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Results for “Exosomal RNA”

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246 records · Page 14Linked to original sources
PubMed2006

The regulation of exosome secretion: a novel function of the p53 protein.

The p53 protein responds to stress signals by regulating the transcription of a variety of genes. Some of these genes encode secreted proteins that may be involved in the communication between adjacent cells. In this study, a proteomics approach was employed to identify proteins secreted by cells in a p53-dependent manner after DNA damage. In addition to the known transcriptional targets of p53, a set of proteins encoded by genes that are not transcriptional targets of p53 were found to increase in the culture medium after p53 activation. These proteins exit the cell via small, secreted vesicles called exosomes and exosome production by cells was found to be regulated by the p53 response. A p53-regulated gene product, TSAP6, was shown to enhance exosome production in cells undergoing a p53 response to stress. Thus, the p53 pathway regulates the production of exosomes into the medium and these vesicles can communicate with adjacent cells and even cells of the immune system.

Alleles↗
PubMed2001

Induced synthesis of albumin-like protein in damaged rat reticulocytes.

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin-like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation. The endogenous origin of rat RBC albumin-like protein was demonstrated by monitoring protein synthesis in reticulocytes and by identification of reticulocyte mRNA for albumin. Haemolytic anaemia, either hereditary (b/b rat) or drug-induced (phenylhydrazine-treated normal rat), results in increased accumulation of the albumin-like protein in rat RBCs as a result of its induced synthesis.

Albumins↗
PubMed1995

Segregation of autoantibodies with disease in monozygotic twin pairs discordant for systemic sclerosis. Three further cases.

OBJECTIVE: To study the serologic status of 3 previously unreported monozygotic twin pairs discordant for systemic sclerosis (SSc). METHODS: Autoantibodies were measured by indirect immunofluorescence, immunodiffusion, and immunoprecipitation of 35S-labeled cell lines. Major histocompatibility complex (MHC) class II allele typing and DNA fingerprinting were used to confirm monozygosity. RESULTS: Anti-PM-Scl, anti-threonyl transfer RNA synthetase, and anti-topoisomerase I antibodies, respectively, were found in each of the twins with SSc. None of the unaffected twin siblings had an identifiable autoantibody, although serum from 1 unaffected twin precipitated several unknown proteins. The MHC class II genotype in each twin was the genotype expected for the autoantibody that was present. CONCLUSION: Autoantibodies of certain defined specificities are intimately linked with the development of SSc, because they segregate with SSc in individuals who start life with identical germline genes.

Autoantibodies↗
PubMed2005

Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export.

In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export. Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription. A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export. Retention depends on the exosome. In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export. Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm. It can exit the nucleus either as cargo of exportin 1 or bound to mRNA. Pab1 is essential but several bypass suppressors have been identified. Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription. Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs. The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.

Active Transport, Cell Nucleus↗
PubMed2003

Interaction between Ski7p and Upf1p is required for nonsense-mediated 3'-to-5' mRNA decay in yeast.

Aberrant mRNAs containing premature termination codons (PTC-mRNAs) are degraded by a conserved surveillance system, referred to as the nonsense- mediated decay (NMD) pathway. Although NMD is reported to operate on the decapping and 5'-to-3' exonucleolytic decay of PTC-mRNAs without affecting deadenylation, a role for an opposite 3'-to-5' decay pathway remains largely unexplored. In this study, we have characterized the 3'-to-5' directed mRNA degradation in the yeast NMD pathway. PTC-mRNAs are stabilized in yeast cells lacking the components of 3'-to-5' mRNA-decay machinery. The 3'-to-5' directed degradation of PTC-mRNAs proceeds more rapidly than that of the PTC-free transcript, in a manner dependent on the cytoplasmic exosome and Upf proteins. Moreover, Upf1p, but not Upf2p, interacts physically with an N-terminal domain of Ski7p, although the interaction requires Upf2p. The efficiency of 3'-to-5' directed degradation of PTC-mRNAs is impaired by overexpression of Ski7p N-domain fragments that contain a sequence of the Upf1p-interaction region. These data suggest that the activation of 3'-to-5' directed NMD is mediated through the interaction between Upf1p and the Ski7p N domain.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.

