RNA surveillance by uridylation-dependent RNA decay inSchizosaccharomyces pombe

Abstract

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3′-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe , Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5′-N x AUUAAAA-3′ RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.

Gld2 activity is regulated by phosphorylation in the N-terminal domain

“…In comparison, a mutation in the active site, D215A, completely abolished enzyme activity on miRNA substrates [4,18]. D215A is part of the conserved catalytic triad responsible for activity [3,38,39], while S116 is found in the disordered N-terminal domain, which was previously shown to be dispensable for catalytic activity in the related uridylyltransferase Cid1 [40][41][42], but we here show that its function lies in the regulation of enzyme activity. Using enzymatic assays and mass spectrometry, we identified and validated Akt1 and PKA as kinases with site-specific phosphorylation activity at S116 in Gld2.…”

Regulation of RNA stability at the 3′ end

“…Further, 3′ oligouridylation signals for recruitment of the 3′-5′ exoribonuclease Dis3L2 (DIS3-like exoribonuclease 2) to the RNA, followed by degradation as part of uridylation dependent decay ( Figure 1B). However, deletion of Dis3L2 alone does not significantly increase the frequency of uridylated transcripts (Chung et al 2019b;Malecki et al 2013), implicating redundancy of this pathway, potentially through minor contributions of other nucleases such as Eri1 (3′-5′ exoribonuclease 1), which has been associated with miRNA homeostasis (Thomas et al 2012).…”

“…Improved RNA-sequencing technologies have recently identified alternate mRNA degradation pathways, diverging from canonical deadenylation dependent decay. 3′-tail sequencing techniques have identified mRNA tails consisting of not only adenosine residues, but combinations of untemplated uridines, cytosines, and guanosines ( Figure 1) (Chung et al 2019b;Chung et al 2017). The most common of theseaside from canonical poly(A) tailsis poly(U) tailing (Lim et al 2018).…”

“…In this recently discovered pathway, untemplated uridine residues are interpreted as an RNA degradation signal by the cell (Figure 1B). While a number of TENTs have been identified in humans, this activity is most commonly attributed to the terminal uridylyltransferase TENT3A (TUT4, ZCCHC11, PAPD3) and its homolog TENT3B (TUT7, ZCCHC6) (Lim et al 2018), along with the TENT3A homolog Cid1 (caffeine induced death protein 1) in Schizosaccharomyces pombe (Chung et al 2019b). Following uridylation, the mRNA is decapped by the Lsm1-7 decapping complex and 5′-3′ degradation is initiated by the exosome (Rissland and Norbury 2009).…”

“…Mammalian histone mRNAs are oligouridylated at their 3′ ends, presumably by TENT3A, and subsequently degraded by Eri1 or Dis3L2 (Hoefig and Heissmeyer 2014). Although there is some disagreement over which enzyme is responsible, uridylation of these transcripts is a clear signal for their degradation, as uridylation has been linked to transcript abundance (Chang et al 2014;Chung et al 2019b). Further, studies using mammalian and vertebrate models have identified a conserved role for TENT3A/B in uridylation-dependent degradation of maternal mRNAs during early zygotic development (Chang et al 2018;Morgan et al 2017).…”