Limits...
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.

Show MeSH

Related in: MedlinePlus

De-repression ofSPO74intfc6and B-box mutants. A) IGV profiles of SPO74 showing apparent de-repression. Schematic diagram showing the relative position of the upstream tDNA, drawn to scale with the IGV profiles. The location of the qRT-PCR primers is shown below the SPO74 ORF. B) Quantitative RT-PCR showing an increase in SPO74 RNA in tfc6 and B-box mutant strains relative to wild type. The B-box mutants were DDY5128 and DDY5129. IGV, integrative genomics viewer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: De-repression ofSPO74intfc6and B-box mutants. A) IGV profiles of SPO74 showing apparent de-repression. Schematic diagram showing the relative position of the upstream tDNA, drawn to scale with the IGV profiles. The location of the qRT-PCR primers is shown below the SPO74 ORF. B) Quantitative RT-PCR showing an increase in SPO74 RNA in tfc6 and B-box mutant strains relative to wild type. The B-box mutants were DDY5128 and DDY5129. IGV, integrative genomics viewer.

Mentions: SPO74 (YGL170C) is required for spore formation and is located on chrVII of S. cerevisiae [43]. As it is a sporulation specific gene, SPO74 is not significantly transcribed in haploid or exponentially dividing S. cerevisiae. The tDNA tK(CUU)G2 terminates approximately 300 bp upstream of the 5′-end of SPO74. Our mapped RNA-Seq reads and DESeq analysis suggested a moderate approximately 11-fold de-repression (padj =3.20E-25) of SPO74 in the mutant strains compared to the low level of reads seen in wild type strains (Figure 5A). Quantitative RT-PCR (qRT-PCR) of SPO74 mRNA levels was performed to confirm this apparent de-repression, and we again constructed strains containing targeted tDNA B-box mutations. Figure 5B shows the results of this analysis. The tfc6 mutants showed an approximately 20-fold increase in transcripts within the SPO74 coding sequence, and the B-box mutants expressed SPO74 transcripts at a 7- to 10-fold higher level compared to wild type.Figure 5


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)

De-repression ofSPO74intfc6and B-box mutants. A) IGV profiles of SPO74 showing apparent de-repression. Schematic diagram showing the relative position of the upstream tDNA, drawn to scale with the IGV profiles. The location of the qRT-PCR primers is shown below the SPO74 ORF. B) Quantitative RT-PCR showing an increase in SPO74 RNA in tfc6 and B-box mutant strains relative to wild type. The B-box mutants were DDY5128 and DDY5129. IGV, integrative genomics viewer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: De-repression ofSPO74intfc6and B-box mutants. A) IGV profiles of SPO74 showing apparent de-repression. Schematic diagram showing the relative position of the upstream tDNA, drawn to scale with the IGV profiles. The location of the qRT-PCR primers is shown below the SPO74 ORF. B) Quantitative RT-PCR showing an increase in SPO74 RNA in tfc6 and B-box mutant strains relative to wild type. The B-box mutants were DDY5128 and DDY5129. IGV, integrative genomics viewer.
Mentions: SPO74 (YGL170C) is required for spore formation and is located on chrVII of S. cerevisiae [43]. As it is a sporulation specific gene, SPO74 is not significantly transcribed in haploid or exponentially dividing S. cerevisiae. The tDNA tK(CUU)G2 terminates approximately 300 bp upstream of the 5′-end of SPO74. Our mapped RNA-Seq reads and DESeq analysis suggested a moderate approximately 11-fold de-repression (padj =3.20E-25) of SPO74 in the mutant strains compared to the low level of reads seen in wild type strains (Figure 5A). Quantitative RT-PCR (qRT-PCR) of SPO74 mRNA levels was performed to confirm this apparent de-repression, and we again constructed strains containing targeted tDNA B-box mutations. Figure 5B shows the results of this analysis. The tfc6 mutants showed an approximately 20-fold increase in transcripts within the SPO74 coding sequence, and the B-box mutants expressed SPO74 transcripts at a 7- to 10-fold higher level compared to wild type.Figure 5

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