Intergenic transcriptional interference is blocked by RNA polymerase III transcription factor TFIIIB in Saccharomyces cerevisiae

Abstract

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such "extratranscriptional" activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467-ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins.

Keywords: RNA polymerase III; TFIIIB; TFIIIC; extratranscriptional effects; noncoding transcription.

Figure 1

Mutation of the tV(UAC)D tRNA gene upstream of ATG31 results in readthrough of the intergenic SUT467 transcript. (A) Schematic of the ATG31–SES1 locus on S. cerevisiae chromosome IV. Colored arrows indicate known annotated transcripts: ATG31, black; SUT467, blue. The overlapping red arrow represents the extended readthrough transcript. (B) Northern blot analysis of ATG31 expression in wild-type and tdnaΔ strains reveals the extended transcript. The ATG31 coding sequence probe hybridized to RNA of ∼800 bp in wild-type strains (black arrow) and to RNA of ∼1200 bp after tDNA deletion (red arrow). The SUT467 probe hybridized to the predicted ∼300-bp transcript in wild-type cells (blue arrow) and to the same ∼1200-bp extended transcript in tdnaΔ strains. The normal ATG31 transcript was absent in tdnaΔ strains. Each pair of lanes contained total RNA from independent wild-type and mutant strains. (C) B-box deletion (B-box∆) or mutation of the invariant cytosine in the B-box (B-box mut) also resulted in extended readthrough transcription. Strains used were: (B) DDY4625 and DDY3 (wild-type); DDY4653 and 4624 (tdnaΔ); (C) DDY3 (wt); DDY4652 (tdnaΔ); DDY4769 (B-boxΔ); and DDY4925 (B-box mut).

Figure 2

Pol III transcription factors are required to block SUT467 readthrough transcription. (A) Northern analysis of temperature-sensitive mutants of the Pol III complex was performed as in Figure 1, except that each culture was shifted from 30° to 37° for 1 hr prior to RNA extraction. Extended ATG31 transcripts are most prominent in TFIIIB and TFIIIC subunit mutants, which also express an intermediate length ATG31 transcript. Strains used in lanes 1–8 were DDY3 (wt); DDY232 (rpc31-236); DDY246 (rpc162–112); DDY416 (brf1 II.9); DDY420 (brf1 II.6); DDY261 (tfc3 G349E); DDY3 (wt); and DDY4300 (tfc6 promoter mutant). (B) 5′-RACE analysis of extended and intermediate ATG31 transcripts. 5′-RACE was performed to map transcriptional start sites (TSS) for the various transcripts observed in the Northern blot analysis of the brf1 II.6 mutant. Colored solid boxes represent the range of alternative TSS, which were observed in three distinct clusters. The exact Saccharomyces Genome Database coordinates of all mapped TSS are given in Figure S1.

Figure 3

Binding of the TFIIIB complex is associated with blocking of SUT467 readthrough transcription. Strains were constructed to recruit the entire Pol III complex, TFIIIB and TFIIIC, or TFIIIC alone to the ATG31–SES1 intergenic region. Each construct was tested for the ability to block readthrough and for binding of Pol III transcription factor complexes to the ectopic locations. (A) Schematic of the modified ATG31 loci and Northern blot of each strain using the ATG31 probe. Lane 1, DDY4816 (tDNA flip); lane 2, DDY4817 (A-box mut); lane 3, DDY4819 (ETC4 replacement); lane 4, DDY4970 (ETC9 replacement); and lane 5, DDY4925 (B-box mut). Replacement of the tDNA by ETC4, or mutating the A-box or B-box, resulted in the presence of the extended transcript (red labels). However, inversion of the tDNA sequence or replacement with the ETC9 sequence still blocked readthrough (black labels). (B and C) Confirmation of expected Pol III transcription factor binding in the above mutants by chromatin immunoprecipitation. Each tDNA mutant strain was crossed to strains containing either BRF1–3X-FLAG or TFC1–3X-FLAG alleles, and then subjected to ChIP analysis using anti-FLAG antibody. (B) The absence of TFIIIB upstream of ATG31 in the A-box mutant, ETC4 replacement, and B-box mutant correlates with the presence of the extended transcript, suggesting that TFIIIB binding is required to block readthrough. Strains used (left to right) were DDY4935, -4943, -4949, -5003, and -4946. (C) ChIP analysis of BRF1–3XFLAG and TFC1–3XFLAG strains demonstrates that TFIIIC but not TFIIIB is bound in ETC4 replacement strains, indicating that TFIIIC binding alone cannot block readthrough transcription. Strains used (left to right) were DDY4938, -5003, -4949, -3860, -5006, and -4917.

Figure 4

Genetic factors involved in tDNA chromatin boundary function have minimal effects on blocking of Pol II progression through tV(UAC)D. (A) RNA from strains containing mutations that weaken tDNA boundary function were analyzed by Northern blotting using the ATG31 probe. Strains in lanes 1–9 were DDY3, -947, -1376, -2236, -2058, -2509, -1631, -1676, and -5010; lane 10, SG154.2; lanes 11–13, ROY1032, -1060, and -1063; lane 14, DDY4925. (B) RT–PCR analysis of readthrough transcription also shows only minimal effects.

Figure 5

Mutations in tV(UAC)D inhibit expression and function of Atg31 protein. (A) Western blot analysis was performed on wild-type and tDNA mutants containing an ATG31–9X-myc tag allele to determine whether the extended transcript affects translation of Atg31p. Compared to wild type, tDNA mutants showed significantly reduced expression of Atg31p. No other bands were observed on the Western blot, demonstrating that the extended transcript does not lead to production of an extended polypeptide. Strains used were DDY5012 (wt), DDY5014 (tdnaΔ), DDY5018 (B-boxΔ), and DDY5020 (B-box mut). Extracts were prepared from cells grown in YPD media. (B) Inhibition of autophagy induction in yeast expressing the extended transcript as measured by the Pho8Δ60 alkaline phosphatase assay. Strains used were: DDY5051 (wt control), DDY5072 (tdnaΔ), DDY5078 (B-boxΔ), DDY5081 (B-box mut), DDY5044 (atg8Δ), and DDY5046 (atg31Δ). (C) Production of the extended transcript is associated with reduced survival under nitrogen starvation conditions. Strains used were: DDY5012 (wt), DDY5081 (B-box mut), and DDY4764 (atg31Δ).