Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

Abstract

The nuclear exosome and the nonsense-mediated mRNA decay (NMD) pathways have been implicated in the degradation of distinct unspliced transcripts in Saccharomyces cerevisiae. In this study we show that these two systems can act sequentially on specific unspliced pre-mRNAs to limit their accumulation. Using steady-state and decay analyses, we show that while specific unspliced transcripts rely mostly on NMD or on the nuclear exosome for their degradation, some unspliced RNAs are stabilized only when both the nuclear exosome and NMD are inactivated. We found that the mechanism of degradation of these unspliced pre-mRNAs is not influenced by promoter identity. However, the specificity in the pre-mRNAs degradation pathways can be manipulated by changing the rate of export or retention of these mRNAs. For instance, reducing the nuclear export of pre-mRNAs mostly degraded by NMD results in a higher fraction of unspliced transcripts degraded by the nuclear exosome. Reciprocally, inactivating the Mlp retention factors results in a higher fraction of unspliced transcripts degraded by NMD for precursors normally targeted by the nuclear exosome. Overall, these results demonstrate that a functional redundancy exists between nuclear and cytoplasmic degradation pathways for unspliced pre-mRNAs, and suggest that the degradation routes of these species are mainly determined by the efficiency of their nuclear export rates. The presence of these two sequential degradation pathways for unspliced pre-mRNAs underscores the importance of limiting their accumulation and might serve as a fail-safe mechanism to prevent the expression of these nonfunctional RNAs.

FIGURE 1.

Steady-state analysis of unspliced transcripts in single and double mutants of RNA degradation pathways. (A) Analysis of RNMD-dependent transcripts. (B) Analysis of transcripts whose turnover depends mostly on one degradation pathway. Northern blots were probed with an intronic (i) riboprobe and either SCR1 or GAPDH was utilized as a loading control. Both the SCR1 and the GAPDH probes were generated via random priming.

FIGURE 2.

Decay analysis of unspliced transcripts and effect of promoter replacement on steady-state levels of unspliced transcripts. (A) Decay analysis. Each of the intron-containing ribosomal protein genes was placed under the control of the conditional galactose promoter ∼50 bp upstream of the translation start site, leading to the creation of five conditionally expressed ribosomal protein genes: GAL::RPP1B, GAL::RPS11A, GAL::RPL16A, GAL::RPS17B, GAL::RPS22B. Each of the knockouts (rrp6Δ, upf1Δ, rrp6Δ upf1Δ) was derived from a single isogenic wild-type strain. Each of the timepoints is indicated in minutes. SCR1 was utilized as a loading control. (B) Comparison of the levels of unspliced pre-mRNAs for intron-containing genes expressed under the control of their endogenous promoters or from the GAL promoter.

FIGURE 3.

Transposition of the RPL16A RNMD-sensitive intron is not sufficient to provide sensitivity of GOT1::RPL16A chimeric unspliced transcripts to both degradation systems. The native GOT1 intron was replaced by the RPL16A or the RPS17B introns and the wild-type, rrp6Δ, upf1Δ, or rrp6Δ upf1Δ strains were derived. A GOT1 exon 2 antisense RNA probe was used to assay for the spliced and chimeric unspliced transcripts. SCR1 or GAPDH was used as a loading control.

FIGURE 4.

RNA export inhibition can result in a higher fraction of unspliced transcripts targeted by the nuclear exosome. RPL22B and RPS17B (A) (or GAL::RPS17B; B) are NMD targets, and RPL18B is predominantly degraded by the nuclear exosome. Cells were grown at a permissive temperature (25°C) until they reached logarithmic phase and shifted to nonpermissive temperature (37°C) for ∼3 h. Northern blot analysis was performed with an intron-spanning antisense RNA probe to detect the accumulation of each of the unspliced transcripts. SCR1 was used as a loading control.

FIGURE 5.

Mlp1p and/or Mlp2p function in retaining the nuclear-degraded unspliced RPL18B transcripts, but not unspliced precursors that are cooperatively degraded by exosome/NMD or NMD. Northern analysis was performed on the RPL18B unspliced transcript (predominantly degraded by the nuclear exosome; A); or RPL22A and RPL16A (cooperatively degraded by exosome and NMD), or RPL22B (predominantly degraded by NMD) in B. SCR1 was used as a loading control.