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PubMed2005

Balance between transcription and RNA degradation is vital for Saccharomyces cerevisiae mitochondria: reduced transcription rescues the phenotype of deficient RNA degradation.

The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3'-to-5' exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3Delta) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3Delta strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation.

Alleles↗

PubMed1999

Functions of the exosome in rRNA, snoRNA and snRNA synthesis.

The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.

Base Sequence↗

PubMed2000

A Sm-like protein complex that participates in mRNA degradation.

In eukaryotes, seven Sm proteins bind to the U1, U2, U4 and U5 spliceosomal snRNAs while seven Smlike proteins (Lsm2p-Lsm8p) are associated with U6 snRNA. Another yeast Sm-like protein, Lsm1p, does not interact with U6 snRNA. Surprisingly, using the tandem affinity purification (TAP) method, we identified Lsm1p among the subunits associated with Lsm3p. Coprecipitation experiments demonstrated that Lsm1p, together with Lsm2p-Lsm7p, forms a new seven-subunit complex. We purified the two related Sm-like protein complexes and identified the proteins recovered in the purified preparations by mass spectrometry. This confirmed the association of the Lsm2p-Lsm8p complex with U6 snRNA. In contrast, the Lsm1p-Lsm7p complex is associated with Pat1p and Xrn1p exoribonuclease, suggesting a role in mRNA degradation. Deletions of LSM1, 6, 7 and PAT1 genes increased the half-life of reporter mRNAs. Interestingly, accumulating mRNAs were capped, suggesting a block in mRNA decay at the decapping step. These results indicate the involvement of a new conserved Sm-like protein complex and a new factor, Pat1p, in mRNA degradation and suggest a physical connection between decapping and exonuclease trimming.

Codon, Nonsense↗

PubMed2002

PNPase activity determines the efficiency of mRNA 3'-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts.

The exoribonuclease polynucleotide phosphorylase (PNPase) has been implicated in mRNA processing and degradation in bacteria as well as in chloroplasts of higher plants. Here, we report the first comprehensive in vivo study of chloroplast PNPase function. Modulation of PNPase activity in Arabidopsis chloroplasts by a reverse genetic approach revealed that, although this enzyme is essential for efficient 3'-end processing of mRNAs, it is insufficient to mediate transcript degradation. Surprisingly, we identified PNPase as also being indispensable for 3'-end maturation of 23S rRNA transcripts. Analysis of tRNA amounts in transgenic Arabidopsis plants suggests a direct correlation of PNPase activity and tRNA levels, indicating an additional function of this exoribo nuclease in tRNA decay. Moreover, the extent of polyadenylated mRNAs in chloroplasts is negatively correlated with PNPase activity. Together, our results attribute novel functions to PNPase in the metabolism of all major classes of plastid RNAs and suggest an unexpectedly complex role for PNPase in RNA processing and decay.

Arabidopsis↗

PubMed2003

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis.

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.

Amino Acid Sequence↗

PubMed1999

Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae.

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

5' Untranslated Regions↗

PubMed1988

Transcriptional terminators in the caa-cal operon and cai gene.

We have analysed by S1 nuclease mapping the in vivo termination sites of transcription of the caa-cal operon and cai gene. The termination region for caa mRNA (T1A terminator) features characteristics of a rho-independent terminator. This terminator is a convergent transcription terminator, its complementary secondary structure being present at the 3'-end of cai mRNA. The caa-cal mRNA terminator (T2A terminator) has a stable potential secondary structure and shows homology with rho-dependent terminators. In vitro transcription of caa-cal operon demonstrated that the two terminators T1A and T2A are efficient. The 3'-ends of the mRNAs which end at T1A and T2A were analysed by S1 mapping with total RNA purified from a mutant strain deficient in exoribonuclease activities, in particular RNase II. The results suggest that the potential secondary structures of T1A and T2A are sufficiently stable to prevent 3'-end degradation by RNase II. On the other hand, the T2A terminator should be efficient enough to stop transcription through the downstream DNA region involved in pColA replication.

Base Sequence↗

PubMed1989

A stable core region of the tra operon mRNA of plasmid R1-19.

The degradation of the polycistronic tra-mRNA of the resistance plasmid R1-19 leads to the accumulation of a well defined series of stable mRNA species. The majority of the most stable mRNAs contains the message for the traA gene only. The differently sized stable mRNAs possess a common 3'terminus within the traL gene but vary at their 5' ends. The 3'terminus probably results from protection against exoribonucleases by a secondary structural feature. We propose that the 5' ends are generated by endoribonucleolytic cleavage. The stability of this part of the tra-mRNA exceeds 30 minutes and probably increases the rate of expression of the traA gene product propilin, the precursor of the sex pilus subunit. The expression of propilin and its processing into a protein of the molecular weight of mature pilin is demonstrated with the isolated gene. The sequence of the so far unknown genes traL and traE of R1-19 is presented.

Amino Acid Sequence↗

PubMed1994

Polynucleotide phosphorylase of Escherichia coli induces the degradation of its RNase III processed messenger by preventing its translation.

