Article

Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies

Department of Molecular and Cellular Biology, University of Arizona, Tucson, 85721, USA.
Cell (Impact Factor: 33.12). 07/2006; 125(6):1095-109. DOI: 10.1016/j.cell.2006.04.037
Source: PubMed

ABSTRACT In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.

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    • "This allows the visualization of the mRNA via coexpression of the MS2 coat protein fused to three green fluorescent proteins (CP–GFP 3 ). Such systems have been used extensively to examine the localization of mRNAs in a wide range of biological systems (Haim et al., 2007; Hamada et al., 2003; Sheth and Parker, 2006). Key advantages of this yeast system are that the control elements associated with mRNA transcription and processing [promoters, UTRs, poly(A) sites and terminators] remain intact, as the MS2-binding sites are inserted directly and precisely into the 39UTR of the endogenous gene at its chromosomal locus (Haim et al., 2007). "
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    ABSTRACT: The relocalization of translationally repressed mRNAs to mRNA Processing bodies (P-bodies) is a key consequence of cellular stress across many systems. P-bodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to P-bodies is poorly understood. We used the MS2-GFP system to follow the movement of specific endogenous mRNAs in live yeast cells after nutritional stress. It appears that the relocalization of mRNA to P-bodies after stress is bi-phasic: some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a late phase localizing P-body protein that is important for the delayed entry of the specific tested mRNAs to P-bodies. Therefore, for the mRNAs tested, relocalization to P-bodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.
    Journal of Cell Science 01/2014; 127(6). DOI:10.1242/jcs.139055 · 5.33 Impact Factor
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    • "Recent evidence of accumulation of NMD substrates as translationally repressed RNAs in P-bodies support the view that translation gets repressed during NMD response, which is followed by the Upf1p-mediated targeting of the NMD substrate to these specific cytoplasmic foci (Sheth and Parker 2006). Finally other Upf factors assemble on these mRNA substrates and the mRNA undergoes degradation in these foci following the events described above (Sheth and Parker 2006; reviewed in Parker 2012). In addition to aberrant mRNAs, NMD pathway was also demonstrated to degrade several specific normal mRNAs (Lelivelt and Culbertson 1999; He and Jacobson 2001; He et al. 2003; Kebaara and Atkin 2009; Rebbapragada and Lykke- Andersen 2009), which is considered as a manifestation of the regulation of cellular abundance of these messages by NMD (see below). "
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    ABSTRACT: Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and posttranscriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant nonfunctional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.
    Journal of Biosciences 09/2013; 38(3):615-40. DOI:10.1007/s12038-013-9337-4 · 1.94 Impact Factor
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    • "Upf1 shows RNA-dependent ATPase and 5 ′ -to-3 ′ RNA helicase activities, both of which, although critical for NMD (Czaplinski et al. 1998; Wang et al. 2001), have unresolved roles in the process. In yeast, ATPase-deficient Upf1 accumulates with NMD substrates in cytoplasmic processing bodies (Sheth and Parker 2006); and in human cells, ATPase-or helicase-deficient Upf1 leads to the accumulation of partially degraded 3 ′ decay intermediates (Franks et al. 2010). These reports and others (Ghosh et al. 2010) suggest a role for Upf1 in disassembling post-termination mRNPs in a mechanism that requires its ATPase and helicase activities. "
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    ABSTRACT: The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins. Upf1 coimmunoprecipitates specifically with epitope-tagged 40S ribosomal subunits, and Upf1 association with high-salt washed or puromycin-released 40S subunits was found to occur without simultaneous eRF1, eRF3, Upf2, or Upf3 association. Two-hybrid analyses and in vitro binding assays identified a specific interaction between Upf1 and Rps26. Using mutations in domains of UPF1 known to be crucial for its function, we found that Upf1:40S association is modulated by ATP, and Upf1:Rps26 interaction is dependent on the N-terminal Upf1 CH domain. The specific association of Upf1 with the 40S subunit is consistent with the notion that this RNA helicase not only triggers rapid decay of nonsense-containing mRNAs, but may also have an important role in dissociation of the premature termination complex.
    RNA 06/2013; 19(8). DOI:10.1261/rna.039396.113 · 4.62 Impact Factor
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