Genome-Wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Posttranscriptional Control of Ribosome Biogenesis Factors

Abstract

Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability. mRNA turnover is an important factor in the regulation of gene expression in eukaryotic cells and complements transcriptional regulation by endowing the cell with the capability to rapidly vary the levels of existing transcripts (7,27). mRNA half-lives range from 3 min to more than 100 min in Saccharomyces cerevisiae (20,58) and from ϳ15 min to more than 10 h in mammals (27). The details of the eukaryotic mRNA decay pathway are best understood in S. cerevisiae, where many of the major proteins involved have been identified and characterized (53, 60). Deadenylation and decapping are the two most important steps in the mRNA degradation pathway and typically occur sequentially (10, 55, 60), although decapping is not absolutely dependent upon deadenylation (54). Deadenylation is carried out by the Pan2/Pan3 and the Ccr4/Caf1 poly(A) nuclease complexes (54), and decapping is carried out by the decapping enzymes Dcp1 and/or Dcp2, all of which are conserved among eukaryotes (33, 44). A deadenylated and decapped mRNA is degraded by the 5Ј33Ј exonuclease Rat1 and/or Xrn1 (22) and by a 3Ј35Ј exonucleolytic protein complex known as the exosome (26).Transcript stability is traditionally thought to be regulated by specific sequences or structures in the 3Ј or 5Ј untranslated region (UTR) and by cognate trans-acting factors that recognize, and in some cases may bind to, these elements (40). The identification of cis-and tran...

Ccr4 Promotes Resolution of the Endoplasmic Reticulum Stress Response during Host Temperature Adaptation in Cryptococcus neoformans

“…In the model yeast Saccharomyces cerevisiae, deletion of CCR4 results in stabilization of transcripts encoding distinct functional classes (ribosome biogenesis, translation initiation, and tRNA synthesis) in response to temperature stress (13). In mammalian cells, subsets of transcripts were destabilized in response to heat shock, and induction of endoplasmic reticulum (ER) stress by treatment with tunicamycin or potentiation of ER calcium release by thapsigargin treatment triggered destabilization of a subset of mRNAs in which are included several transcripts encoding ribosomal proteins (8).…”

Repeated elements coordinate the spatial organization of the yeast genome

“…For 3C and 4C analyses, strains were cultured to A 600 = 0.566 ± 0.07. Where required, growing S. cerevisiae 4741 cells (OD 600 = 0.6) were inhibited with phenanthroline (150 µg/ml) for 1 h. Phenanthroline causes changes to yeast transcription and mRNA stability, which mirror the rpb1-1-mediated inactivation of RNA polymerase II (POL-II) (Grigull et al, 2004).…”

Evaluating the Control of mRNA Decay in Fission Yeast

“…Much less is known about the utility of such methods in S. pombe, although some studies using phenanthroline have provided evidence for inhibition of transcription (Kim et al, 2000;Kim et al, 2002). The utility of such chemical inhibitors in yeasts is somewhat limited, since a wide range of "off-target" effects may be observed that include increases in the expression of certain genes (Grigull et al, 2004) and stimulation of the stress transcriptional response (Adams and Gross, 1991). In S. pombe, temperature sensitive mutants of RNA polymerase II have been described (Mitobe et al, 2001;Mitobe et al, 1999), but studies of mRNA decay in these mutant strains have not yet been published, to our knowledge.…”

“…The mechanisms through which mRNAs are degraded in yeasts have been reviewed (Amrani et al, 2006;Coller and Parker, 2004;Garneau et al, 2007). A genome wide scan of mRNA decay that used various methods of inhibiting transcription in S. cerevisiae, including temperature-sensitive mutants of yeast RNA polymerase II and chemical inhibitors of transcription such as phenanthroline and thiolutin, has been published (Grigull et al, 2004). Much less is known about the utility of such methods in S. pombe, although some studies using phenanthroline have provided evidence for inhibition of transcription (Kim et al, 2000;Kim et al, 2002).…”

“…We used a commercially available version of the nmt promoter-based expression vector (Invitrogen, Carlsbad, CA) that has a mutated TATA box, which reduces expression levels to ~15% of the unmodified nmt promoter (Basi et al, 1993); various other versions of the nmt vector, with different promoter strengths, are also available. The nmt expression system has been used to examine mRNA decay in S. pombe (Daga et al, 2003), and appeared to be an attractive alternative to other approaches for inhibiting transcription in yeast (Adams and Gross, 1991;Grigull et al, 2004;Herruer et al, 1988;Kim et al, 2002;Li et al, 1999) for many reasons, including the rapid nature of transcription repression and the ability to avoid stressful conditions that alter the levels of many mRNAs (Adams and Gross, 1991;Herruer et al, 1988;Li et al, 1999).…”