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

Qing Wang Chance M Nowak 0 Asawari Korde 0 Dong-Ha Oh Maheshi Dassanayake David Donze 0 Equal contributors Department of Biological Sciences, Louisiana State University , Baton Rouge, LA 70803 , USA 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. - Background In eukaryotes, there are three major types of RNA polymerase designated as Pol I, II, and III (with additional polymerase complexes in plants), which function to transcribe the vast array of RNA species that contribute to the highly complex and heterogeneous eukaryotic transcriptome. Pol I transcribes the majority of ribosomal RNAs, and Pol II is mainly dedicated to protein coding genes. RNA polymerase III (Pol III) transcribes genes encoding small non-translated RNAs, which in the budding yeast Saccharomyces cerevisiae includes transfer RNAs (tRNAs), 5S ribosomal RNA (5S rRNA), 7SL RNA, U6 spliceosome RNA, snR52 small nucleolar RNA as well as the RNA component of RNaseP [1-3]. These diverse genes contain three types of promoter element arrangements. The tRNA genes (tDNAs) utilize what is referred to as a type 2 internal promoter, and the transcription factor binding sites within these genes are referred to as internal control regions (ICRs). Type 2 promoters contain conserved A-box and B-box ICR elements separated by a variable distance. These sequences serve as binding sites for the multi-subunit transcription factor TFIIIC [4-6]. In yeast, Pol III transcription of tDNAs requires binding of three multimeric protein complexes TFIIIC (six polypeptides), TFIIIB (three polypeptides) and Pol III enzyme (seventeen polypeptides). Pol III complex assembly at tDNAs is initiated by the binding of TFIIIC, which then recruits TFIIIB followed by Pol III [4]. The binding affinity of TFIIIC is primarily determined by B-box interactions, and mutation of an invariant cytosine residue in the B-box consensus sequence GWTCRANNC severely diminishes TFIIIC binding affinity and subsequent transcriptional activity of the mutated tDNA [3,7,8]. In addition to Pol III transcribed genes, TFIIIC complexes appear to be bound to other chromosomal locations in the absence of TFIIIB and Pol III [9,10], and in S. cerevisiae such locations have been referred to as extra-TFIIIC (ETC) sites [11]. In addition to promoting small RNA transcription, Pol III complexes assembled on eukaryotic chromosomes are responsible for what has been termed product independent or extra-transcriptional functions [5,12]. Characterized extra-transcriptional effects of Pol III complexes, mainly studied in S. cerevisiae, include targeting yeast Ty retroelement integration [13-15], phasing of local nucleosome positioning [16-18] and pausing of DNA polymerase progression as replication forks encounter tDNAs [19,20]. Additional effects include inhibition of transcription from nearby Pol II promoters, referred to as tRNA gene mediated (tgm) silencing [21] or position effects [22], and also include both barrier and insulator types of chromatin boundary activities [23,24]. Sequences that recruit the TFIIIC complex have also been shown to have chromatin boundary-like activities in other eukaryotes [25-29]. Most recently, our lab demonstrated in S. cerevisiae that a tDNA acts as a roadblock to cryptic intergenic transcription [30]. This latest study showed that either mutation of the tDNA upstream of ATG31 or global impairment of Pol III complex formation allowed readthrough of the SUT467 noncoding intergenic transcript through the tDNA region. Readthrough at this site resulted in the production of extended SUT467-ATG31 hybrid transcripts. These transcripts are defective for translation of Atg31p due to the extended 5-untranslated region (5-UTR), which results in reduced fitness under nitrogen starvation conditions due to under-expression of this critical autophagy protein. A previous study was performed to assess genomewide extra-transcriptional effects of assembled Pol III complexes on Pol II transcribed genes by comparing coding sequence microarray expression levels of wild type versus a variety of Pol III defective mutant yeast strains. Mutant subunits of TFIIIC, TFIIIB or Pol III resulted in minimal effects on expression levels of genes adjacent to tDNAs and ETC sites, and most of the differences observed were due to secondary effects mediated by activation of Gcn4p transcription factor activity in response to reduced initiator tRNAMet levels [31]. Since we observed changes in intergenic transcription upstream of ATG31 upon mutation of the adjacent tDNA and in mutants under-expressing the TFIIIC subunit Tfc6p, we revisited the genome-wide analysis of Pol III complex mediated extra-transcriptional effects using high-throughput RNA-Sequencing (RNA-Seq). We reasoned that RNA-Seq would identify differences in intergenic transcription that were missed in the previous microarray analysis. The results presented here comparing wild type yeast to Tfc6p under-expressing mutants recapitulate the Gcn4p mediated effects from the Conesa et al. [31] study. Additionally, numerous alterations in intergenic transcription in close proximity to tDNAs and other Pol III complex binding sites are observed in tfc6 mutants. Analysis of loci adjacent to Pol III complex binding sites that were significantly altered in the tfc6 mutants reveal both (...truncated)