We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1⌬ and xrn1⌬ mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5 to 3 decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression. Molecular & Cellular Proteomics 3:1154 -1169, 2004.An initial step in the systematic investigation of cellular processes is the identification and measurement of expression levels of relevant sets of proteins. Recently, quantitative approaches utilizing MS and a host of stable isotope-labeling chemistries have emerged (reviewed in Refs. 1 and 2), offering a departure from traditional techniques employing comparative two-dimensional gel electrophoresis. The ICAT quantitative labeling strategy (3, 4) is perhaps the best-characterized method for relative protein quantitation using MS. Other elegant approaches use cell-culture enrichment with a stable isotope-labeled amino acid, including arginine (5), lysine (6), tyrosine (7), and leucine (8), for in vivo incorporation of a mass difference to support relative quantitation. This circumvents potential difficulties surrounding chemical labeling downstream in a comparative experiment. All of these methods impart a mass difference as the basis for quantitation by measurement of relative peak areas of MS and/or MS/MS mass spectra. There are, however, a number of limitations imposed by mass-difference labeling. The mass-difference concept for many practical purposes is limited to a binary (2-plex) set of reagents, and this makes comparison of multiple states (e.g. several experimental controls or time-course studies) difficult to undertake. Multiple 2-plex datasets can be combined after separate analyses, but there is a high likelihood that different sets of peptides and proteins will be identified between each experiment. In addition, the use of massdifference labels increases MS complexity, and this problem increases with numbers of a multiplexed set. Finally, the cysteine-selective affinity strategy for reduction of sample complexity (ICAT) is not amenable to identification of post-translationally modified peptides, as the majority of posttranslational modification (PTM) 1 -containing peptides are discarded at the affinity step.We have developed a multiplexed set of reagents for quantitative protein analysis that place isobaric mass label...

Nonsense-mediated messenger RNA decay (NMD) is triggered by premature translation termination, but the features distinguishing premature from normal termination are unknown. One model for NMD suggests that decay-inducing factors bound to mRNAs during early processing events are routinely removed by elongating ribosomes but remain associated with mRNAs when termination is premature, triggering rapid turnover. Recent experiments challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination. Here we use a primer extension inhibition (toeprinting) assay to delineate ribosome positioning and find that premature translation termination in yeast extracts is indeed aberrant. Ribosomes encountering premature UAA or UGA codons in the CAN1 mRNA fail to release and, instead, migrate to upstream AUGs. This anomaly depends on prior nonsense codon recognition and is eliminated in extracts derived from cells lacking the principal NMD factor, Upf1p, or by flanking the nonsense codon with a normal 3'-untranslated region (UTR). Tethered poly(A)-binding protein (Pab1p), used as a mimic of a normal 3'-UTR, recruits the termination factor Sup35p (eRF3) and stabilizes nonsense-containing mRNAs. These findings indicate that efficient termination and mRNA stability are dependent on a properly configured 3'-UTR.

While the potential importance of mRNA stability to the regulation of gene expression has been recognized, the structures and mechanisms involved in the determination of individual mRNA decay rates have just begun to be elucidated, particularly in mammalian systems and yeast. It is now well established that mRNA decay is not a default process, in which an array of nonspecific nucleases degrades indiscriminately based on target size or ribosome protection of the substrate. Rather, like transcription, RNA processing, and translation, mRNA decay is a precise process dependent on a variety of specific cis-acting sequences and trans-acting factors. Entry into the pathways of mRNA decay is triggered by at least three types of initiating event: poly(A) shortening, arrest of translation at a premature nonsense codon, and endonucleolytic cleavage. Steps subsequent to poly(A) shortening or premature translational termination converge in a pathway that progresses from removal of the 5' cap to exonucleolytic digestion of the body of the mRNA. mRNA fragments generated by endonucleolytic cleavage are most likely removed by exonucleolytic decay as well, but these events have not been characterized in detail. Nucleases and other factors (including mRNA sequence elements and autoregulatory proteins) required for the promotion or inhibition of these pathways have been identified by both biochemical and genetic methods and systematic attempts to understand their respective roles have begun. mRNA sequences whose presence or absence has marked effects on mRNA decay rates include the ubiquitous cap and poly(A) tail, sequences that comprise endonuclease cleavage sites, and sequences that promote poly(A) shortening. The latter are found in the 3'-UTR (untranslated region) and in coding regions. Evidence that poly(A) stimulates translation initiation, that some destabilization sequences must be translated in order to function, and that premature translation termination promotes rapid mRNA decay indicates a close linkage between the elements regulating mRNA decay and components of the protein synthesis apparatus. This linkage, and other data, leads us to propose a model for a functional mRNP. In this model, interactions between factors associated with opposite ends of an mRNA stimulate translation initiation and minimize the rate of entry into the pathways of mRNA decay. Events that initiate mRNA decay are postulated to be those that can disrupt this functional complex and create substrates for exonucleolytic digestion.

