scholarly journals RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sofia Battaglia ◽  
Michael Lidschreiber ◽  
Carlo Baejen ◽  
Phillipp Torkler ◽  
Seychelle M Vos ◽  
...  
Keyword(s):  
Rna Recognition ◽  
Elongation Factors ◽  
Elongation Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Recognition Motifs ◽  
Pol Ii ◽  
Recognition Motifs ◽  
Rna Interaction

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex.

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.

Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

1999 ◽  
Vol 112 (24) ◽  
pp. 4501-4512 ◽  
Author(s):  
Y.M. Yannoni ◽  
K. White
Keyword(s):  
Nuclear Localization ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Localization Sequence ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Localization Sequence ◽  
Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

scholarly journals A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

2020 ◽  
Vol 48 (6) ◽  
pp. 2897-2911 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Giovanni L Burke ◽  
Ziyan Chen ◽  
Jack F Greenblatt ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Nascent Transcripts ◽  
Target Rna

Abstract The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

scholarly journals The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs.

1991 ◽  
Vol 113 (1) ◽  
pp. 1-12 ◽  
Author(s):  
W C Lee ◽  
Z X Xue ◽  
T Mélèse
Keyword(s):  
Nuclear Localization ◽  
Partial Purification ◽  
Nuclear Localization Sequence ◽  
Affinity Column ◽  
Rna Recognition ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Recognition Motifs ◽  
Localization Sequence ◽  
Recognition Motifs ◽  
Nuclear Localization Sequences

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.

scholarly journals An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

1997 ◽  
Vol 8 (1) ◽  
pp. 97-108 ◽  
Author(s):  
K E McGrath ◽  
J F Smothers ◽  
C A Dadd ◽  
M T Madireddi ◽  
M A Gorovsky ◽  
...  
Keyword(s):  
Casein Kinase Ii ◽  
Ribosomal Gene ◽  
Casein Kinase ◽  
Electron Microscopic ◽  
Rna Recognition Motifs ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Recognition Motifs

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

scholarly journals Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 777-788 ◽  
Author(s):  
Yi Pei ◽  
Hongyan Du ◽  
Juliet Singer ◽  
Courtney St. Amour ◽  
Selena Granitto ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Growth Defect ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Elongation Complex ◽  
Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

scholarly journals Bioassaying Putative RNA-Binding Motifs in a Protein Encoded by a Gene That Influences Courtship and Visually Mediated Behavior in Drosophila: In Vitro Mutagenesis of nonA

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 259-275
Author(s):  
Ralf Stanewsky ◽  
Thomas A Fry ◽  
Ingolf Reim ◽  
Harald Saumwebert ◽  
Jeffrey C Hall
Keyword(s):  
Rna Binding ◽  
Courtship Song ◽  
In Vitro Mutagenesis ◽  
Rna Recognition ◽  
Null Mutant ◽  
Rna Recognition Motifs ◽  
Binding Motifs ◽  
Recognition Motifs ◽  
Transgenic Strains

Abstract The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nod-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromcsomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nod-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA' s ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele.

scholarly journals An essential RNA-binding lysine residue in the Nab3 RRM domain undergoes mono and trimethylation

2019 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Kyle Biggar ◽  
Marc D. Meneghini
Keyword(s):  
Rna Binding ◽  
Growth Defect ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Target Rna

AbstractThe Nrd1-Nab3-Sen1 (NNS) complex integrates molecular inputs to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by methylation of histone H3 lysine-4 as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3, and Sen1 that are mono-, di-, or trimethylated, suggesting novel molecular inputs for NNS regulation. One of these methylated residues, Nab3 lysine-363 (K363), resides within its RRM, and is known to physically contact target RNA. Although mutation of Nab3-K363 to arginine (Nab3-K363R) causes a severe growth defect, it nevertheless produces a stable protein that is incorporated into the NNS complex, suggesting that RNA binding through Nab3-K363 is crucial for NNS function. Consistent with this hypothesis, K363R mutation decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects in vivo. These findings reveal crucial roles for Nab3-K363 and suggest that methylation of this residue may modulate NNS activity through its impact on Nab3 RNA binding.

scholarly journals In vitro analysis of RNA polymerase II elongation complex dynamics

2019 ◽  
Author(s):  
Yoo Jin Joo ◽  
Scott B. Ficarro ◽  
Yujin Chun ◽  
Jarrod A. Marto ◽  
Stephen Buratowski
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Complex Dynamics ◽  
Nuclear Extract ◽  
Initiation Factor ◽  
Elongation Factors ◽  
Elongation Complex

AbstractRNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 levels remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD), and the factors that recognize them, change as a function of post-initiation time, rather than distance elongated. Chemical inhibition of Kin28/Cdk7 blocks both Serine 5 and Serine 2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, Cap Binding Complex, Set2, and the PAF1 complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated core elongation factors, chromatin regulators, and RNA processing factors change at each step of transcription.

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