Autoantibodies as probes for the molecular architecture of the nucleus

1991 ◽  
Vol 49 ◽  
pp. 18-19
Author(s):  
David L. Spector ◽  
Gayle Lark ◽  
Mika Sovak ◽  
Sui Huang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Types ◽  
Detectable Effect ◽  
Molecular Architecture ◽  
Small Nuclear Ribonucleoprotein ◽  
Highly Sensitive ◽  
Polymerase I ◽  
Nuclear Ribonucleoprotein

Autoantibodies to a variety of nuclear components including DNA, histones, DNA polymerases, DNA topoisomerases, lamins, coiled bodies, RNA polymerase I, and several classes of ribonucleoproteins have been identified in the sera of individuals with autoimmune disorders. We have taken advantage of autoantibodies to several major classes (U1, U2, U4/U6, U5) of small nuclear ribonucleoprotein particles (snRNPs) which play a crucial role in the processing of pre-mRNA molecules. We have used these antibodies to evaluate the organization of factors which participate in pre-mRNA splicing in the mammalian cell nucleus.We have previously shown snRNPs to be localized to discrete immunostained regions, “speckles”, which form a latticework within the interphase nuclei of several mammalian cell types. Since RNA polymerase II, which is involved in the synthesis of pre-mRNA, is highly sensitive to a-amanitin we were interested in the effect of an inhibition of pre-mRNA synthesis on the snRNP distribution pattern. Cells incubated in drug for 5 hours (Fig. 1d) showed no detectable effect on the organization of snRNPs nor any effect on the level of transcription.

scholarly journals Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

2007 ◽  
Vol 178 (6) ◽  
pp. 937-949 ◽  
Author(s):  
Snehal Bhikhu Patel ◽  
Natalya Novikova ◽  
Michel Bellini
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Messenger Rnas ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Small Nuclear Rnas ◽  
Transcriptional Units ◽  
Necessary And Sufficient ◽  
Nuclear Ribonucleoprotein ◽  
Nascent Transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

scholarly journals Coilin participates in the suppression of RNA polymerase I in response to cisplatin-induced DNA damage

2011 ◽  
Vol 22 (7) ◽  
pp. 1070-1079 ◽  
Author(s):  
Andrew S. Gilder ◽  
Phi M. Do ◽  
Zunamys I Carrero ◽  
Angela M. Cosman ◽  
Hanna J. Broome ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Γ Irradiation ◽  
Small Nuclear Ribonucleoprotein ◽  
Pol I ◽  
Upstream Binding Factor ◽  
Polymerase I ◽  
Nuclear Ribonucleoprotein

Coilin is a nuclear phosphoprotein that concentrates within Cajal bodies (CBs) and impacts small nuclear ribonucleoprotein (snRNP) biogenesis. Cisplatin and γ-irradiation, which cause distinct types of DNA damage, both trigger the nucleolar accumulation of coilin, and this temporally coincides with the repression of RNA polymerase I (Pol I) activity. Knockdown of endogenous coilin partially overrides the Pol I transcriptional arrest caused by cisplatin, while both ectopically expressed and exogenous coilin accumulate in the nucleolus and suppress rRNA synthesis. In support of this mechanism, we demonstrate that both cisplatin and γ-irradiation induce the colocalization of coilin with RPA-194 (the largest subunit of Pol I), and we further show that coilin can specifically interact with RPA-194 and the key regulator of Pol I activity, upstream binding factor (UBF). Using chromatin immunoprecipitation analysis, we provide evidence that coilin modulates the association of Pol I with ribosomal DNA. Collectively, our data suggest that coilin acts to repress Pol I activity in response to cisplatin-induced DNA damage. Our findings identify a novel and unexpected function for coilin, independent of its role in snRNP biogenesis, establishing a new link between the DNA damage response and the inhibition of rRNA synthesis.

scholarly journals Structure of a transcribing RNA polymerase II–U1 snRNP complex

2020 ◽  
Author(s):  
Suyang Zhang ◽  
Shintaro Aibara ◽  
Seychelle M. Vos ◽  
Dmitry E. Agafonov ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Splice Site ◽  
Loop Formation ◽  
Mrna Isoforms ◽  
Pol Ii ◽  
Small Nuclear Ribonucleoprotein ◽  
Starting Point ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein

