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Compromised RNA polymerase III complex assembly leads to local alterations of intergenic RNA polymerase II transcription in Saccharomyces cerevisiae.

Wang Q, Nowak CM, Korde A, Oh DH, Dassanayake M, Donze D - BMC Biol. (2014)

Bottom Line: Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast.The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as 'extra-transcriptional' effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.

Results: Given our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5'- and 3'-extended transcripts, and de-repression of adjacent genes and intergenic regions.

Conclusions: The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

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5′-UTR extension at non-tDNA/non-ETC Pol III associated locusZOD1-RPM2. A) IGV profiles showing extended 5′-UTR of RPM2 in tfc6 mutants. B) Schematic of locus and location of primers. As before, extended transcripts are enriched in tfc6 mutant and B-box mutant strains relative to wild type. Strains used were wild type DDY3630, tfc6 DDY4300, and B-box mutants DDY5164 and DDY5165. IGV, integrative genomics viewer; Pol III, Polymerase III.
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Fig6: 5′-UTR extension at non-tDNA/non-ETC Pol III associated locusZOD1-RPM2. A) IGV profiles showing extended 5′-UTR of RPM2 in tfc6 mutants. B) Schematic of locus and location of primers. As before, extended transcripts are enriched in tfc6 mutant and B-box mutant strains relative to wild type. Strains used were wild type DDY3630, tfc6 DDY4300, and B-box mutants DDY5164 and DDY5165. IGV, integrative genomics viewer; Pol III, Polymerase III.

Mentions: Figure 6 shows the effect of compromising Pol III complex assembly at the ZOD1 locus. Here again, we observe an extended 5′-UTR in RPM2 transcripts (red arrow in Figure 6A) in the tfc6 mutants. These altered transcripts in one tfc6 mutant were confirmed by RT-PCR, and are much more highly enriched in two strains containing specific mutations in the ZOD1 B-box (Figure 6B). Although not confirmed by B-box mutagenesis, observed effects (determined by inspection of IGV profiles and DESeq analysis) at other non-tDNA loci in tfc6 mutants include: SNR6, increased level of adjacent Ty1 LTR transcripts; RPR1, increased level of the adjacent SUT088 intergenic transcript; and SCR1, increased level and 5′-extension of the adjacent uncharacterized gene YER137C. 5′-Extension of adjacent RAD14 transcripts was confirmed in tfc6 and RNA170 B-box mutants (Q. Wang, unpublished data).Figure 6


Compromised RNA polymerase III complex assembly leads to local alterations of intergenic RNA polymerase II transcription in Saccharomyces cerevisiae.

Wang Q, Nowak CM, Korde A, Oh DH, Dassanayake M, Donze D - BMC Biol. (2014)

5′-UTR extension at non-tDNA/non-ETC Pol III associated locusZOD1-RPM2. A) IGV profiles showing extended 5′-UTR of RPM2 in tfc6 mutants. B) Schematic of locus and location of primers. As before, extended transcripts are enriched in tfc6 mutant and B-box mutant strains relative to wild type. Strains used were wild type DDY3630, tfc6 DDY4300, and B-box mutants DDY5164 and DDY5165. IGV, integrative genomics viewer; Pol III, Polymerase III.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4228148&req=5

Fig6: 5′-UTR extension at non-tDNA/non-ETC Pol III associated locusZOD1-RPM2. A) IGV profiles showing extended 5′-UTR of RPM2 in tfc6 mutants. B) Schematic of locus and location of primers. As before, extended transcripts are enriched in tfc6 mutant and B-box mutant strains relative to wild type. Strains used were wild type DDY3630, tfc6 DDY4300, and B-box mutants DDY5164 and DDY5165. IGV, integrative genomics viewer; Pol III, Polymerase III.
Mentions: Figure 6 shows the effect of compromising Pol III complex assembly at the ZOD1 locus. Here again, we observe an extended 5′-UTR in RPM2 transcripts (red arrow in Figure 6A) in the tfc6 mutants. These altered transcripts in one tfc6 mutant were confirmed by RT-PCR, and are much more highly enriched in two strains containing specific mutations in the ZOD1 B-box (Figure 6B). Although not confirmed by B-box mutagenesis, observed effects (determined by inspection of IGV profiles and DESeq analysis) at other non-tDNA loci in tfc6 mutants include: SNR6, increased level of adjacent Ty1 LTR transcripts; RPR1, increased level of the adjacent SUT088 intergenic transcript; and SCR1, increased level and 5′-extension of the adjacent uncharacterized gene YER137C. 5′-Extension of adjacent RAD14 transcripts was confirmed in tfc6 and RNA170 B-box mutants (Q. Wang, unpublished data).Figure 6

Bottom Line: Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast.The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Assembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as 'extra-transcriptional' effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.

Results: Given our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5'- and 3'-extended transcripts, and de-repression of adjacent genes and intergenic regions.

Conclusions: The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.

Show MeSH
Related in: MedlinePlus