Anti-PM-Scl antibodies are associated with polymyositis-scleroderma overlap or either disease alone. Among sera from 39 patients with anti-PM-Scl, 23 recognized the 100-kD band in immunoblot against HeLa cell extract, 16 of which also stained the 70-kD band. A human thymocyte lambda gt11 cDNA expression library was screened with anti-PM-Scl serum, and two clones were identified whose products reacted with 33 and 37 of 39 anti-PM-Scl sera, respectively, but none of 26 negative control sera. Affinity-purified antibody reacting specifically with plaques of the clone stained the 100-kD band on immunoblot, reacted with nucleoli of HEp-2 cells, and immunoprecipitated the PM-Scl protein complex. Partial sequences of both inserts were identical. One insert was fully sequenced, and additional 5' and 3' sequence was obtained using a gene-specific primer to form a cDNA with HeLa cell RNA as template followed by PCR. The complete nucleotide sequence included 2,739-bp coding for a predicted full-length protein of 98,088 D. There was no homology with the PM-Scl 75-kD protein and no significant homology with other proteins. A mixed-charge cluster was identified, with 22 charged amino acids of 37. In conclusion, the full-length cDNA sequence was determined coding for the PM-Scl 100-kD protein, the most commonly antigenic protein of the PM-Scl complex.

Amino Acid Sequence↗
PubMed2004

Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.

Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

Drug Evaluation, Preclinical↗
PubMed2006

Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance.

Cryptic unstable transcripts (CUTs) are widely distributed in the genome of S. cerevisiae. These RNAs generally derive from nonannotated regions of the genome and are degraded rapidly and efficiently by the nuclear exosome via a pathway that involves degradative polyadenylation by a new poly(A) polymerase borne by the TRAMP complex. What is the share of significant information that is encrypted in CUTs and what distinguishes a CUT from other Pol II transcripts are unclear to date. Here we report the dissection of the molecular mechanism that leads to degradation of a model CUT, NEL025c. We show that the Nrd1p-Nab3p-dependent pathway, involved in transcription termination of sno/snRNAs, is required, albeit not sufficient, for efficient degradation of NEL025c RNAs and at least a subset of other CUTs. Our results suggest an important role for the Nrd1p-Nab3p pathway in the control of gene expression throughout the genome.

Base Sequence↗
PubMed2003

Coordinate expression of NADPH-dependent flavin reductase, Fre-1, and Hint-related 7meGMP-directed hydrolase, DCS-1.

A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors. Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear. To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme. We used the novel substrate, 7meGpppBODIPY, to demonstrate that DCS-1 has low micromolar specificity for guanine ribonucleotides with the 7me modification, whereas trimethylated G substrates are poor competitors. Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases. These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts. We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have a nearly identical expression profile in human tissues. Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure. Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses.

Amino Acid Sequence↗
PubMed2006

Yeast transcripts cleaved by an internal ribozyme provide new insight into the role of the cap and poly(A) tail in translation and mRNA decay.

It has been proposed that the 7-methylguanosine cap and poly(A) tail of mRNAs have important functions in translation and transcript stability. To directly test these roles of the cap and poly(A) tail, we have constructed plasmids with a ribozyme within the coding region or 3' UTR of reporter genes. We show that the unadenylated 5' cleavage product is translated and is rapidly degraded by the cytoplasmic exosome. This exosome-mediated decay is independent of the nonstop mRNA decay pathway, and, thus, reveals an additional substrate for exosome-mediated decay that may have physiological equivalents. The rapid decay of this transcript in the cytoplasm indicates that this unadenylated cleavage product is rapidly exported from the nucleus. We also show that this cleavage product is not subject to rapid decapping; thus, the lack of a poly(A) tail does not always trigger rapid decapping of the transcript. We show that the 3' cleavage product is rapidly degraded by Xrn1p in the cytoplasm. We cannot detect any protein from this 3' cleavage product, which supports previous data concluding that the 5' cap is required for translation. The reporter genes we have utilized in these studies should be generally useful tools in studying the importance of the poly(A) tail and 5' cap of a transcript for export, translation, mRNA decay, and other aspects of mRNA metabolism in vivo.

Base Sequence↗
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↗
PubMed2004

Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination.

Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.

Amino Acid Sequence↗