20091028

Wilson et al. BBA-Review mRNA surveillance pathways

Pathways of normal mRNA degradation

2 major cytoplasmic degradation pathways for normal mRNAs. All of the enzymes required for these two pathways are conserved in other eukaryotes, suggesting that the pathways of mRNA decay are also conserved. Messenger RNA degradation is initiated by gradual removal of the poly(A) tail, a process which is normally carried out by the 3′ exonuclease Ccr4, but also by Pan2, at a slower rate. In the absence of both Ccr4 and Pan2, poly(A) tails are stable, suggesting that there are no other enzymes that can substitute for this function. Removal of the poly(A) tail triggers two mRNA degradation pathways. In the first pathway, the 5′ cap is removed by the Dcp1/Dcp2 complex. Decapping of the mRNa triggers degradation of the transcript from the 5′ end by Xrn1, a 5′ exoribonuclease. In the second pathway, the body of the transcript is degraded from the 3′ end by a multi-subunit 3′ exoribonuclease termed the exosome. The exosome has both nuclear and cytoplasmic functions, both requiring additional factors. For example, Ski2, Ski3, Ski7 and Ski8 are required for all cytoplasmic exosome functions.



NSD-Nonstop mRNA decay

which selectively degrades transcripts that lack all in-frame termination codons. The translation ribosome translates to the end of the poly(A) tail of a nonstop mRNA and stalls. The stalled ribosome is hypothesized to be recognized by Ski7, possibly because of the absence of a codon in the A-site of the ribosome. Recognition by Ski7 recruits the exosome to the nonstop mRNA, resulting in degradation of the transcript.

Several lines of evidence support the current model. First, it is likely that the translating ribosome stalls at the end of  a nonstop transcript. Nonstop mRNAs remain physically associated with ribosomes when used in in vitro translation reactions, while ribosomes dissocieate from mRNAs that contain a stop codon. Stalled ribosome-mRNA complexes have been useful tools in studying the sorting of nascent protiens, and are surprisingly stable, as they can be purified by sucrose gradient centrifugation or gel filtration. Thus, unlike DNA or RNA polymerases, ribosomes do not simply dissociate when they reach the end of a temlate, which implies that a specific facgtor (perhaps Ski7) is necessary for disassembly of the ribosome. Similarly, the bacterial ribosome needs trans-acting factors to dissociate when it reaches the end of a nonstop mRNA (i.e. tmRNA and SmpB)

Second, nonstop mRNA decay requires active translation. Nonstop mRNAs are stabilized in wild type yeast cells treated with a translational inhibitor, and in mutant cells depleted of charged tRNAs. More importantly, translation of the nonstop mRNA itself is needed: nonstop mRNA decay is prevented when a stop codon is inserted close to the poly(A) tail, and when a stable structure in the 5’UTR prevents its translation.

Third, a nonstop PGK1pG reporter mRNA was stabilized in yeast strains lacking cytoplasmic exosome function, suggesting that the exosome degrades nonstop mRNA. In contrast, mutations inactivating the decapping enzyme or the 5′ to 3′ exoribonuclease, xrn1p, have large effects on the degradation of normal mRNAs, but do not detectably affect the stability of the nonstop PGK1pG mRNA.

Fourth, based on sequence similarity, Ski7 is a likely candidate for recognizing the stalled ribosome at the end of a nonstop mRNA. The C-terminal domain of Ski7 is homologous to translation factors eEF1A and eRF3. Because eEF1A interacts with the ribosome when the A-site contains a sense codon and eRF3 interacts with the ribosome when the A-site contains a stop codon, it is proposed that the homologous domain of Ski7 interacts with the ribosome when the A-site is empty. This hypothesis is supported by the observation that deletion of the C-terminal domain of Ski7 inactivates the nonstop mRNA decay pathway without affecting other exosome functions.

Fifth, the N-terminal domain of Ski7 inteacts with the exosome, and with additional exosome cofactors. This domain is required for both nonstop mRNA deacay and other cytoplasmic exosome functions. Importantly, a point mutation in the exosome that disrupts its interaction with Ski7 also blocks nonstop mRNA decay, confirming that the interaction between Ski7 and the exosome is functioally important.

The exosome degrades the poly(A) tail on nonstop mRNAs is surprising since the exosome is incapable of degrading the poly(A) tail on normal mRNAs, as shown by a complete absence of deadenylation in a ccr4Δpan2Δ double mutant. The dependence on translation and the independence of a distinct deadenylation phase appear to be conserved aspects of nonstop mRNA decay.

