scholarly journals Coupling of Termination, 3′ Processing, and mRNA Export

2002 ◽  
Vol 22 (18) ◽  
pp. 6441-6457 ◽  
Author(s):  
C. M. Hammell ◽  
Stefan Gross ◽  
Daniel Zenklusen ◽  
Catherine V. Heath ◽  
Francoise Stutz ◽  
...  
Keyword(s):  
Mrna Export ◽  
Mrna Processing ◽  
Nonpermissive Temperature ◽  
Pol Ii ◽  
The Core ◽  
In Vitro Processing ◽  
Novel Alleles ◽  
Processing Factors

ABSTRACT In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3′ processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3′ processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3′ processed transcripts at the nonpermissive temperature. 3′ maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3′ processing. The similarity of the phenotypes of some mRNA export mutants and 3′ processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3′ processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3′ maturation. Of the core 3′ processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3′ processing and mRNA export defects found in xpo1-1 cells.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

scholarly journals A U2-snRNP–independent role of SF3b in promoting mRNA export

2019 ◽  
Vol 116 (16) ◽  
pp. 7837-7846 ◽  
Author(s):  
Ke Wang ◽  
Changping Yin ◽  
Xian Du ◽  
Suli Chen ◽  
Jianshu Wang ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mrna Export ◽  
Mrna Processing ◽  
Dependent Manner ◽  
U2 Snrnp ◽  
Dose Dependent ◽  
Antisense Morpholino

To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3′ processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b–THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP–independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.

scholarly journals Role for PSF in Mediating Transcriptional Activator-Dependent Stimulation of Pre-mRNA Processing In Vivo

2005 ◽  
Vol 25 (15) ◽  
pp. 6734-6746 ◽  
Author(s):  
Emanuel Rosonina ◽  
Joanna Y. Y. Ip ◽  
John A. Calarco ◽  
Malina A. Bakowski ◽  
Andrew Emili ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Mrna Processing ◽  
Pol Ii ◽  
293 Cells ◽  
Two Factors ◽  
Elevated Expression ◽  
Strong Activator ◽  
Stimulation Of

ABSTRACT In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3′-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator- and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54nrb/NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3′-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3′-end cleavage. Moreover, PSF and p54nrb/NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator- and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.

scholarly journals The histone methyltransferase SETD2 couples transcription and splicing by engaging pre-mRNA processing factors through its SHI domain

2020 ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Histone Methyltransferase ◽  
Mrna Processing ◽  
Key Factors ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

SUMMARYHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP L Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2 by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

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 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.

scholarly journals In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

1990 ◽  
Vol 10 (8) ◽  
pp. 3868-3872 ◽  
Author(s):  
C M Shumard ◽  
C Torres ◽  
D C Eichler
Keyword(s):  
18S Rrna ◽  
Precise Determination ◽  
In Vivo Studies ◽  
Rrna Sequence ◽  
S1 Nuclease ◽  
In Vitro Processing ◽  
Rrna Maturation ◽  
A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

BAF is required for emerin assembly into the reforming nuclear envelope

2001 ◽  
Vol 114 (24) ◽  
pp. 4575-4585 ◽  
Author(s):  
Tokuko Haraguchi ◽  
Takako Koujin ◽  
Miriam Segura-Totten ◽  
Kenneth K. Lee ◽  
Yosuke Matsuoka ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Core Region ◽  
Lamin B ◽  
The Core ◽  
Double Stranded Dna ◽  
Recessive Form ◽  
Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

1989 ◽  
Vol 9 (10) ◽  
pp. 4479-4487
Author(s):  
M Cotten ◽  
G Schaffner ◽  
M L Birnstiel
Keyword(s):  
Antisense Rna ◽  
Nuclear Extract ◽  
Mrna Processing ◽  
Rna Molecules ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Processing ◽  
Antisense Dna ◽  
Nuclear Ribonucleoprotein ◽  
Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

Sign in / Sign up

Export Citation Format

Share Document