scholarly journals Participation of the C-Terminal Domain of RNA Polymerase II in Exon Definition during Pre-mRNA Splicing

2000 ◽  
Vol 20 (21) ◽  
pp. 8290-8301 ◽  
Author(s):  
Changqing Zeng ◽  
Susan M. Berget
Keyword(s):  
Rna Polymerase Ii ◽  
Repeat Sequence ◽  
Splicing Factors ◽  
Exon Definition ◽  
Pol Ii ◽  
Terminal Domain ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Precursor Rna

ABSTRACT Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3′ and 5′ splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.

scholarly journals Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

1989 ◽  
Vol 9 (12) ◽  
pp. 5273-5280 ◽  
Author(s):  
G C Conway ◽  
A R Krainer ◽  
D L Spector ◽  
R J Roberts
Keyword(s):  
Rna Splicing ◽  
De Novo ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Large Structures ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

scholarly journals EWS, but Not EWS-FLI-1, Is Associated with Both TFIID and RNA Polymerase II: Interactions between Two Members of the TET Family, EWS and hTAFII68, and Subunits of TFIID and RNA Polymerase II Complexes

1998 ◽  
Vol 18 (3) ◽  
pp. 1489-1497 ◽  
Author(s):  
Anne Bertolotti ◽  
Thomas Melot ◽  
Joël Acker ◽  
Marc Vigneron ◽  
Olivier Delattre ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromosomal Translocation ◽  
Binding Studies ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Tfiid Complex

ABSTRACT The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of theEWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.

scholarly journals TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II

2009 ◽  
Vol 29 (20) ◽  
pp. 5455-5464 ◽  
Author(s):  
Kira Glover-Cutter ◽  
Stéphane Larochelle ◽  
Benjamin Erickson ◽  
Chao Zhang ◽  
Kevan Shokat ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Histone H4 ◽  
Pol Ii ◽  
Terminal Domain ◽  
Histone H4 Acetylation ◽  
Flanking Regions

ABSTRACT The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7 as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

scholarly journals Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing

1989 ◽  
Vol 9 (12) ◽  
pp. 5273-5280
Author(s):  
G C Conway ◽  
A R Krainer ◽  
D L Spector ◽  
R J Roberts
Keyword(s):  
Rna Splicing ◽  
De Novo ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Large Structures ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

scholarly journals The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme.

1997 ◽  
Vol 17 (4) ◽  
pp. 1817-1823 ◽  
Author(s):  
T P Cujec ◽  
H Cho ◽  
E Maldonado ◽  
J Meyer ◽  
D Reinberg ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Step ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Immunodeficiency Virus ◽  
Rna Hairpin ◽  
Rna Polymerase Ii Holoenzyme

The human immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV.

scholarly journals Formation of a Carboxy-Terminal Domain Phosphatase (Fcp1)/TFIIF/RNA Polymerase II (pol II) Complex in Schizosaccharomyces pombe Involves Direct Interaction between Fcp1 and the Rpb4 Subunit of pol II

2002 ◽  
Vol 22 (5) ◽  
pp. 1577-1588 ◽  
Author(s):  
Makoto Kimura ◽  
Hisako Suzuki ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Schizosaccharomyces Pombe ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Gene Encoding ◽  
Pol Ii ◽  
Terminal Domain ◽  
Ctd Phosphatase

ABSTRACT In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIFα, TFIIFβ, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

2007 ◽  
Vol 82 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Jinhong Chang ◽  
Xingcao Nie ◽  
Ho Eun Chang ◽  
Ziying Han ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Cell System ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Delta Antigen ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

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