Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae

Abstract

The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3′ to 5′ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5′ monophosphate ends on mRNAs in wild-type and dcp2Δ xrn1Δ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5′ monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5′ exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.D egradation of mRNA plays a crucial role in the control and fidelity of gene expression. In eukaryotes, the general mRNA decay pathway initiates with shortening of the 3′ poly(A) tail, followed by 3′ to 5′ exonucleolytic degradation and/or removal of the 5′ 7-methylguanosine cap by the Dcp2 decapping enzyme allowing degradation by the Xrn1 5′ to 3′ exonuclease (1, 2).In some organisms, the decay of specific mRNAs can be initiated by endonucleolytic cleavage (3, 4). In plants, mRNA decay mediated by small interfering (si)RNAs and micro-RNAs (miRNAs) often occurs via endonucleolytic cleavage (5), leading to 5′ to 3′ decay by the Xrn1 homolog XRN4 (6, 7). Moreover, in mammalian cells miRNA dependent and independent endonuclease cleavage sites in mRNAs have been identified (8, 9). Endonucleolytic cleavage also initiates mRNA decay in quality control mechanisms, such as nonsense-mediated decay (NMD) in metazoans and no-go decay (NGD) in yeast (10-13). In both NGD and NMD pathways, the 3′ endonuclease cleavage product with a 5′ monophosphate end is rapidly degraded by Xrn1. Endonucleases can also function in cytoplasmic RNA processing events. For example, during the unfolded protein response (UPR), the IRE1 endonucleolytically cleaves XBP1 mRNA (a metazoan homolog of HAC1 in S. cerevisiae) to cause its unconventional splicing and production of the UPR-specific transcription factor encoded by the mRNA (14).In this work, we set out to determine the contribution of endonucleases to mRNA degradation in Saccharomyces cerevisiae and to determine whether any other processes contribute to 5′ to 3′ degradation of mRNAs. Although our analysis revealed few endonucleolytic cleavage events at appreciable levels, we identified debranched lariat intermediates arising from endogenous intron-containing genes that were subject to degradation by Xrn1. This observation identifies a discard pathway for natural pre-mRNA splicing substrates and ra...

Spliceosome-mediated decay (SMD) regulates expression of nonintronic genes in budding yeast

“…Sequencing of the spliced product revealed that the 59ss reported here and a more downstream 39ss at nucleotide +1672 were used ( Fig. 4A; Harigaya and Parker 2012). A BP consensus located 12 nucleotides (nt) upstream of this 39ss was proposed; however, usage of this element was not experimentally validated.…”

“…However, the biological relevance of this splicing event had not been addressed (Harigaya and Parker 2012). Sequencing of the spliced product revealed that the 59ss reported here and a more downstream 39ss at nucleotide +1672 were used ( Fig.…”

“…Surprisingly, these studies revealed that spliceosomal components are also recruited to some genes whose transcripts are not known to be spliced. It was recently shown that several nonintronic transcripts can be spliced; however, it remained unclear whether this had any regulatory role or was the result of stochastic splicing events that occur due to the lack of specificity in the spliceosome recruitment (Harigaya and Parker 2012).…”

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

“…Specific PARE has been developed for the study of plant miRNA processing by specifically amplifying miRNA processing intermediates (Bologna et al, 2013). Although these approaches have been used to profile mRNA degradation intermediates in mutants impaired in XRNs or proteins with endonucleolytic activity (German et al, 2008;Harigaya and Parker, 2012;Schmidt et al, 2015), the interpretations of some results remain challenging because the complexity of the RNA degradome is currently still largely uncharacterized.…”

Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo

“…Cap substitution by lariat capping is presumably a stabilizing modification of mRNA because decapping followed by 5 ′ -3 ′ decay is the predominant cytoplasmic degradation pathway and because much of the 3 ′ -5 ′ degradation is dependent on translation competence and thus also indirectly involves the m 7 G cap (Harigaya and Parker 2012). In addition, the lariat cap is resistant in vitro to yeast debranching enzyme (Nielsen et al 2005) and the Xrn1p 5 ′ -3 ′ exonuclease ( Fig.…”