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PubMed2004

Posttranscriptional regulation of the karyogamy gene by Kem1p/Xrn1p exoribonuclease and Rok1p RNA helicase of Saccharomyces cerevisiae.

The major biochemical activities ascribed to Kem1p/Xrn1p of Saccharomyces cerevisiae are 5'-3' exoribonuclease functioning in RNA turnover and a microtubule-binding protein. Mutational analysis has shown that Kem1p/Xrn1p participates in microtubule-related functions such as nuclear fusion (karyogamy) during mating, chromosome transmission, and spindle pole body duplication. Here, evidence is presented that Kem1p plays a specific role in nuclear fusion by affecting, at the posttranscriptional level, the pheromone induction of the karyogamy-specific transcription factor Kar4p and the expression of Rok1p, a putative RNA helicase. We found that Rok1p itself also affects the pheromone induction of Kar4p and thereby participates in nuclear fusion. Analysis of the active-site mutations, xrn1-D206A or D208A, shows that nuclear fusion as well as the Rok1p synthesis do not require the exoribonuclease activity of Kem1p. Our data provide an important insight into the gene-specific regulatory function mediated by the general RNA-modulating enzymes.

Base Sequence↗

PubMed2001

Exoribonucleases and their multiple roles in RNA metabolism.

In recent years there has been a dramatic shift in our thinking about ribonucleases (RNases). Although they were once considered to be nonspecific, degradative enzymes, it is now clear that RNases play a central role in every aspect of cellular RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms, and end-turnover of certain RNAs. Recognition of the importance of this class of enzymes has led to an explosion of work and the establishment of significant new concepts. Thus, we now realize that RNases, both endoribonucleases and exoribonucleases, can be highly specific for particular sequences or structures. It has also become apparent that a single cell can contain a large number of distinct RNases, approaching as many as 20 members, often with overlapping specificities. Some RNases also have been found to be components of supramolecular complexes and to function in concert with other enzymes to carry out their role in RNA metabolism. This review focuses on the exoribonucleases, both prokaryotic and eukaryotic, and details their structure, catalytic properties, and physiological function.

Bacterial Proteins↗

PubMed1997

The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases.

We identified a complex in S. cerevisiae, the "exosome," consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p). Remarkably, four of these proteins are homologous to characterized bacterial 3'-->5' exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH. Recombinant Rrp4p, Rrp44p, and Rrp41p are 3'-->5' exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively. All components of the exosome are required for 3' processing of the 5.8S rRNA. Human Rrp4p is found in a comparably sized complex, and expression of the hRRP4 gene in yeast complements the rrp4-1 mutation. We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.

Amino Acid Sequence↗

PubMed1999

Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T.

Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 3' terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 3' end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 3' residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 3' end has no effect on 5' maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T- cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 5' and 3' termini of most stable RNAs.

Blotting, Northern↗

PubMed1984

Isolation and properties of a single-strand 5'----3' exoribonuclease from Ehrlich ascites tumor cell nucleoli.

A single-strand-specific, nucleolar exoribonuclease from Ehrlich ascites tumor cells has been isolated and purified free from other nucleases. The exonuclease degraded single-stranded RNA processively from either a 5'-hydroxyl or a 5'-phosphorylated end and released 5'-mononucleotides. The enzyme digested single-strand poly(C), poly(U), and poly(A) equally well but did not degrade duplex poly(C).poly(I) or poly(A).poly(U). Less than 0.2% of duplex DNA or 1.5% of heat-denatured DNA was degraded under the conditions which resulted in greater than 26% degradation of RNA. The ribonuclease required Mg2+ (0.2 mM) for optimum activity and was inhibited by ethylenediaminetetraacetic acid but not by human placental RNase inhibitor. The native enzyme had a Stokes radius of 42 A and a sedimentation coefficient (S20,w) of 4.3 S. From these values, an apparent molecular weight of 76 000 was derived by using the Svedberg equation. The localization and unique mode of degradation suggest a role for the 5'----3' exoribonuclease in ribosomal RNA processing.

Animals↗

PubMed2004

Two exoribonucleases act sequentially to process mature 3'-ends of atp9 mRNAs in Arabidopsis mitochondria.

In plant mitochondria, transcription proceeds well beyond the region that will become mature 3' extremities of mRNAs, and the mechanisms of 3' maturation are largely unknown. Here, we show the involvement of two exoribonucleases, AtmtPNPase and AtmtRNaseII, in the 3' processing of atp9 mRNAs in Arabidopsis thaliana mitochondria. Down-regulation of AtmtPNPase results in the accumulation of pretranscripts of several times the size of mature atp9 mRNAs, indicating that 3' processing of these transcripts is performed mainly exonucleolytically by AtmtPNPase. This enzyme is however not sufficient to completely process atp9 mRNAs, because with down-regulation of another mitochondrial exoribonuclease, AtmtRNaseII, about half of atp9 transcripts exhibit short 3' nucleotide extensions compared with mature mRNAs. These short extensions can be efficiently removed by AtmtRNaseII in vitro. Taken together, these results show that 3' processing of atp9 mRNAs in Arabidopsis mitochondria is, at least, a two-step phenomenon. First, AtmtPNPase is involved in removing 3' extensions that may reach several kilobases. Second, AtmtRNaseII degrades short nucleotidic extensions to generate the mature 3'-ends.

Arabidopsis↗

PubMed2006

Substrate recognition and catalysis by the exoribonuclease RNase R.

RNase R is a processive, 3' to 5' hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3' overhang is present. Using a variety of specifically designed substrates, we show here that a 3' overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5' overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3' to 5' polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3' termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.

Catalysis↗

PubMed2001

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast.