AbstractTo initiate co-transcriptional splicing, RNA polymerase II (Pol II) recruits U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent pre-mRNA. Here we report the cryo-EM structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the 5’ splice site in pre-mRNA near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5’ splice site, leading to formation of an intron loop. Loop formation may facilitate scanning of the nascent pre-mRNA for the 3’ splice site and enable prespliceosome assembly and functional pairing of distant intron ends. Our results provide a starting point for a mechanistic analysis of co-transcriptional splicing and the biogenesis of mRNA isoforms during alternative splicing.

scholarly journals Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types

2016 ◽  
Vol 17 (1) ◽  
Author(s):  
Daniel S. Day ◽  
Bing Zhang ◽  
Sean M. Stevens ◽  
Francesco Ferrari ◽  
Erica N. Larschan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cell ◽  
Cell Types ◽  
Comprehensive Analysis

Control points in eucaryotic ribosome biogenesis

1991 ◽  
Vol 69 (1) ◽  
pp. 5-22 ◽  
Author(s):  
D. E. Larson ◽  
P. Zahradka ◽  
B. H. Sells
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Polymerase Iii ◽  
Ribosomal Subunits ◽  
Rrna Species ◽  
Polymerase I ◽  
Ribosomal Precursor ◽  
Precursor Molecules

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.

Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA

1993 ◽  
Vol 13 (7) ◽  
pp. 4331-4341
Author(s):  
W R Widner ◽  
R B Wickner
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Copy Number ◽  
Dsrna Virus ◽  
Cdna Clones ◽  
Viral Mrnas ◽  
Nucleolar Proteins ◽  
Toxin Secretion ◽  
Polymerase I ◽  
Beta Galactosidase

The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X dsRNA, of the L-BC dsRNA virus, and of the single-stranded replicon 20S RNA. We show that SKI2 encodes a 145-kDa protein with motifs characteristic of helicases and nucleolar proteins and is essential only in cells carrying M dsRNA. Unexpectedly, Ski2p does not repress M1 dsRNA copy number when M1 is supported by aN L-A cDNA clone; nonetheless, it did lower the levels of M1 dsRNA-encoded toxin produced. Since toxin secretion from cDNA clones of M1 is unaffected by Ski2p, these data suggest that Ski2p acts by specifically blocking translation of viral mRNAs, perhaps recognizing the absence of cap or poly(A). In support of this idea, we find that Ski2p represses production of beta-galactosidase from RNA polymerase I [no cap and no poly(A)] transcripts but not from RNA polymerase II (capped) transcripts.

scholarly journals The Swi/Snf Chromatin Remodeling Complex Is Required for Ribosomal DNA and Telomeric Silencing in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (18) ◽  
pp. 8227-8235 ◽  
Author(s):  
Vardit Dror ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Ribosomal Dna ◽  
Transcriptional Activation ◽  
Rdna Locus ◽  
Detectable Effect ◽  
Chromatin Remodeling Complex ◽  
Two Factors

ABSTRACT The Swi/Snf chromatin remodeling complex has been previously demonstrated to be required for transcriptional activation and repression of a subset of genes in Saccharomyces cerevisiae. In this work we demonstrate that Swi/Snf is also required for repression of RNA polymerase II-dependent transcription in the ribosomal DNA (rDNA) locus (rDNA silencing). This repression appears to be independent of both Sir2 and Set1, two factors known to be required for rDNA silencing. In contrast to many other rDNA silencing mutants that have elevated levels of rDNA recombination, snf2Δ mutants have a significantly decreased level of rDNA recombination. Additional studies have demonstrated that Swi/Snf is also required for silencing of genes near telomeres while having no detectable effect on silencing of HML or HMR.

scholarly journals Regulation of ribosomal DNA transcription by insulin

1998 ◽  
Vol 275 (1) ◽  
pp. C130-C138 ◽  
Author(s):  
Katherine M. Hannan ◽  
Lawrence I. Rothblum ◽  
Leonard S. Jefferson
Keyword(s):  
Rna Polymerase ◽  
Cell Types ◽  
Rna Polymerase I ◽  
Model Systems ◽  
Serum Starvation ◽  
Swiss Albino Mouse ◽  
Rdna Transcription ◽  
Cellular Content ◽  
Upstream Binding Factor ◽  
Polymerase I

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.

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