Biomedical subjects

Stuart W Peltz

Publications and source records attributed to Stuart W Peltz.

13 recordsLinked to original sources

PubMed2006

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site.

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.

Archaea↗

PubMed2006

Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.

Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.

Base Sequence↗

PubMed2006

Role for Upf2p phosphorylation in Saccharomyces cerevisiae nonsense-mediated mRNA decay.

Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A newly discovered function for RNase L in regulating translation termination.

The antiviral and antiproliferative effects of interferons are mediated in part by the 2'-5' oligoadenylate-RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2'-5' oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.

Carrier Proteins↗

PubMed2005

Human immunodeficiency virus type 1 latency model for high-throughput screening.

Human immunodeficiency virus type 1 (HIV-1) is not eliminated from patients even after years of antiretroviral therapy, apparently due to the presence of latently infected cells. Here we describe the development of a cell-based system of latency that can be used for high-throughput screening aimed at novel drug discovery to eradicate HIV-1 infection.

Anti-HIV Agents↗

PubMed2005

Global analysis of Pub1p targets reveals a coordinate control of gene expression through modulation of binding and stability.

Regulation of mRNA turnover is an important cellular strategy for posttranscriptional control of gene expression, mediated by the interplay of cis-acting sequences and associated trans-acting factors. Pub1p, an ELAV-like yeast RNA-binding protein with homology to T-cell internal antigen 1 (TIA-1)/TIA-1-related protein (TIAR), is an important modulator of the decay of two known classes of mRNA. Our goal in this study was to determine the range of mRNAs whose stability is dependent on Pub1p, as well as to identify specific transcripts that directly bind to this protein. We have examined global mRNA turnover in isogenic PUB1 and pub1delta strains through gene expression analysis and demonstrate that 573 genes exhibit a significant reduction in half-life in a pub1delta strain. We also examine the binding specificity of Pub1p using affinity purification followed by microarray analysis to comprehensively distinguish between direct and indirect targets and find that Pub1p significantly binds to 368 cellular transcripts. Among the Pub1p-associated mRNAs, 53 transcripts encoding proteins involved in ribosomal biogenesis and cellular metabolism are selectively destabilized in the pub1delta strain. In contrast, genes involved in transporter activity demonstrate association with Pub1p but display no measurable changes in transcript stability. Characterization of two candidate genes, SEC53 and RPS16B, demonstrate that both Pub1p-dependent regulation of stability and Pub1p binding require 3' untranslated regions, which harbor distinct sequence motifs. These results suggest that Pub1p binds to discrete subsets of cellular transcripts and posttranscriptionally regulates their expression at multiple levels.

3' Untranslated Regions↗

PubMed2005

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript.

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.

3' Untranslated Regions↗

PubMed2004

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation.

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.

Amino Acid Sequence↗

PubMed2004

The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy.

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.

Anti-HIV Agents↗

PubMed2003

Nuclear mRNA surveillance.

Recent studies demonstrate that the factors involved in mRNA processing specify the fate of a transcript. The mRNA is either committed for export to the cytoplasm or accumulates in the nucleus, where it may be degraded. These studies reveal crosstalk among the nuclear events faced by the pre-mRNA. It is becoming evident that the components of the mRNA synthesis machinery interact with each other to establish a distinct surveillance mechanism that determines release of the transcript from the transcription site for further export and utilisation. Recent advances suggest that the major nuclear decay machinery, the nuclear exosome, and an Rrp6p-specific complex coordinate with processing factors to perform a unique regulatory function that determines its fate: either confinement of the defective mRNA at its transcription site, or release from its site of transcription for further processing and export or decay. Furthermore, message-specific regulatory mechanisms correspond with the nuclear mRNA synthesis machinery to control gene expression.

Animals↗

PubMed2003

Toward the development of a virus-cell-based assay for the discovery of novel compounds against human immunodeficiency virus type 1.

The emergence of human immunodeficiency virus type 1 (HIV-1) strains resistant to highly active antiretroviral therapy necessitates continued drug discovery for the treatment of HIV-1 infection. Most current drug discovery strategies focus upon a single aspect of HIV-1 replication. A virus-cell-based assay, which can be adapted to high-throughput screening, would allow the screening of multiple targets simultaneously. HIV-1-based vector systems mimic the HIV-1 life cycle without yielding replication-competent virus, making them potentially important tools for the development of safe, wide-ranging, rapid, and cost-effective assays amenable to high-throughput screening. Since replication of vector virus is typically restricted to a single cycle, a crucial question is whether such an assay provides the needed sensitivity to detect potential HIV-1 inhibitors. With a stable, inducible vector virus-producing cell line, the inhibitory effects of four reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, and didanosine) and one protease inhibitor (indinavir) were assessed. It was found that HIV-1 vector virus titer was inhibited in a single cycle of replication up to 300-fold without affecting cell viability, indicating that the assay provides the necessary sensitivity for identifying antiviral molecules. Thus, it seems likely that HIV-1-derived vector systems can be utilized in a novel fashion to facilitate the development of a safe, efficient method for screening compound libraries for anti-HIV-1 activity.

Anti-HIV Agents↗

PubMed2003

A yeast homologue of Hsp70, Ssa1p, regulates turnover of the MFA2 transcript through its AU-rich 3' untranslated region.

Many eukaryotic mRNAs exhibit regulated decay in response to cellular signals. AU-rich elements (AREs) identified in the 3' untranslated region (3'-UTR) of several such mRNAs play a critical role in controlling the half-lives of these transcripts. The yeast ARE-containing mRNA, MFA2, has been studied extensively and is degraded by a deadenylation-dependent mechanism. However, the trans-acting factors that promote the rapid decay of MFA2 have not been identified. Our results suggest that the chaperone protein Hsp70, encoded by the SSA family of genes, is involved in modulating MFA2 mRNA decay. MFA2 is specifically stabilized in a strain bearing a temperature-sensitive mutation in the SSA1 gene. Furthermore, an AU-rich region within the 3'-UTR of the message is both necessary and sufficient to confer this regulation. Stabilization occurs as a result of slower deadenylation in the ssa1(ts) strain, suggesting that Hsp70 is required for activation of the turnover pathway.

3' Untranslated Regions↗

PubMed2002

Non-stop decay--a new mRNA surveillance pathway.

Gene expression is an inherently complex process and errors often occur during the transcription and processing of mRNAs. Several surveillance mechanisms have evolved to check the fidelity at each step of mRNA manufacture. Two recent reports describe the identification of a novel pathway in eukaryotes that recognizes and degrades mRNAs that lack a stop codon. The non-stop decay mechanism releases ribosomes stalled at the 3' end of a mRNA and stimulates the exosome to rapidly degrade the transcript.

3' Untranslated Regions↗