The Schizosaccharomyces pombe dhp1(+) gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5'-->3' exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5'-->3' exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)(+) RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1(+) is required for proper chromosome segregation as well as for poly(A)(+) RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1(+). We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

Cell Division↗

PubMed1993

Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo.

Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the enzymes necessary for defining the complete processing pathway for E. coli tRNA precursors.

Amino Acyl-tRNA Synthetases↗

PubMed1991

Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1.

The portion of the internal transcribed spacer 1 found on 20S pre-rRNA accumulates in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1, showing that an endonucleolytic cleavage at the 3' terminus of 18S rRNA is involved in the 20S pre-rRNA to 18S mature rRNA conversion. Smaller fragments of the spacer sequence are also found. The exoribonuclease may be involved as a cytoplasmic RNase in the hydrolysis of the spacer.

Exoribonucleases↗

PubMed1997

An essential function for the phosphate-dependent exoribonucleases RNase PH and polynucleotide phosphorylase.

Escherichia coli cells lacking both polynucleotide phosphorylase (PNPase) and RNase PH, the only known P(i)-dependent exoribonucleases, were previously shown to grow slowly at 37 degrees C and to display a dramatically reduced level of tRNA(Tyr)su3+ suppressor activity. Here we show that the RNase PH-negative, PNP-negative double-mutant strain actually displays a reversible cold-sensitive phenotype and that tRNA biosynthesis is normal. In contrast, ribosome structure and function are severely affected, particularly at lower temperatures. At 31 degrees C, the amount of 50S subunit is dramatically reduced and 23S rRNA is degraded. Moreover, cells that had been incubated at 42 degrees C immediately cease growing and synthesizing protein upon a shift to 31 degrees C, suggesting that the ribosomes synthesized at the higher temperature are defective and unable to function at the lower temperature. These data indicate that RNase PH and PNPase play an essential role that affects ribosome metabolism and that this function cannot be taken over by any of the hydrolytic exoribonucleases present in the cell.

Cold Temperature↗

PubMed1993

An essential yeast gene with homology to the exonuclease-encoding XRN1/KEM1 gene also encodes a protein with exoribonuclease activity.

An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomyces cerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as well as activities involving chromosomal DNA pairing and mechanics. Preliminary analysis of an hke1ts mutant reveals a precipitous decline in the translation of mRNA at the nonpermissive temperature. Sporulation of heterozygous HKE1/hke1::URA3 diploids reveals that this gene, unlike the highly related XRN1/KEM1 gene, is essential for cell viability. Overexpression of the homologous gene product, p175, failed to rescue cells lacking a functional p116. In vitro studies demonstrate that p116 is a protein with 5'-->3' exoribonuclease activity, a major activity of the related p175. An immunoreactive RNase activity of 116 kDa is abolished with antiserum against p116. Both the level of this protein and the RNase activity correlate with HKE1 gene dosage. The RNase activity purifies coincidentally with a previously described 116-kDa RNase having 5'-->3' exoribonuclease activity.

Cloning, Molecular↗

PubMed2000

Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p.

The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5. 8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

Amino Acid Sequence↗

PubMed1991

mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo.

Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes. For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript. We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro. Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo. These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo. The implications for the regulation of mRNA stability are discussed.

Amino Acid Sequence↗

PubMed1984

A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN.

A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction. Extracts of the mutant strain were lacking the aforementioned RNase activities. The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages. In addition, RNA synthesis was normal and no precursor RNA accumulation was observed. The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E. coli. The implications of these findings to our understanding of RNA processing and degradation are discussed.

Endoribonucleases↗

PubMed1980

Purification and characterization of a poly(A)-specific exoribonuclease from calf thymus.

An exoribonuclease from calf thymus which specifically cleaves poly(A) in the single- or in the double-stranded form has been isolated and purified to homogeneity. The enzyme has a molecular weight of about 80,000 as estimated by gel filtration, and consists of two subunits with molecular weights of 58,000 and 31,000 as analyzed by sodium dodecyl sulfate-gel electrophoresis. For optimal activity, the poly(A)-specific exoribonuclease requires divalent cations, alkaline pH, and 39 degrees C. The enzyme is inhibited at 0.2 ionic strength and is sensitive to reagents for thiol groups. The only product formed by the action of the enzyme is 5'-AMP. It is suggested that this enzyme plays a role in the degradation of the poly(A) sequence of mRNA in the nucleus.

Animals↗

PubMed1985

Purification and mode of action of a microsomal exoribonuclease from rat liver.

An exoribonuclease has been purified nearly to homogeneity from rat liver microsomes and its mode of action and general properties were studied. The molecular weight values for the enzyme, as estimated by gel filtration and SDS-polyacrylamide gel electrophoresis, were 88 000 and 92 000, respectively. The enzyme produced, via a processive mechanism Ado5'P as the only product from poly(A). The results of the hydrolysis of 4 S (Ado5'P)n and (Ado3'P)n by the exoribonuclease with or without alkaline phosphatase and the inhibition of the enzymatic activity by oligonucleotides having a 3'-phosphate group in the 3'-terminus suggested that the degradation proceeds in the 3' to 5' direction. These findings were confirmed by the analysis of hydrolyzed products of various oligoadenylates and Ado3'PUrdPGuo and by the comparison of the rates of hydrolysis of (Ado3'P)2Ado by the enzyme in the presence of varying amounts of (Ado3'P)3. Mg2+ was required for the enzymatic activity, and Mn2+ partially substituted for Mg2+. The activity of the enzyme was stimulated by K+ and spermine.

Alkaline Phosphatase↗

PubMed2005

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing.

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.

Amino Acid Motifs↗