Identification of a quality-control mechanism for mRNA 5′-end capping

Jiao, Xuan; Xiang, Song; Oh, Chan-Seok; Martin, Charles E.; Tong, Liang; Kiledjian, Megerditch

doi:10.1038/nature09338

Cited by 155 publications

(218 citation statements)

References 36 publications

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“…29 Thus, its implication in this alternative nuclear mRNP QC is conceivable. In contrast, our data indicate clearly that Rai1p, the Rat1p cofactor specialized in removing unmethylated caps, 23,24 is not involved in the production of uncapped HXK1 mRNPs since no transcript recovery could be observed in the rai1D strain (Fig. 5A).…”

Section: Discussioncontrasting

confidence: 52%

“…This possibility was explored first by performing RT-qPCR measurements with RNAs extracted from cells lacking the catalytic subunit of the decapping enzyme (dcp2D) or lacking Rai1p (rai1D), a known cofactor of Rat1p that possesses also a decapping endonuclease activity specifically toward unmethylated caps. 24 As shown in Fig. 5A, the removal of Dcp2p leads to a substantial rescue of the HXK1 transcripts under Rho inducing conditions (mRNA level around 60% in dcp2D as compared to the 30% level for the wild-type strain), whereas the absence of Rai1p did not lead to any rescue effect.…”

Section: Rho-induced Aberrant Hxk1 Mrnps Are Decapped Prior To Degradmentioning

confidence: 95%

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Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

2016

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The co-transcriptional biogenesis of export-competent messenger ribonucleoprotein particles (mRNPs) in yeast is under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from inappropriate or inefficient processing and packaging reactions are detected by the QC system and retained in the nucleus with ensuing elimination of their mRNA component by a mechanism that requires the catalytic activity of Rrp6p, a 3 0 -5 0 exonuclease associated with the RNA exosome. In previous studies, we implemented a new experimental approach in which the production of aberrant mRNPs is massively increased upon perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor expressed in S. cerevisiae. The analyses of a subset of transcripts such as PMA1 led us to substantiate the essential role of Rrp6p in the nuclear mRNP QC and to reveal a functional coordination of the process by Nrd1p. Here, we extended those results by showing that, in contrast to PMA1, Rho-induced aberrant HXK1 mRNPs are targeted for destruction by an Nrd1p-and Rrp6p-independent alternative QC pathway that relies on the 5 0 -3 0 exonuclease activity of Rat1p. We show that the degradation of aberrant HXK1 mRNPs by Rat1p occurs cotranscriptionally following decapping by Dcp2p and leads to premature transcription termination. We discuss the possibility that this alternative QC pathway might be linked to the well-known specific features of the HXK1 gene transcription such as its localization at the nuclear periphery and gene loop formation.

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Section: Discussioncontrasting

confidence: 52%

Section: Rho-induced Aberrant Hxk1 Mrnps Are Decapped Prior To Degradmentioning

confidence: 95%

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

2016

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“…S3B), we used a dcp2Δ xrn1Δ rai1Δ triple mutant expressing a Rai1 mutant with amino acid substitution E221A D223A, which corresponds to the E199A D201A mutation in Schizosaccharomyces pombe Rai1 that was shown previously to inactivate the catalytic activity (Fig. S3A) (26). We observed that the 5′ RLM-RACE signals from ERG13, CWP2, and RPS31 were still detectable in a dcp2Δ xrn1Δ rai1EADA mutant (Fig.…”

Section: ′ P Ends Located In Intronsmentioning

confidence: 99%

“…Previous work has identified the Rai1 protein as an enzyme that can function to remove the cap structure from mRNA in S. cerevisiae (25,26). In contrast to the Dcp1/Dcp2 complex that preferentially functions on a cap with an N7 methyl moiety and release m 7 Gpp, Rai1 preferentially targets mRNAs with unmethylated caps and releases the entire cap structure GpppN, although it can function on a methylated cap to release m 7 GpppN to a lesser extent (26). Moreover, Rai1 also possesses the activity to hydrolyze the 5′ triphosphate of an uncapped RNA to release diphosphate and a monophosphorylated 5′-end RNA (25).…”

