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At least 271 records · Page 15Linked to original sources
PubMed2001

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Animals↗
PubMed2000

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested.

In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The "protection model" states that the three viral RNAs must have a few coat protein subunits at their 3' termini in order to protect them in the host cell against degradation by 3'- to- 5' exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883-887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published "messenger release model" for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617-621] is discussed.

Alfalfa mosaic virus↗
PubMed1999

Genome activation in alfamo- and ilarviruses.

Alfamo- and ilarviruses are characterized by the deficiency of their genomes (three messenger-sense RNAs) to start an infection cycle. The RNAs are in capsids built from a single species of protein of about 24 kD. A few dimers of this coat protein per RNA molecule are sufficient to activate the genome. Since the first description of genome activation [Bol JF, van Vloten-Doting L, Jaspars EMJ (1971) Virology 46: 73-85] three models have been proposed concerning its mechanism: the protection, the replicase and the messenger release hypotheses. The first two models make use of the fact that in these genera of RNA viruses the 3' termini of the RNAs bind the coat protein very strongly. The resulting structure would provide protection against 3'- to 5' exoribonucleases, or would permit correct initiation of minus-strand synthesis, respectively. However, naked inoculated RNAs of alfalfa mosaic virus appear to be quite stable in the cell, and in vitro the coat protein is inhibiting rather than stimulating initiation of minus-strand synthesis. The messenger release hypothesis states that the coat protein is needed for the release of viral messenger RNAs from membranous replication complexes throughout the whole viral replication cycle. This is supported by in vivo and in vitro observations, but as yet a detailed molecular mechanism is difficult to give.

Alfamovirus↗
PubMed1984

Base-specific ribonucleases potentially involved in heterogeneous nuclear RNA processing and poly(A) metabolism.

Polyadenylation and splicing of heterogeneous nuclear RNA, two crucial steps in mRNA processing, are apparently enzymatically mediated processes. This contribution summarizes the properties and the presumed functions of the known poly(A) catabolic enzymes (endoribonuclease IV and V, 2',3'- exoribonuclease ) as well as those of the pyrimidine-specific endoribonucleases associated with snRNP -hnRNP complexes (endoribonuclease VII, acidic pI 4.1 endoribonuclease and poly(U)-specific U1 snRNP -nuclease).

Animals↗
PubMed1993

Evidence for age-dependent impairment of antiviral 2',5'-oligoadenylate synthetase/ribonuclease L-system in tissues of rat.

The 2',5'-oligoadenylate system (2-5A system) has an essential role in the establishment of the antiviral state of cells exposed to virus infection. The effects of 2-5A are mediated by a 2-5A-dependent ribonuclease (RNase L) which cleaves viral RNA. A study of 2-5A metabolism in different tissues of rats of different age (newborn: 1-day-old; young adult: 2- to 3-month-old; middle-aged adult: 12-month-old; and old: 32- to 33-month-old) revealed that the activities of the 2-5A metabolic enzymes alter during aging and development. We demonstrate that soluble 2-5A synthetase activity strongly increases after birth, reaching maximal levels in young adult and middle-aged adult animals and then significantly decreases with age; the age-dependent decrease was found also for the nuclear matrix-associated enzyme. In contrast, the activity of 2',3'-exoribonuclease which inactivates 2-5A increases by 3-fold with age. The decrease in 2-5A synthetase activity and increase in 2-5A nuclease activity were found to result in a decrease in the cellular 2-5A content with age. The RNase L which is activated by 2-5A also changes age-dependently. The amount and activity of this enzyme were determined in cross-linking experiments, in nitrocellulose binding assays and in the ribosomal RNA cleavage assay. The livers of old rats displayed a 5- to 6-fold decrease in RNase L activity compared to the adult animal groups, whilst the amount of the enzyme did not change significantly during aging with the exception of a drop by 30% in the nuclear matrix fraction. From these results we conclude that the antiviral activity of the 2-5A system is impaired in old cells with the consequences that virus production cannot be efficiently suppressed.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript.

We report that the light-harvesting and reaction center genes in the rxcA locus of R. capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript. The 3' portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides. The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins. The unstable 3' portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3' exoribonucleases and thereby protect the upstream RNA segment. When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E. coli, the long precursor transcript was processed to two short messages of greater stability, as in R. capsulata.

Bacterial Proteins↗
PubMed1988

An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability.

Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capsulatus. The comparatively stable 5' segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5' puf mRNA segment but that its insertion at the 3' end of the puf operon transcripts fails to stabilize the labile 3' puf mRNA segment. Evidence is presented that decay of the 3' segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3' exoribonucleases.

Bacterial Proteins↗
PubMed1988

Mechanisms of mRNA decay in bacteria: a perspective.

Messenger RNA decay plays an important role in prokaryotic gene expression. The disparate stabilities of bacterial messages in vivo are a consequence of their differential susceptibility to degradation by cellular endoribonucleases and 3' -exoribonucleases, which in turn results from differences in mRNA sequence and structure. RNase II and polynucleotide phosphorylase, the major bacterial exonucleases involved in mRNA turnover, rapidly degrade single-stranded RNA from the 3' end, but are impeded by 3' stem-loop structures. At present, the identify and substrate specificity of the endonucleases that control mRNA decay rates are relatively poorly defined. Ribosomes and antisense RNA also can influence the stability of transcripts with which they associate. Differences in mRNA stability can contribute to differential expression of genes within polycistronic operons and to modulation of gene expression in response to changes in bacterial growth conditions.