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Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that monitors cytoplasmic mRNA translation and targets mRNAs undergoing premature translation termination for rapid degradation. From yeasts to humans, activation of NMD requires the function of the three conserved Upf factors: Upf1, Upf2, and Upf3. Here, we summarize the progress in our understanding of the molecular mechanisms of NMD in several model systems and discuss recent experiments that address the roles of Upf1, the principal regulator of NMD, in the initial targeting and final degradation of NMD-susceptible mRNAs. We propose a unified model for NMD in which the Upf factors provide several functions during premature termination, including the stimulation of release factor activity and the dissociation and recycling of ribosomal subunits. In this model, the ultimate degradation of the mRNA is the last step in a complex premature termination process.

SummaryMost eukaryotic mRNAs are subject to considerable post-transcriptional modification, including capping, splicing, and polyadenylation. The process of polyadenylation adds a 3 poly(A) tail and provides the mRNA with a binding site for a major class of regulatory factors, the poly(A)-binding proteins (PABPs). These highly conserved polypeptides are found only in eukaryotes; single-celled eukaryotes each have a single PABP, whereas humans have five and Arabidopis has eight. They typically bind poly(A) using one or more RNA-recognition motifs, globular domains common to numerous other eukaryotic RNA-binding proteins. Although they lack catalytic activity, PABPs have several roles in mediating gene expression. Nuclear PABPs are necessary for the synthesis of the poly(A) tail, regulating its ultimate length and stimulating maturation of the mRNA. Association with PABP is also a requirement for some mRNAs to be exported from the nucleus. In the cytoplasm, PABPs facilitate the formation of the 'closed loop' structure of the messenger ribonucleoprotein particle that is crucial for additional PABP activities that promote translation initiation and termination, recycling of ribosomes, and stability of the mRNA. Collectively, these sequential nuclear and cytoplasmic contributions comprise a cycle in which PABPs and the poly(A) tail first create and then eliminate a network of cis-acting interactions that control mRNA function.

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

The poly(A) tail of an mRNA is believed to influence the initiation of translation, and the rate at which the poly(A) tail is removed is thought to determine how fast an mRNA is degraded. One key factor associated with this 3-end structure is the poly(A)-binding protein (Pab1p) encoded by the PAB1 gene in Saccharomyces cerevisiae. In an effort to learn more about the functional role of this protein, we used a two-hybrid screen to determine the factor(s) with which it interacts. We identified five genes encoding factors that specifically interact with the carboxy terminus of Pab1p. Of a total of 44 specific clones identified, PBP1 (for Pab1p-binding protein) was isolated 38 times. Of the putative interacting genes examined, PBP1 promoted the highest level of resistance to 3-aminotriazole (>100 mM) in constructs in which HIS3 was used as a reporter. We determined that a fraction of Pbp1p cosediments with polysomes in sucrose gradients and that its distribution is very similar to that of Pab1p. Disruption of PBP1 showed that it is not essential for viability but can suppress the lethality associated with a PAB1 deletion. The suppression of pab1⌬ by pbp1⌬ appears to be different from that mediated by other pab1 suppressors, since disruption of PBP1 does not alter translation rates, affect accumulation of ribosomal subunits, change mRNA poly(A) tail lengths, or result in a defect in mRNA decay.Rather, Pbp1p appears to function in the nucleus to promote proper polyadenylation. In the absence of Pbp1p, 3 termini of pre-mRNAs are properly cleaved but lack full-length poly(A) tails. These effects suggest that Pbp1p may act to repress the ability of Pab1p to negatively regulate polyadenylation.