Section: ′ P Ends Located In Intronsmentioning

confidence: 99%

Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae

Harigaya

Parker

2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Cytoplasmic Xrn1 and nuclear Rat1 belong to a large family of conserved 5′-3′ exoribonucleases (33). Interestingly, Rat1 also forms a complex with a pyrophosphatase, Rai1, that was recently shown to cleave unmethylated capped RNA (34). Rai1 helps the exoribonuclease activity by stabilizing Rat1, and the catalytic activity of Rai1 is also dispensable for the activation of Rat1.…”

Section: Table 1 Dcs Family Proteins and Activation Of Exoribonucleamentioning

confidence: 99%

Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast

Sinturel

Bréchemier-Baey

Kiledjian

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The scavenger decapping enzyme Dcs1 has been shown to facilitate the activity of the cytoplasmic 5′-3′ exoribonuclease Xrn1 in eukaryotes. Dcs1 has also been shown to be required for growth in glycerol medium. We therefore wondered whether the capacity to activate RNA degradation could account for its requirement for growth on this carbon source. Indeed, a catalytic mutant of Xrn1 is also unable to grow in glycerol medium, and removal of the nuclear localization signal of Rat1, the nuclear homolog of Xrn1, restores glycerol growth. A cytoplasmic 5′-3′ exoribonuclease activity is therefore essential for yeast growth on glycerol, suggesting that Xrn1 activation by Dcs1 is physiologically important. In fact, Xrn1 is essentially inactive in the absence of Dcs1 in vivo. We analyzed the role of Dcs1 in the control of exoribonuclease activity in vitro and propose that Dcs1 is a specific cofactor of Xrn1. Dcs1 does not stimulate the activity of other 5′-3′ exoribonucleases, such as Rat1, in vitro. We demonstrate that Dcs1 improves the apparent affinity of Xrn1 for RNA and that Xrn1 and Dcs1 can form a complex in vitro. We examined the biological significance of this regulation by performing 2D protein gel analysis. We observed that a set of proteins showing decreased levels in a DCS deletion strain, some essential for respiration, are also systematically decreased in an XRN1 deletion mutant. Therefore, we propose that the activation of Xrn1 by Dcs1 is important for respiration.RNA turnover | ribonuclease | post-transcriptional control | porin T urnover of messenger RNA (mRNA) is a regulated process and a key step in the control of gene expression (1). In eukaryotic cells, most cytoplasmic mRNAs are degraded through two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Subsequently, the cap (5′-m 7 GpppN) structure is removed by the decapping complex Dcp1/Dcp2, and the mRNA is degraded in the 5′ to 3′ direction by the major cytoplasmic enzyme Xrn1. Alternatively, deadenylated mRNAs can be degraded from their 3′-ends by the exosome, a multimeric complex possessing 3′ to 5′ exoribonuclease activity (2). The cap structure resulting from 3′-end decay is hydrolyzed by conserved scavenger decapping enzyme Dcs1 (3). Dcs1 is also necessary for the 5′ to 3′ exonucleolytic activity of Xrn1, a requirement that functionally links these two alternative degradation pathways (4). The 5′-3′ exoribonuclease Xrn1 is highly conserved in eukaryotes and has been extensively described for its role in the degradation of cytoplasmic mRNAs (5, 6). Xrn1 also participates in the degradation of nonfunctional mRNAs (7) and noncoding RNAs (8,9).In addition to causing direct defects in RNA turnover, it has been known for a long time that a deletion of XRN1 is detrimental to other cellular functions. XRN1 mutants exhibit pleiotropic phenotypes, including slow growth, loss of viability upon nitrogen starvation, meiotic arrest, defective sporulation, defects in microtubule-related processes, telomere sho...

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Identification of a quality-control mechanism for mRNA 5′-end capping

Cited by 155 publications

References 36 publications

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

Co-transcriptional degradation by the 5′-3′ exonuclease Rat1p mediates quality control of HXK1 mRNP biogenesis in S. cerevisiae

Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae

Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast

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