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Allan Jacobson

Publications and source records attributed to Allan Jacobson.

14 recordsLinked to original sources
PubMed2006

Early nonsense: mRNA decay solves a translational problem.

Gene expression is highly accurate and rarely generates defective proteins. Several mechanisms ensure this fidelity, including specialized surveillance pathways that rid the cell of mRNAs that are incompletely processed or that lack complete open reading frames. One such mechanism, nonsense-mediated mRNA decay, is triggered when ribosomes encounter a premature translation-termination--or nonsense--codon. New evidence indicates that the specialized factors that are recruited for this process not only promote rapid mRNA degradation, but are also required to resolve a poorly dissociable termination complex.

3' Untranslated Regions↗
PubMed2005

Ribosome occupancy of the yeast CPA1 upstream open reading frame termination codon modulates nonsense-mediated mRNA decay.

Saccharomyces cerevisiae CPA1 mRNA contains an upstream open reading frame (uORF) encoding the arginine attenuator peptide (AAP). Negative translational regulation of CPA1 occurs when the nascent AAP responds to arginine (Arg) by stalling ribosomes at the uORF termination codon. CPA1 expression is also controlled by nonsense-mediated mRNA decay (NMD). Using wild-type and decay-defective strains expressing CPA1-LUC, we determined how this uORF contributes to NMD control. Arg addition to media rapidly destabilized the CPA1 transcript in wild-type but not upf1delta cells. The wild-type uORF exerted translational control and induced NMD of CPA1-LUC; the mutated D13N uORF, which eliminates stalling and regulation, did not. Thus, regulation by NMD was not governed simply by ribosomes encountering the uORF terminator but appeared dependent on the AAP's ribosome-stalling ability. Improving the D13N uORF initiation context also promoted NMD. Hence, NMD appears to be triggered by increased ribosomal occupancy of the uORF termination codon.

Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzin↗
PubMed2004

A faux 3'-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay.

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.

3' Untranslated Regions↗
PubMed2004

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.

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 upf1Delta and xrn1Delta 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.

Cations↗
PubMed2004

Regulation of mRNA decay: decapping goes solo.

In this issue of Molecular Cell, Badis et al. demonstrate that autoregulated destabilization of the RPS28B mRNA is mediated by Edc3p interaction with an Rps28p/3'UTR mRNP complex. Subsequent recruitment of Dcp1p/Dcp2p to the complex appears to ensure rapid, deadenylation-independent decapping of the mRNA.

3' Untranslated Regions↗
PubMed2004

Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.

As a test case for optimizing how to perform proteomics experiments, we chose a yeast model system in which the UPF1 gene, a protein involved in nonsense-mediated mRNA decay, was knocked out by homologous recombination. The results from five complete isotope-coded affinity tag (ICAT) experiments were combined, two using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) and three using electrospray MS/MS. We sought to assess the reproducibility of peptide identification and to develop an informatics structure that characterizes the identification process as well as possible, especially with regard to tenuous identifications. The cleavable form of the ICAT reagent system was used for quantification. Most proteins did not change significantly in expression as a consequence of the upf1 knockout. As expected, the Upf1 protein itself was down-regulated, and there were reproducible increases in expression of proteins involved in arginine biosynthesis. Initially, it seemed that about 10% of the proteins had changed in expression level, but after more thorough examination of the data it turned out that most of these apparent changes could be explained by artifacts of quantification caused by overlapping heavy/light pairs. About 700 proteins altogether were identified with high confidence and quantified. Many peptides with chemical modifications were identified, as well as peptides with noncanonical tryptic termini. Nearly all of these modified peptides corresponded to the most abundant yeast proteins, and some would otherwise have been attributed to "single hit" proteins at low confidence. To improve our confidence in the identifications, in MALDI experiments, the parent masses for the peptides were calibrated against nearby components. In addition, five novel parameters reflecting different aspects of identification were collected for each spectrum in addition to the Mascot score that was originally used. The interrelationship between these scoring parameters and confidence in protein identification is discussed.

Amino Acid Sequence↗
PubMed2004

Identification of factors regulating poly(A) tail synthesis and maturation.

Posttranscriptional maturation of the 3' end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3'-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1 Delta and pbp1 Delta cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

Base Sequence↗
PubMed2004

Positive and negative regulation of poly(A) nuclease.

PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Amino Acid Sequence↗
PubMed2003

Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression.

Most 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.

Active Transport, Cell Nucleus↗
PubMed2003

Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5' to 3' mRNA decay pathways in yeast.

Transcripts regulated by the yeast nonsense-mediated and 5' to 3' mRNA decay pathways were identified by expression profiling of wild-type, upf1Delta, nmd2Delta, upf3Delta, dcp1Delta, and xrn1Delta cells. This analysis revealed that inactivation of Upf1p, Nmd2p, or Upf3p has identical effects on global RNA accumulation; inactivation of Dcp1p or Xrn1p exhibits both common and unique effects on global RNA accumulation but causes upregulation of only a small fraction of transcripts; and the majority of transcripts upregulated in upf/nmd strains are also upregulated to similar extents in dcp1Delta and xrn1Delta strains. Our results define the core transcripts regulated by NMD, identify several novel structural classes of NMD substrates, demonstrate that nonsense-containing mRNAs are primarily degraded by the 5' to 3' decay pathway even in the absence of functional NMD, and indicate that 3' to 5' decay, not 5' to 3' decay, may be the major mRNA decay activity in yeast cells.

Cluster Analysis↗
PubMed2003

Nonsense-containing mRNAs that accumulate in the absence of a functional nonsense-mediated mRNA decay pathway are destabilized rapidly upon its restitution.

Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins. Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm. Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway. To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter. The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced. Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min. These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs.

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.

Base Sequence↗
PubMed2002

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.

3' Untranslated Regions↗