Bacteria↗
PubMed2005

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach.

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.

Endoribonucleases↗
PubMed2005

Processing the message: structural insights into capping and decapping mRNA.

The 5' m7GpppN cap plays an essential role in the life cycle of eukaryotic mRNA and is required for efficient pre-mRNA splicing, export, stability and translation. Nascent pre-mRNA is capped through a series of three enzymatic steps that result in a 5' N7-methyl guanosine linked by an inverted 5'-5' triphosphate bridge to the first nucleotide of the transcript. Early structural studies revealed the mechanisms employed in the first two steps of capping, and more recent structural studies have completed the suite of capping activities and shed light on the mechanisms that target the capping apparatus to the phosphorylated C-terminal domain of RNA polymerase II. RNA decay pathways also target the RNA cap structure. After deadenylation of polyadenylated mRNA, enzymes of the 5'-3' decay pathway hydrolyze the mRNA cap to expose the 5' RNA end to 5'-3' exoribonuclease activities. In the 3'-5' decay pathway, exosome-mediated degradation of RNA occurs from the 3' end after deadenylation, ultimately generating a cap structure that is hydrolyzed by enzymes of this pathway. Recent structural studies have illuminated the mechanisms employed for decapping mRNA in both 5'-3' and 3'-5' decay pathways.

Acid Anhydride Hydrolases↗
PubMed1999

Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail.

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs cooperate to promote translation initiation but whether this functional interaction benefits certain classes of mRNAs has not been investigated. In this study, we investigate whether a structured 5'-leader or 3'-untranslated region (UTR) affects the cap/poly(A) tail interaction. A structured leader reduced the degree to which the 5'-cap promoted translation in plant cells and inhibited translation from capped and uncapped mRNAs equally in yeast. Secondary structure within the 3'-UTR reduced translational efficiency when adjacent to the stop codon but had little effect on the cap/poly(A) tail synergy. The functional interaction between the cap and poly(A) tail was as important for an mRNA with a structured leader or 3'-UTR as it was for an unstructured mRNA in either species, suggesting that these structures can reduce translation without affecting the functional interaction between the cap and poly(A) tail. However, the loss of Xrn1p, the major 5'-->3' exoribonuclease in yeast, abolished cap-dependent translation and the functional interaction between the cap and poly(A) tail, suggesting that the cap/poly(A) tail synergy is of particular importance under conditions of active RNA turnover.

3' Untranslated Regions↗
PubMed2002

Mutational analysis of polynucleotide phosphorylase from Escherichia coli.

Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.

Amino Acid Sequence↗
PubMed2002

Polynucleotide phosphorylase binds to ssRNA with same affinity as to ssDNA.

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is a multifunctional protein, with a 3'-5' processive exoribonuclease, a Pi exchange, an RNA polymerase and an autoregulatory activity. The interaction between this enzyme and the mRNA target is crucial for its activities. In the present study, we characterized the interaction of PNPase with its mRNA regulatory region and ssRNA, as well as with ssDNA and dsDNA by determining K(d). Our results indicate that PNPase has high affinity for its mRNA, ssRNA and for ssDNA (K(d) approximately 10-20 nM). However, this enzyme exhibits a lower affinity for dsDNA (K(d) approximately 200-1400 nM). Possible implications of these results on the molecular mechanisms by which PNPase is regulated and degrades mRNA are discussed.

5' Untranslated Regions↗
PubMed1999

Degrading chloroplast mRNA: the role of polyadenylation.

Chloroplast development involves changes in the stability of specific plastid mRNAs. To understand how the half-lives of these mRNAs are modified, several laboratories are investigating how plastid mRNAs are degraded. This has led to the isolation of a high-molecular-weight complex that contains an endoribonuclease and a 3'-5' exoribonuclease, and the discovery that efficient mRNA degradation requires polyadenylation. These findings are similar to recent discoveries in Escherichia coli. However, an important difference between the two systems is that chloroplast mRNA degradation involves nuclear-encoded proteins. Modification of these proteins could provide the mechanism for altering plastid-mRNA half-lives in response to developmental stimuli.

Chloroplasts↗
PubMed2000

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo.

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.

Antiviral Agents↗
PubMed2001

The specificity of nucleotide removal during RNA editing in Trypanosoma brucei.

RNA editing in Trypanosoma brucei produces mature mRNAs by posttranscriptional insertion and deletion of uridylates (Us) by a series of catalytic steps, which include endoribonucleolytic cleavage, 3' terminal addition or removal of Us, and RNA ligation. Preedited mRNA (pre-mRNA) and guide RNA (gRNA) that are mutated at or near the editing site (ES) were used to examine the effects on the specificity of in vitro editing. Sequences that are not predicted to form a gRNA/pre-mRNA base pair immediately 5' to the ES still supported accurate editing. Substitution of a non-U nucleotide at various positions within a stretch of Us that are normally removed from the ES resulted in deletion of only the Us that were 3' to the substituted nucleotide. Overall, ES selection by the endoribonuclease, the specificity of the 3' exoribonuclease for Us, and ligation appear to act in concert to ensure the production of accurately edited RNA.

Animals↗
PubMed1992

Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli.

Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.

Bacterial Outer Membrane Proteins↗