2 questions à Résoudre:

1. Whether the translation rate of a nonstop mRNA is the same as for a control mRNA?

2. The fate of the protein produced from a nonstop mRNA is also unclear.

Reports from 2 groups indicate that nonstop proteins may be targeted for proteolysis by the proteasome. Inhibitors of the proteasome increased the levels of the protein encoded by nonstop mRNAs, and a screen to identify trans-acting factors in nonstop mRNA decay uncovered three mutations that inactivate the proteasome. Interestingly, the effects of the proteasome and Ski7 are additive, suggesting that recognition of the stalled ribosome by Ski7 is not required for the rapid degradation of the encoded protein.

Premature polyadenylation generates nonstop mRNAs

1.2% of random yeast cDNA clones and 0.7% of human cDNAs were prematurely polyadenylated. Premature polyadenylation can also serve as a method of gene-specific regulation. In S. cerevisiae, 0.8% of all open reading frames contain a cryptic plyadenylation site upstream of the normal termination codon. Cryptic sites could be favored over the normal poly(A) site under various conditions to down-regulate the expression of the normal mRNA.This implies that premature polyadenylation, coupled with nonstop mRNA decay, could serve to down-regulated many genes.

NGD-No Go mRNA decay

Pseudoknots, rare codons, or stem-loops within an open reading frame cause the translating ribosome to stall. Transcripts with stalled ribosomes are degraded by the no-go mRNA surveillance pathways. A stalled ribosome within the coding region of the no-go transcript is thought to recruit Dom34 and Hbs1. Dom34 and Hbs1 are homologous to eRF1 and eRF3, respectively, suggesting that they may function to recognize stalled ribosome. Interaction of Dom34 and Hbs1 with the stalled riboosme is thought to trigger endonculeolytic cleavage of the no-go mRNA. The resulting 5″ cleavage product is degraded by the cytoplasmic exosome, while the 3′ cleavage product is degraded by the 5′ exoribonuclease, Xnr1. Consistent with this model, an xrn1Δ strain accumulates the expected 3′ cleavage product, but deleting DOM34 from this strain prevents this accumulation. Likewise, a strin lacking cytoplasmic exosome activity accumulates the expected 5′ cleavage product, and deletion DOM34 from this strin prevents this accumulation. Similarly, mutants in HBS1 show drastically reduced levels of no-go mRNA decay intermediates, but hbs1Δ is not as effective as dom34Δ, implying that Hbs1 may not play as great a role as Dom34 in this pathway. Consistent with this observation, recent structrural work suggests that Dom34 is responsbile for endonucleolytic cleavage of no-go mRNA.

Ribosomes can also stalled because of aberrancies within the ribosome. Mutations that are prefentially degraded through the nonfunctional ribosomes that are preferentially degraded through the nonfunctional ribosome decay pathway (NRD). Thus, in the cas of no-go decay, a stalled ribosome leads to mRNA decay, while in NRD, a stalled ribosome leads to rRNA decay. Yet in other cases of ribosomal stalling, yeast mRNAs are stabilized ( e.g. treatment with cycloheximide or depletion of charged tRNA in a cca1-1 stain). Thus, an important question yet to be addressed is how the cellular machinery distinguishes between whether the mRNA or rRNA in a stalled complex is defective, or whether in both cases both mRNA and rRNA are degraded.

An interesting aspect of the relationship between nonstop and no-go decay is that Ski7 and Hbs1 are a pair of duplicated genes in yeast, whereas most other eukaryotes have only one homolog. The single Ski7/Hbs1 homolog from the related yeast Saccharomyces kluyveri could complement the growth phenotypes of an HBS1 mutant and a SKI7 mutant in S. cerevisiae. In contrast, the human homolog of Hbs1/Ski7 is unable to complement either an HBS1 mutant or a SKI7 mutant in S. c. the most likely explanation is that human eRFS is nonfunctional when expressed in yeast, leaving the question whether eRFS can function in nonstop and/or no-go decay unanswered. The evolutionary history of Hbs1p and Ski7 suggests the possibility taht in most eukaryotes, the single Ski7/Hbs1 protein recognizes stalled ribosomes within the coding region and at the end of an mRNA. This also suggests taht in other eukaryotes, nonstop and no-go mRNA decay may be a mixture of endonucleolytic decay and exosome-mediated decay.