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At least 523 records · Page 29Linked to original sources
PubMed1997

Cloning and characterization of mouse Dhm2 cDNA, a functional homolog of budding yeast SEP1.

We have isolated mouse Dhm2 cDNAs encoding a homolog of budding yeast SEP1, whose product is involved in many cellular processes including meiosis, cellular senescence, and telomere maintenance. The putative Dhm2 protein (Dhm2p), which consists of 1687 amino acids and whose molecular weight is 191,400, matches the size of Sep1p and shares extensive homology with Sep1p especially in their N-terminal regions. A multicopy plasmid containing of the Dhm2 cDNA complements the slow growth phenotype, sporulation defect, and DNA recombination defect caused by the sep1 mutation in yeast, indicating that Dhm2 is a functional homolog of SEP1. Since Dhm1, another SEP1 homolog we reported previously, only partially compensates for the sep1 mutation, we conclude that Dhm2 is a true homolog of SEP1. Northern analysis revealed that 5.8 kb mRNA corresponding to Dhm2 open reading frame is produced highly in testis. These results strongly suggest that Dhm2p participates in gametogenesis in mouse.

Animals↗
PubMed1997

sep1+ encodes a transcription-factor homologue of the HNF-3/forkhead DNA-binding-domain family in Schizosaccharomyces pombe.

We report on the cloning of sep1+, a gene whose mutation causes filamentous growth in Schizosaccharomyces pombe. Since cell growth and propagation are not affected by the mutation, it could not be cloned using selective conditions for the identification of the positive transformants. Instead, we cloned it from a cosmid of a contig (Hoheisel et al., Cell 73, 109-1120, 1993) supposed to cover the chromosomal region where the sep1-1 mutation mapped. The 1761 bp long ORF codes for a protein containing a sequence similar to the DNA-binding domains of the HNF-3/forkhead family of transcription factors.

Amino Acid Sequence↗
PubMed1996

Antinuclear antibody in systemic sclerosis (scleroderma).

The presence of autoantibodies to intracellular molecules is the hallmark immunologic finding of SSc. Recent sophisticated methods have contributed to characterization of unidentified antigens of ANA in sera from patients with SSc. Antibodies to RNA polymerases are the third major SSc-specific ANA, in addition to anti-topo I and anticentromere antibodies, and it is now possible to identify over 85% of SSc patients. These antibodies have proved helpful in diagnosis of this disease. An immunogenetic predisposition to synthesis of the ANAs has been unveiled. Both antigen-driven and molecular mimicry hypotheses have been proposed for ANA production in SSc. Although the role of ANAs in the pathogenesis of SSc is not yet known, it is logical to assume that the origin of ANAs is linked to the causes of this disease because each antibody is associated with its own unique constellation of clinical features.

Antibodies, Antinuclear↗
PubMed2003

Nocturnin, a deadenylase in Xenopus laevis retina: a mechanism for posttranscriptional control of circadian-related mRNA.

BACKGROUND: Different types of regulation are utilized to produce a robust circadian clock, including regulation at the transcriptional, posttranscriptional, and translational levels. A screen for rhythmic messages that may be involved in such circadian control identified nocturnin, a novel gene that displays high-amplitude circadian expression in the Xenopus laevis retina, with peak mRNA levels in the early night. Expression of nocturnin mRNA is confined to the clock-containing photoreceptor cell layer within the retina. RESULTS: In these studies, we show that nocturnin removes the poly(A) tail from a synthetic RNA substrate in a process known as deadenylation. Nocturnin nuclease activity is magnesium dependent, as the addition of EDTA or mutation of the residue predicted to bind magnesium disrupts deadenylation. Substrate preference studies show that nocturnin is an exonuclease that specifically degrades the 3' poly(A) tail. While nocturnin is rhythmically expressed in the cytoplasm of the retinal photoreceptor cells, the only other described vertebrate deadenylase, PARN, is constitutively present in most retinal cells, including the photoreceptors. CONCLUSIONS: The distinct spatial and temporal expression of nocturnin and PARN suggests that there may be specific mRNA targets of each deadenylase. Since deadenylation regulates mRNA decay and/or translational silencing, we propose that nocturnin deadenylates clock-related transcripts in a novel mechanism for posttranscriptional regulation in the circadian clock or its outputs.

Animals↗
PubMed1998

The exosome: a versatile RNA processing machine.

A conserved complex of multiple 3'-to-5' exonucleases has been discovered recently in eukaryotes. This essential complex, the exosome, is required for diverse RNA processing events.

Exoribonucleases↗
PubMed2002

The yin and yang of the exosome.

Recent studies of the eukaryotic ribosomal RNA processing pathway have identified a complex of ten riboexonucleases called the exosome that plays a central role in the precise formation of the 3' ends of several types of RNAs. The exosome also destroys excess ribosomal RNA precursors and unused intermediates and degrades poly(A)-mRNAs in the cytoplasm. In the nucleus, the complex appears to function in a regulated mRNA surveillance system that degrades transcripts in response to defects in the mRNA processing and export pathways. How the cell regulates the nucleolytic prowess of the exosome to ensure correct and timely synthesis and destruction of RNAs is a central focus of current research.

Animals↗
PubMed1995

Effect of TAP on the generation and intracellular trafficking of peptide-receptive major histocompatibility complex class I molecules.

