckles. We report here that a hyperphosphorylated form of the RNA polymerase II largest subunit is the major MPM-2 interphase antigen. These findings were made possible by the availability of another monoclonal antibody, CC-3, that was previously used to identify a 255 kDa nuclear matrix protein associated with spliceosomal components as a hyperphosphorylated form of the RNA polymerase II largest subunit. MPM-2 recognizes a phosphoepitope of the large subunit that becomes hyperphosphorylated upon heat shock in contrast to the phosphoepitope defined by CC-3, whose reactivity is diminished by the heat treatment. Therefore, these two antibodies may discriminate between distinct functional forms of RNA polymerase II. We also show that RNA polymerase II large subunit interacts with Pin1 in HeLa cells. Pin1 may thus regulate transcriptional and post-transcriptional events by catalyzing phosphorylation-dependent conformational changes of the large RNA polymerase II subunit. (+info)
A protein-protein interaction map of yeast RNA polymerase III.
(52/4175)
The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex. (+info)
RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo.
(53/4175)
Biochemical evidence indicates that pre-mRNA splicing factors physically interact with the C-terminal domain of the largest subunit of RNA polymerase II. We have investigated the in vivo function of this interaction. In mammalian cells, truncation of the CTD of RNA pol II LS prevents the targeting of the splicing machinery to a transcription site. In the absence of the CTD, pre-mRNA splicing is severely reduced. The presence of unspliced RNA alone is not sufficient for the accumulation of splicing factors at the transcription site, nor for its efficient splicing. Our results demonstrate a critical role for the CTD of RNA pol II LS in the intranuclear targeting of splicing factors to transcription sites in vivo. (+info)
Mechanism and regulation of transcriptional elongation by RNA polymerase II.
(54/4175)
Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it. Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged. (+info)
Expressed sequence tag (EST) phenotyping of HT-29 cells: cloning of ser/thr protein kinase EMK1, kinesin KIF3B, and of transcripts that include Alu repeated elements.
(55/4175)
To study the mechanisms that control epithelial commitment and differentiation we have used undifferentiated HT-29 colon cancer cells and a subpopulation of mucus secreting cells obtained by selection of HT-29 cells in 10-6 M methotrexate (M6 cells) as experimental models. We isolated cDNAs encoding transcripts overexpressed in early confluent M6 cells regarding steady-state levels in HT-29 cells by subtractive hybridisation. Fifty-one cDNA clones, corresponding to 34 independent transcripts, were isolated, partially sequenced by their 5' end, and classified into four groups according to their identity: transcripts that included a repeated sequence of the Alu family (10 clones, among them those encoding ribonucleoprotein RNP-L and E-cadherin), transcripts encoded by the mitochondrial genome (nine clones), transcripts encoding components of the protein synthesis machinery (23 clones, including the human ribosomal protein L38 not previously cloned in humans) and nine additional cDNAs that could not be classified in the previous groups. These last included ferritin, cytokeratin 18, translationally controlled human tumour protein (TCHTP), mt-aldehyde dehydrogenase, as well as unknown transcripts (three clones), and the human homologues of the molecular motor kinesin KIF3B and of the ser/thr protein kinase EMK1. Spot dot and Northern blot analyses showed that ser/thr protein kinase EMK1 was differentially expressed in M6 cells when compared with parental HT-29 cells. Steady-state levels of EMK1 were higher in proliferating, preconfluent, M6 and HT-29 cells than in 2 days post confluence (dpc) and 8dpc M6 and HT-29 cells. Transcripts that included an Alu repeat were also shown to be differentially expressed and accumulated in differentiating M6 cells when analysed by Northern blot. The significance of the transcripts cloned is discussed in the context of the commitment and differentiation of the M6 cells to the mucus secreting lineage of epithelial cells. (+info)
The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins.
(56/4175)
We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity. (+info)