Defects in the NC2 repressor affect both canonical and non-coding RNA polymerase II transcription initiation in yeast

Abstract

Background: The formation of the pre-initiation complex in eukaryotic genes is a key step in transcription initiation. The TATA-binding protein (TBP) is a universal component of all pre-initiation complexes for all kinds of RNA polymerase II (RNA pol II) genes, including those with a TATA or a TATA-like element, both those that encode proteins and those that transcribe non-coding RNAs. Mot1 and the negative cofactor 2 (NC2) complex are regulators of TBP, and it has been shown that depletion of these factors in yeast leads to defects in the control of transcription initiation that alter cryptic transcription levels in selected yeast loci.

Results: In order to cast light on the molecular functions of NC2, we performed genome-wide studies in conditional mutants in yeast NC2 essential subunits Ydr1 and Bur6. Our analyses show a generally increased level of cryptic transcription in all kinds of genes upon depletion of NC2 subunits, and that each kind of gene (canonical or ncRNAs, TATA or TATA-like) shows some differences in the cryptic transcription pattern for each NC2 mutant.

Conclusions: We conclude that NC2 plays a general role in transcription initiation in RNA polymerase II genes that is related with its known TBP interchange function from free to promoter bound states. Therefore, loss of the NC2 function provokes increases in cryptic transcription throughout the yeast genome. Our results also suggest functional differences between NC2 subunits Ydr1 and Bur6.

Fig. 1

Dubious ORFs are up-regulated under Ydr1 depletion conditions. Histogram of the number ORFs classified as “dubious” by SGD, included in each group of 500 ORF, detected by yeast ORF macroarrays and ranked in decreasing order of expression level in the ydr1 mutant vs. the wild type

Fig. 2

NC2 shutoff provokes an increase in non-coding RNA transcripts. Metagene representation of the average transcription profiles for each analyzed gene class. Ten units correspond to 200 bp. In between TSS (transcription start site) and polyA (polyadenylation site), the entire gene lengths are normalized to 20 units (20–40 on the abscissa scale). The ordinates scale represents the ratio between the mutant and wild-type signals. a–b) Canonical ORFs show an increase in both the short sense transcripts in the 5′ and 3′ flanks and in antisense transcripts when blocking the transcription of both NC2 subunits. c–d) Only minor differences are seen between TATA and TATA-like genes when blocking NC2 transcripts. e–h) Cryptic mapped transcripts (SUTs and CUTs) show similar effects to those observed in canonical genes after shutting down NC2 subunits. a, c, e, g: pGAL-YDR1 shutoff; b, d, f, h: pGAL-BUR6 shutoff. i) Interpretation of the observed results. The cryptic transcripts (red arrows) over a scheme for a canonical yeast gene with its typical nucleosomal organization. Putative SRT (Ssu72-restricted transcripts) and RRT (Rco1-restricted transcripts) are marked. See the main text for a discussion

Fig. 3

Selected examples of verified ORF-genes showing cryptic transcription in their promoters. Screenshots from the tiling analysis software (TAS) are shown as example of the different cryptic transcripts detected after shutting down either BUR6 or YDR1. Intensity profiles on an arbitrary, but identical, scale for the mutants and wild type are shown. In (a, b and d), only the forward signal is shown, and both the forward and reverse intensity signals from the strand-specific microarray are shown in c. The SGD map for canonical genes is represented below and indicates the sense of transcription with an arrowhead. a) Cryptic transcription in the 5′ flank of the YOL039w/RRP2A gene in both NC2 mutants. b) Cryptic transcription in the 3′ flank of the YHR163w/SOL3 gene in both NC2 mutants. c) Cryptic transcription in the 5′ flank of the YDL133w/SRF1 gene in both NC2 mutants in both forward and reverse (antisense). d) Cryptic transcription in the 5′ flank of the YDR510w/SMT3 gene in both NC2 mutants. In this case the cryptic transcript could be detected with a probe from the YDR509w dubious ORF as in the macroarray experiment described in Fig. 1 and in the text

Fig. 4

Short transcripts are detected upstream the YOR184W/SER1 and YOL039W/RPP2A genes in conditions of Ydr1 depletion. Yeast strains FY86 (+Ydr1) and P _GAL1-YDR1 (−Ydr1) were grown in YPGal in the early exponential phase and then transferred to YPD and incubated for 4 h. a) Total RNA was prepared and analyzed by Northern blot using probes corresponding to the ORF or the 5′ upstream region. Ethidium bromide staining of ribosomal RNA (rRNA) was used as a loading control for the total amount of RNA. b) A scheme of the probes used is shown