Transcript selection and the recruitment of mRNA decay factors for NMD in Saccharomyces cerevisiae

RNA (Impact Factor: 4.94). 10/2005; 11(9):1333-9. DOI: 10.1261/rna.2113605
Source: PubMed


In Saccharomyces cerevisiae, nonsense-mediated mRNA decay (NMD) requires Upf1p, Upf2p, and Upf3p to accelerate the decay rate of two unique classes of transcripts: (1) nonsense mRNAs that arise through errors in gene expression, and (2) naturally occurring transcripts that lack coding errors but have built-in features that target them for accelerated decay (error-free mRNAs). NMD can trigger decay during any round of translation and can target Cbc-bound or eIF-4E-bound transcripts. Extremely low concentrations of the Upf proteins relative to the total pool of transcripts make it difficult to understand how nonsense transcripts are selectively recruited. To stimulate debate, we propose two alternative mechanisms for selecting nonsense transcripts for NMD and for assembling components of the surveillance complex, one for the first (pioneer) round of translation, called "nuclear marking," and the other for subsequent rounds, called "reverse assembly." The model is designed to accommodate (1) the low abundance of NMD factors, (2) the role of nucleocytoplasmic shuttling proteins in NMD, (3) the independent and nonobligate order of assembly of two different subcomplexes of NMD factors, and (4) the ability of NMD to simultaneously reduce or eliminate the synthesis of truncated proteins produced by nonsense transcripts while down-regulating but not completely eliminating functional proteins produced from error-free NMD-sensitive transcripts

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    • "Comparison of Upf1 sequences from eukaryotes shows that the SQ domain is much less conserved than the CH domain (31,32). The SQ domain is notably absent in lower eukaryotic organisms such as yeasts. "
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    ABSTRACT: The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.
    Nucleic Acids Research 12/2012; 41(4). DOI:10.1093/nar/gks1320 · 9.11 Impact Factor
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    • "Changes in the third position were slightly less tolerated than changes in the first position. The same experiment done in a yeast strain (upf1D) deficient for nonsense-mediated decay (nmd) (Culbertson and Neeno- Eckwall 2005) produced the same results (Fig. 3), indicating that these effects are not due to nonsense-mediated decay of the mRNAs lacking normal start codons. "
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    ABSTRACT: Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA(i)*eIF2*GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S*mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43S*mRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.
    RNA 12/2008; 15(1):138-52. DOI:10.1261/rna.1318509 · 4.94 Impact Factor
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    • "Furthermore, even in mammalia, at least in some cases, NMD depends on 3 0 UTR length (Buhler et al., 2006) or on the nature of the 3 0 UTR mRNP (Weil and Beemon, 2006). It has even been suggested that the scenario in mammals might be similar if not identical to that in yeast and that the influence of EJCs on NMD lies simply in the fact that they enhance the translation rate and thus simultaneously the turnover rate of nonsense-mutated mRNAs (Culbertson and Neeno-Eckwall, 2005; Ford et al., 2006; Hilleren and Parker, 1999). However, even though EJCs appear to promote initial polysome formation and translation (Nott et al., 2004), many nonsense-mutated mRNAs would have sufficient EJCs in their ORFs 5 0 to the PTC to fulfill that requirement ; nevertheless, at least one EJC 3 0 to the PTC is necessary for its recognition by the NMD machinery. "
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    ABSTRACT: Gene expression is a highly specific and regulated multilayer process with a plethora of interconnections as well as safeguard and feedback mechanisms. Messenger RNA, long neglected as a mere subcarrier of genetic information, is more recently recognized as a linchpin of regulation and control of gene expression. Moreover, the awareness of not only proteins but also mRNA as a modulator of genetic disorders has vastly increased in recent years. Nonsense-mediated mRNA decay (NMD) is a posttranscriptional surveillance mechanism that uses an intricate network of nuclear and cytoplasmic processes to eliminate mRNAs, containing premature termination codons. It thus helps limit the synthesis of potentially harmful truncated proteins. However, recent results suggest functions of NMD that go far beyond this role and affect the expression of wild-type genes and the modulation of whole pathways. In both respects--the elimination of faulty transcripts and the regulation of error-free mRNAs--NMD has many medical implications. Therefore, it has earned increasing interest from researchers of all fields of the life sciences. In the following text, we (1) present current knowledge about the NMD mechanism and its targets, (2) define its relevance in the regulation of important biochemical pathways, (3) explore its medical significance and the prospects of therapeutic interventions, and (4) discuss additional functions of NMD effectors, some of which may be networked to NMD. The main focus of this chapter lies on mammalian NMD and resorts to the features and factors of NMD in other organisms if these help to complete or illuminate the picture.
    Advances in genetics 02/2008; 62:185-243. DOI:10.1016/S0065-2660(08)00604-4 · 6.76 Impact Factor
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