Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

Abstract

RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5 ′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3′ -extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3 ′ -extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5 ′ -exonuclease Rat1.[Supplemental material is available for this article.]Transcription of nuclear DNA in eukaryotes is carried out by at least three different RNA polymerases. RNA polymerase III (RNAPIII) is specialized for high-level synthesis of small noncoding RNAs. The most abundant products of RNAPIII-dependent transcription are the 5S rRNA and the many pre-tRNA species. In addition, RNAPIII synthesizes numerous, less abundant small RNAs that are involved in diverse cellular processes, including protein translocation and the processing of pre-rRNA and pre-tRNAs (Dieci et al. 2013). The nuclear genome of Saccharomyces cerevisiae contains 275 actively transcribed tRNA genes (including a tRNA of undetermined specificity [tX(XXX)D]). These are grouped into 20 isotypes, each charged with a single amino acid, which are subdivided into 41 isoacceptors that each recognize the same anticodon sequence(s) (Hani and Feldmann 1998; Chan and Lowe 2009). The reported lengths of the primary transcripts vary between 72 and 133 nucleotides (nt), and ∼25% of pre-tRNAs include introns. Primary pre-tRNA transcripts undergo 5 ′ and 3 ′ maturation and intron excision to generate the mature tRNAs. In tRNA genes, the transcription machinery recognizes conserved promoter elements, termed box A and box B, which are located within the transcribed region and form a bipartite binding site for the six-subunit basal transcription factor TFIIIC (Acker et al. 2013). Box A starts at position +8 of the mature tRNA, and the transcription start site is most frequently located 18-20 nt upstream (Dieci et al. 2013). Within yeast tRNA genes, boxes A and B are localized 31-93 nt apart and correspond to the universally conserved D-and T-loops in the tRNA structure. Internally located A and B boxes are...

Common genomic elements promote transcriptional and DNA replication roadblocks

“…The second peak occurs at the 3 ′ -end of tRNA genes and may indicate a collision between DNA Pol δ and TFIIIC subunits bound at box B. The lack of a significant peak at this location for Pol II may be due to the presence of actively transcribing Pol III at tRNA genes preventing Pol II from approaching TFIIIC, consistent with a recent report that Pol III termination typically occurs over a broad region downstream from tDNA (Turowski et al 2016). As DNA synthesis by Pol δ occurs in the context of replication fork movement, it is possible that rapid TFIIIC and TFIIIB assembly on newly synthesized DNA prior to the initiation of Pol III transcription blocks Pol δ.…”

Evolutionarily Conserved Polyadenosine RNA Binding Protein Nab2 Cooperates with Splicing Machinery To Regulate the Fate of Pre-mRNA

“…Since snoRNAs are often located in introns in mammals, understanding whether Nab2 functions directly in snoRNA processing or the splicing events that liberate introns containing snoRNAs could provide insights into the coupling of splicing and snoRNA processing. Recent work also uncovers a potential function for Nab2 in the surveillance of tRNAs (111), suggesting that Nab2 could play a general role in the regulation of noncoding RNAs.…”

Transcription by RNA polymerase III: insights into mechanism and regulation

“…Events following transcription initiation are less well characterized (Figure 1B). Recent analyses in yeast revealed surprisingly uneven distribution of RNAPIII along individual transcription units [10,11]. In particular, a strong 5′-peak of RNAPIII occupancy was observed on all tRNA genes at the position of box A.…”

“…Minimum lengths are reported to be A 4 for human and A 5 or A 6 for yeast [14–16], although recent analyses indicate that in vivo termination is significantly more effective with A 7 –A 8 [11]. During termination, the weak base-pairing interactions between oligo(dA) in the template strand and oligo(U) in the nascent transcript act as a principal destabilizing signal.…”