scholarly journals Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation.

1987 ◽  
Vol 7 (10) ◽  
pp. 3602-3612 ◽  
Author(s):  
S S Sisodia ◽  
B Sollner-Webb ◽  
D W Cleveland
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Rnase H ◽  
Intact Cells ◽  
S1 Nuclease ◽  
Polymerase Iii ◽  
Hybrid Genes ◽  
Rna Maturation

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.

Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation

1987 ◽  
Vol 7 (10) ◽  
pp. 3602-3612
Author(s):  
S S Sisodia ◽  
B Sollner-Webb ◽  
D W Cleveland
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Rnase H ◽  
Intact Cells ◽  
S1 Nuclease ◽  
Polymerase Iii ◽  
Hybrid Genes ◽  
Rna Maturation

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.

scholarly journals Sen1 is required for RNA Polymerase III transcription termination in an R-loop independent manner

2019 ◽  
Author(s):  
Julieta Rivosecchi ◽  
Marc Larochelle ◽  
Camille Teste ◽  
Frédéric Grenier ◽  
Amélie Malapert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Rna Polymerases ◽  
Independent Manner ◽  
Polymerase Iii

ABSTRACTR-loop disassembly by the human helicase Senataxin contributes to genome stability and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin-mediated R-loop disassembly also contributes to transcription termination at other classes of genes has remained unclear. Here we show in fission yeast that SenataxinSen1promotes efficient termination of RNA Polymerase III (RNAP3) transcriptionin vivo. In the absence of SenataxinSen1, RNAP3 accumulates downstream of the primary terminator at RNAP3-transcribed genes and produces long exosome-sensitive 3’-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that SenataxinSen1acts as an ancillary factor for RNAP3 transcription terminationin vivochallenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Senataxin is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

scholarly journals Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

1982 ◽  
Vol 2 (12) ◽  
pp. 1595-1607 ◽  
Author(s):  
Timothy J. Miller ◽  
Janet E. Mertz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Structural Requirements ◽  
Infected Monkey ◽  
Full Complement ◽  
Sv40 Dna

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei ofXenopus laevisoocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II

Cell ◽  
1987 ◽  
Vol 51 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Philippe Carbon ◽  
Sylvie Murgo ◽  
Jean-Pierre Ebel ◽  
Alain Krol ◽  
Graham Tebb ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Octamer Motif

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

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