Cytoplasmic decay of intergenic transcripts in Saccharomyces cerevisiae

Abstract

Eukaryotes produce a number of noncoding transcripts from intergenic regions. In Saccharomyces cerevisiae, such cryptic unstable transcripts (CUTs) are thought to be degraded in the nucleus by a process involving polyadenylation and 3'-to-5' degradation by the nuclear exosome. In this work, we examine the degradation pathway of the RNA SRG1, which is produced from an intergenic region and contributes to the regulation of the SER3 gene by promoter occlusion during SRG1 transcription. Although there is some effect on SRG1 transcript levels when the nuclear exosome is compromised, the bulk of the SRG1 RNA is degraded in the cytoplasm by decapping and 5'-to-3' exonucleolytic digestion. Examination of other CUTs suggests that individual CUTs can be degraded by a variety of different mechanisms, including nuclear decay, cytoplasmic decapping and 5'-to-3' decay, and nonsense-mediated decay. Moreover, some CUTs appear to be associated with polyribosomes. These results indicate that some CUTs can be exported from the nucleus and enter translation before being degraded, identifying a potential mechanism for the evolution of new protein-encoding genes.

FIG. 1.

Three forms of SRG1 RNA. A. Agarose Northern blot showing that three transcripts are produced from the SRG1 locus. B. RNase H-directed cleavage of SRG1 using oligo(dT). The probe used and the expected sizes of cleavage products following treatment with oligo(dT) are shown in panel C. L, SRG1-l; S, SRG1-s. Positions of the size markers (M) are indicated in nucleotides. This experiment was repeated three times; a representative blot is shown. C. Schematic of SRG1 transcripts, their approximate sizes, and their 3′ ends (black octagons) relative to SER3. The SER3 promoter is represented by the gray arrow. The schematic is not to scale.

FIG. 2.

Accumulation of SRG1 transcripts in mutant strains. A. Accumulation of SRG1 RNAs in various mutant strains at steady state. The short (S), long (L), and read-through (RT) forms of SRG1 are indicated. 7s is used as a loading control. At least three independent samples per mutant strain were examined; a representative blot is shown. B. Accumulation of SRG1-l and SRG1-s RNAs in various mutant strains. Bars represent the averages of at least three independent experiments, normalized to the 7s loading control. C. RNase H-directed cleavage with (+) or without (−) oligo(dT) of SRG1 RNA in indicated strains. 7s is used as a loading control. Positions of the size markers (M) are indicated in nucleotides. “S” denotes SRG1-s following cleavage; “C” indicates a constant band which does not change in size. This experiment was repeated three times; a representative blot is shown. D. Accumulation of SRG1-rt RNA in various mutant strains. Bars represent the averages of at least three independent experiments, normalized to the 7s loading control.

FIG. 3.

Decay of SRG1 transcripts in mutant strains. A to E. Representative Northern blots showing SRG1 RNA levels in indicated strains following transcriptional shutoff. 7s is used as a loading control. Half-lives indicated are averages calculated from at least three experiments. The half-lives of SRG1-l and SRG1-s (relative to one another) did not significantly differ in any mutant strain tested. F. SRG1-l and SRG1-s decay curves, semilog plot. Curves are calculated from normalized values of at least three experiments per strain. Error bars represent 1 standard deviation from the mean.

FIG. 4.

Microarray predictions of CUT accumulation. The pie chart shows results for 207 CUTs that showed increased accumulation compared to wild type in the indicated decay mutant strains or strain combinations by microarray analysis.

FIG. 5.

Accumulation and decay of CUTs in the rrp6Δ or xrn1Δ mutant strain. A. Representative Northern blots showing NEL025c RNA levels in rpb1-1 (wild-type), rrp6Δ rpb1-1, and xrn1Δ rpb1-1 strains following transcriptional shutoff. 7s is used as a loading control. Decay in xrn1Δ rpb1-1 was not determined precisely, due to a low signal-to-noise ratio. B. The experiment was done as described for panel A but with a probe for NMR026w. Equal exposures of blot sets are shown to facilitate comparison of relative accumulation. Three independent samples from each mutant strain were analyzed; representative examples are shown.

FIG. 6.

Polysome analysis of the CUT NMR026w. A. Polysome profile of wild-type cells, with Northern blot of RNA isolated from indicated fractions, probed for NMR026w and SRG1. Twenty-eight percent of NMR026w transcript is present in fractions 1 to 5. B. Polysome profile of wild-type cells treated with 1 M KCl for 30 min to disrupt polysome formation, with Northern blot of RNA isolated from indicated fractions, probed for NMR026w. Fifty-one percent of NMR026w is present in fractions 1 to 5.

FIG. 7.

Model for new ORF evolution from intergenic transcripts. Shown is the potential mechanism for the evolution of new protein-encoding ORFs from intergenic transcripts. The process begins with adventitious transcription, followed by gain of a proper 3′ end to allow for nuclear export, evolution of a start codon and engagement of the translation machinery, and natural selection of a desirable polypeptide. IT, intergenic transcript.