Using a fluorescein-conjugated antigenic peptide, peptide-receptive H-2Kb MHC class I molecules were found throughout the secretory pathways of RMA cells and peptide transporter (TAP)-deficient derivative cells (RMA/S). RMA/S cells displayed higher levels of intracellular peptide-receptive molecules, while, surprisingly, RMA cells expressed 3- to 5-fold more cell surface peptide-receptive molecules. Metabolic radiolabeling of Kb-associated oligosaccharides with [1-3H]galactose demonstrated that despite a large difference in the fraction of Kb molecules in native conformation in detergent extracts, Kb transport rates from the trans-Golgi complex to the surfaces of RMA and RMA/S cells were similar. Thus, although considerable numbers of class I alpha chains reach the RMA/S cell surface, they are a less productive source of peptide-receptive molecules than class I molecules synthesized by TAP-expressing RMA cells, suggesting paradoxically that TAP functions to increase the amount of peptide-receptive molecules at the cell surface.

Biological Transport↗
PubMed1997

14-3-3sigma is a p53-regulated inhibitor of G2/M progression.

Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3sigma is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3sigma into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.

14-3-3 Proteins↗
PubMed2000

Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro.

We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.

Eukaryotic Initiation Factor-4E↗
PubMed2002

Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs.

Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination. Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p. The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype. Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation. Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation. The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media.

3' Untranslated Regions↗
PubMed2003

RNAse III-mediated degradation of unspliced pre-mRNAs and lariat introns.

Double-stranded RNA (dsRNA) has emerged as a modulator of gene expression, from gene silencing to antiviral responses. Here we show that dsRNA stem-loop structures found in intronic regions of the S. cerevisiae RPS22B and RPL18A transcripts trigger degradation of unspliced pre-mRNAs and lariat introns and can control the level of mRNA produced from these intron-containing genes. The dsRNA regions are cleaved by Rnt1p, the yeast ortholog of RNase III, which creates an entry site for complete degradation by the Xrn1p and Rat1p exonucleases and by the nuclear exosome. These results identify an alternative discard pathway for precursors and products of the splicing machinery and a physiological function for dsRNA in eukaryotic RNA catabolism.

Base Sequence↗
PubMed2003

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation.

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.

3' Untranslated Regions↗
PubMed2003

mRNA decay: x (XRN1) marks the spot.

Degradation of mRNA is a vital aspect of gene expression. In yeast, Dcp1p, Dcp2p, Lsm1-7p, and Xrn1p are required for mRNA decay and are localized within discrete cytoplasmic foci; in the May 2 issue of Science, Sheth and Parker provide compelling evidence that these foci represent sites for mRNA decay.

Animals↗
PubMed2003

Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities.

Nonsense-mediated mRNA decay (NMD) is a mechanism by which cells recognize and degrade mRNAs that prematurely terminate translation. To date, the polarity and enzymology of NMD in mammalian cells is unknown. We show here that downregulating the Dcp2 decapping protein or the PM/Scl100 component of the exosome (1) significantly increases the abundance of steady-state nonsense-containing but not nonsense-free mRNAs, and (2) significantly slows the decay rate of transiently induced nonsense-containing but not nonsense-free mRNA. Downregulating poly(A) ribonuclease (PARN) also increases the abundance of nonsense-containing mRNAs. Furthermore, NMD factors Upf1, Upf2, and Upf3X coimmunopurify with the decapping enzyme Dcp2, the putative 5'-->3' exonuclease Rat1, the proven 5'-->3' exonuclease Xrn1, exosomal components PM/Scl100, Rrp4, and Rrp41, and PARN. From these and other data, we conclude that NMD in mammalian cells degrades mRNAs from both 5' and 3' ends by recruiting decapping and 5'-->3' exonuclease activities as well as deadenylating and 3'-->5' exonuclease activities.

Animals↗
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↗
PubMed2000

The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases.

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Base Sequence↗
PubMed2001

The mechanism and regulation of deadenylation: identification and characterization of Xenopus PARN.

In Xenopus oocytes, the deadenylation of a specific class of maternal mRNAs results in their translational repression. Here we report the purification, characterization, and molecular cloning of the Xenopus poly(A) ribonuclease (xPARN). xPARN copurifies with two polypeptides of 62 kDa and 74 kDa, and we provide evidence that the 62-kDa protein is a proteolytic product of the 74-kDa protein. We have isolated the full-length xPARN cDNA, which contains the tripartite exonuclease domain conserved among RNase D family members, a putative RNA recognition motif, and a domain found in minichromosome maintenance proteins. Characterization of the xPARN enzyme shows that it is a poly(A)-specific 3' exonuclease but does not require an A residue at the 3' end. However, the addition of 25 nonadenylate residues at the 3' terminus, or a 3' terminal phosphate is inhibitory. Western analysis shows that xPARN is expressed throughout early development, suggesting that it may participate in the translational silencing and destabilization of maternal mRNAs during both oocyte maturation and embryogenesis. In addition, microinjection experiments demonstrate that xPARN can be activated in the oocyte nucleus in the absence of cytoplasmic components and that nuclear export of deadenylated RNA is impeded. Based on the poly(A) binding activity of xPARN in the absence of catalysis, a model for substrate specificity is proposed.

Amino Acid Sequence↗