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At least 19 recordsLinked to original sources

PubMed1994

The role of individual exoribonucleases in processing at the 3' end of Escherichia coli tRNA precursors.

We have used an in vitro Escherichia coli tRNA processing system to investigate the specific role of individual exoribonucleases in the 3' maturation of tRNA precursors. The processing of pre-tRNA(Tyr)su3+ and pre-tRNA(2Arg) was studied using extracts from cells lacking one or multiple exoribonucleases or using purified RNases. Earlier genetic studies had suggested that multiple exoribonucleases contributed to the maturation of tRNA precursors, and this was proven directly in the studies described here. Complete 3' processing required the combined action of multiple exoribonucleases, and each RNase showed distinct specificities for maturation of the different parts of the 3' precursor segment. RNase II and polynucleotide phosphorylase were most effective in shortening long 3' trailer sequences to intermediates with 2-4 extra 3' residues. Final trimming of the last few 3' nucleotides of these precursors was carried out most efficiently by RNases T and PH, but the two enzymes differed in their specificity for individual nucleotide positions. Depending on the tRNA precursor, the relative importance of the various RNases to the overall maturation process differed. We also showed that purified exoribonucleases can completely complement mutant extracts and that tRNA maturation can be totally reconstructed in vitro using purified enzymes. These studies provide the first detailed information about the specific role of individual exoribonucleases in tRNA processing, and bring us closer to defining a complete E. coli tRNA maturation pathway.

Escherichia coli↗

PubMed1984

Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA.

Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA. This prompted a search for and identification of a new exoribonuclease, termed RNase T. RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases. RNase T is optimally active at pH 8-9 and requires a divalent cation for activity. The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C. Its preferred substrate is tRNA-C-C-[14C]A, with much less activity shown against tRNA-C-C. RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis. The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.

Base Sequence↗

PubMed2005

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W.

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.

Adenosine Triphosphate↗

PubMed2001

Exoribonuclease superfamilies: structural analysis and phylogenetic distribution.

Exoribonucleases play an important role in all aspects of RNA metabolism. Biochemical and genetic analyses in recent years have identified many new RNases and it is now clear that a single cell can contain multiple enzymes of this class. Here, we analyze the structure and phylogenetic distribution of the known exoribonucleases. Based on extensive sequence analysis and on their catalytic properties, all of the exoribonucleases and their homologs have been grouped into six superfamilies and various subfamilies. We identify common motifs that can be used to characterize newly-discovered exoribonucleases, and based on these motifs we correct some previously misassigned proteins. This analysis may serve as a useful first step for developing a nomenclature for this group of enzymes.

Amino Acid Sequence↗

PubMed1989

Exoribonuclease activity of purified reverse transcriptase preparations from retroviruses.

Highly purified and commercially available preparations of reverse transcriptases from retroviruses contain a 3' to 5' exoribonuclease activity capable of hydrolyzing synthetic homopolyribonucleotides having a 3'-OH end. The exoribonuclease activity of reverse transcriptase preparations from Rous associated virus-2 was further characterized. This exoribonuclease activity cleaves poly(C) and poly(U) exonucleolytically from the 3'-OH end to produce nucleoside 5'-phosphates. Poly(A), poly(G), circular polyribonucleotide, and double-stranded polyribonucleotide were not hydrolyzed by the activity. This is a novel type of exoribonuclease activity.

Buffers↗

PubMed1993

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure.

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

Base Sequence↗

PubMed1985

Simultaneous isolation of cytoplasmic endoribonuclease and exoribonuclease of Trypanosoma brucei.

An endoribonuclease and an exoribonuclease have been isolated simultaneously from the cytoplasm of Trypanosoma brucei by hydroxyapatite column chromatography. The endoribonuclease produced oligonucleotides from poly(adenylic acid) with 5'-phosphate and 3'-OH termini. The exoribonuclease produced only ribonucleoside 5'-phosphates from poly(adenylic acid). The relative rates of degradation of synthetic homopolynucleotides by the endoribonuclease under standard conditions were in the order poly(adenylic acid) greater than poly(uridylic acid) poly(cytidylic acid); for the exoribonuclease the order was poly(adenylic acid) poly(uridylic acid) greater than poly(cytidylic acid). Natural transfer and ribosomal RNAs were also degraded by both enzymes, while DNA was resistant to them. The optimal pH of activity for each enzyme was 7.5-8.0. Both ribonucleases require Ca2+ for maximum enzymatic activity.

Animals↗

PubMed2004

Identification of an exoribonuclease homolog, CaKEM1/CaXRN1, in Candida albicans and its characterization in filamentous growth.

KEM1/XRN1 and RAT1 are two known exoribonuclease genes in Saccharomyces cereivsiae and encode a cytoplasmic and nuclear exoribonuclease, respectively. CaKEM1/CaXRN1 and CaRAT1, the Candida albicans homologs of 5'-->3' exoribonuclease genes, were identified by protein sequence comparisons and by functional complementation of the S. cerevisiae kem1/xrn1 null mutation. The deduced amino acid sequences of CaKEM1 and CaRAT1 show 51% and 55% identities to those of the S. cerevisiae KEM1 and RAT1, respectively. The exonuclease motifs were found to be highly conserved in CaKem1p and CaRat1p. We disrupted two chromosomal copies of CaKEM1 in a diploid C. albicans strain and demonstrate that C. albicans kem1/kem1 mutants are defective in filamentous growth on filamentous-inducing media. These results imply that CaKEM1 is involved in filamentous growth of C. albicans.

Amino Acid Sequence↗

PubMed1998

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster.

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

Amino Acid Sequence↗

PubMed1997

A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates.

Exoribonucleases are important enzymes for the turnover of cellular RNA species. We have isolated the first mammalian cDNA from mouse demonstrated to encode a 5'-3' exoribonuclease. The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in cytoplasmic mRNA turnover and pre-rRNA processing similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast. Therefore, a key component of the mRNA decay system in S. cerevisiae has been conserved in evolution from yeasts to mammals. The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex-containing substrates demonstrated in binding and hydrolysis experiments. mXRN1p is the first RNA turnover function that has been localized in the cytoplasm of mammalian cells. mXRN1p was distributed in small granules and was highly enriched in discrete, prominent foci. The specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have a role in RNA turnover.

Animals↗

PubMed1991

A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates.

We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.

Adenosine Triphosphate↗

PubMed1992

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

Bacterial Proteins↗

PubMed1995

Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli.

Oligoribonuclease, an exoribonuclease specific for small oligoribonucleotides, was initially characterized 20 years ago (S. K. Niyogi and A. K. Datta, J. Biol. Chem. 250:7307-7312, 1975) and shown to be different from RNase II and polynucleotide phosphorylase. Here we demonstrate, using mutant strains and purified enzymes, that oligoribonuclease is not a manifestation of RNases D, BN, T, PH, and R, exoribonucleases discovered subsequently. Thus, oligoribonuclease is the eighth distinct exoribonuclease discovered in Escherichia coli. We also show that oligoribonuclease copurifies with polynucleotide phosphorylase.

Escherichia coli↗

PubMed2000

The yvaJ gene of Bacillus subtilis encodes a 3'-to-5' exoribonuclease and is not essential in a strain lacking polynucleotide phosphorylase.

Studies of Bacillus subtilis RNases that are involved in mRNA degradation reveal a different pattern from that of Escherichia coli. A strain lacking polynucleotide phosphorylase, the major 3'-to-5' exoribonuclease activity in cell extracts, is viable. Here, we show that the B. subtilis yvaJ gene encodes a second 3'-to-5' exoribonuclease. A strain lacking both of these RNases grows slowly but is viable. The existence of another, as yet unknown, 3'-to-5' exoribonuclease in B. subtilis is suggested.

Bacillus subtilis↗

PubMed1997

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.

Amino Acid Sequence↗

PubMed1994

Purification of a human polyribosome-associated 3' to 5' exoribonuclease.

In cells and cell-free extracts, the early steps in histone mRNA decay occur at the 3' terminus and appear to be catalyzed by a polysome-associated 3' to 5' exoribonuclease. We describe the purification of a polysomal 3' to 5' exoribonuclease that is magnesium-dependent, active at pH 7-8 in salt concentrations below 200 mM, and resistant to the inhibitor of the RNase A family of RNases. The purified enzyme is inactive with 3'-phosphorylated RNA substrates and with DNA but can degrade duplex RNA in the absence of added ATP. The enzyme migrates at approximately 37 kDa by native state gel filtration and at 33 kDa in a SDS-polyacrylamide gel. It degrades poly(A) but not a complex of poly(A) with poly(A) binding protein, and it accelerates histone mRNA decay in high salt-washed (enzyme-depleted) polysomes. Similarities between the purified exoribonuclease and the activity that degrades histone mRNA in vitro suggest that the enzyme might be a mammalian messenger ribonuclease.

Exoribonucleases↗

PubMed1996

A chloroplast transcript lacking the 3' inverted repeat is degraded by 3'-->5' exoribonuclease activity.

Chlamydomonas reinhardtii strains harboring deletions of the chloroplast atpB 3' inverted repeat (IR) are weakly phototrophic due to reduced accumulation of discrete atpB transcripts and the chloroplast ATPase beta-subunit protein. A sequence of 18 guanosine residues, which can impede a 3'-->5' exoribonuclease in vitro, is able to substitute for the atpB IR in vivo. Strains containing the poly-guanosine tract in place of the atpB 3' IR are phototrophic and accumulate near wild-type levels of discrete atpB transcripts and the ATPase beta-subunit protein. Because these atpB transcripts contain the 18 guanosine residues, and the poly-guanosine tract is not a terminator of transcription, the accumulation of discrete atpB transcripts is likely the result of impediment of 3'-->5' exoribonuclease activity. These findings support a model in which atpB transcripts lacking the 3' IR are degraded by 3'-->5' exoribonuclease activity, and demonstrate that the poly-guanosine tract can be used to study chloroplast RNA metabolism in vivo.

Adenosine Triphosphatases↗

PubMed2005

TbMP42, a protein component of the RNA editing complex in African trypanosomes, has endo-exoribonuclease activity.

RNA editing in trypanosomatids is catalyzed by a high molecular mass RNP complex, which is only partially characterized. TbMP42 is a 42 kDa protein of unknown function that copurifies with the editing complex. The polypeptide is characterized by two Zn fingers and a potential barrel structure/OB-fold at its C terminus. Using recombinant TbMP42, we show that the protein can bind to dsRNA and dsDNA but fails to recognize DNA/RNA hybrids. rTbMP42 degrades ssRNA by a 3' to 5' exoribonuclease activity. In addition, rTbMP42 has endoribonuclease activity, which preferentially hydrolyzes non-base-paired uridylate-containing sequences. Gene silencing of TbMP42 inhibits cell growth and is ultimately lethal to the parasite. Mitochondrial extracts from TbMP42-minus trypanosomes have only residual RNA editing activity and strongly reduced endo-exoribonuclease activity. However, all three activities can be restored by the addition of rTbMP42. Together, the data suggest that TbMP42 contributes both endo- and exoribonuclease activity to the editing reaction cycle.

Animals↗