Polynucleotide phosphorylase, a 3' to 5' processive exoribonuclease is post-transcriptionally autocontrolled and it was previously shown that this control is dependent on a 5' processing by RNase III. In this paper, the mechanism of regulation is analyzed by studying the properties of a pnp-lacZ translational gene fusion. It is shown that this message is stable, even when processed by RNase III, and that the degradation rate is directly linked to the intracellular concentration of polynucleotide phosphorylase or to the pnp-lacZ messenger translation rate. Mutations able to decrease the level of repression are all located in the ribosome loading site. Taken together, these results suggest that polynucleotide phosphorylase is able to recognize specifically the processed messenger and to prevent its translation, thus allowing degradation of the message.

Base Sequence↗

PubMed1997

The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains.

Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain.

Amino Acid Sequence↗

PubMed1999

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA.

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.

Base Sequence↗

PubMed2026

Structural insights into RNA phosphorylation by the RNase PNK module of the human rixosome complex.

The mammalian rixosome complex is a large multi-subunit complex that plays essential roles in ribosome assembly and heterochromatin maintenance. Three structural proteins form the stable core of the rixosome to which three enzymatic modules are flexibly tethered including an RNA processing module, AAA-ATPase, and SUMO protease. The RNA processing module is formed by RNase PNK, a tetrameric assembly comprising two copies each of the LAS1L endoribonuclease (RNase) and the NOL9 polynucleotide kinase (PNK). Using single particle cryo-EM, we determined ATPγS and AMP-PNP/RNA-bound structures of human RNase PNK. The structures revealed the overall butterfly-like architecture of the complex and provide new insights into the mechanism of RNA accommodation and 5' hydroxyl phosphorylation within the NOL9 active site. Through reconstitution studies and molecular modeling, we establish how RNase PNK is incorporated into the larger rixosome complex by a distinct domain of LAS1L. Finally, we show that the human 5'-3' exoribonuclease XRN2 directly associates with RNase PNK and selectively degrades NOL9-phosphorylated RNA in vitro, thereby linking ITS2 processing by the rixosome to processive exonucleolytic decay. Collectively this work establishes an updated model for how the rixosome integrates its diverse enzymatic activities to regulate ITS2 processing.

Humans↗

PubMed2006

Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis.

Plastid isoprenoids (including hormones and photosynthetic pigments) are essential for plant growth and development, but relatively little is known of how the production of their metabolic precursors via the recently elucidated methylerythritol phosphate (MEP) pathway is regulated. We have identified an Arabidopsis (Arabidopsis thaliana) mutant that survives an otherwise lethal block of the MEP pathway with fosmidomycin (FSM). In rif10 (resistant to inhibition with FSM 10) plants, the accumulation of flux-controlling enzymes of the pathway is posttranscriptionally up-regulated. Strikingly, this phenotype is linked to a lower accumulation of plastidial isoprenoid pigments such as chlorophylls and carotenoids, resulting in mutant plants that are paler and smaller than the wild type. The rif10 mutant is impaired in plastid RNA processing due to a T-DNA insertion in the coding region of the At3g03710 gene encoding the chloroplast-targeted exoribonuclease polyribonucleotide phosphorylase. FSM resistance and other rif10-like phenotypes were also observed in wild-type Arabidopsis, tomato (Lycopersicon esculentum), and rice (Oryza sativa) seedlings grown in the presence of sublethal concentrations of chloramphenicol (an inhibitor of protein synthesis in plastids). By contrast, treatment with norflurazon (an inhibitor of carotenoid biosynthesis causing a similar pale cotyledon phenotype) did not result in FSM resistance. Together, the results support that plastome-encoded proteins are involved in negatively regulating the posttranscriptional accumulation of specific nuclear-encoded MEP pathway enzymes in chloroplasts. Regulation of the MEP pathway by a mechanism dependent on plastid cues might function under physiological conditions to finely adjust plastidial isoprenoid biosynthesis to the metabolic capabilities or requirements of plastids.

Arabidopsis↗

PubMed2004

Antisense transcript and RNA processing alterations suppress instability of polyadenylated mRNA in chlamydomonas chloroplasts.

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Delta26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Delta26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3' poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS-, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Delta26, in which atpB mRNA is unstable because of the lack of a 3' stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3'-->5' exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

Animals↗

PubMed1993

Initiation of mRNA decay in Bacillus subtilis.

Ribosome stalling in the leader region of ermC mRNA results in a 10-15-fold increase in ermC mRNA half-life in Bacillus subtilis. Fusion of the ermC 5' regulatory region to several B. subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5' region acts as a general '5' stabilizer'. RNA products of an ermC-lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3'-to-5' exoribonuclease activity. Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5' and 3' ends. It can be concluded from this study that initiation of mRNA decay in B. subtilis generally occurs at or near the 5' terminus.

Bacillus subtilis↗

PubMed1994

A protein complex mediating mRNA degradation in Escherichia coli.

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribonucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional polypeptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.

Amino Acid Sequence↗

PubMed1994

mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures.

Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases. The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo. In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro. This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo. Its mechanism of action, and possible role in regulating mRNA degradation, is discussed.

Bacterial Proteins↗

PubMed1997

Inert chromium and cobalt complexes as probes of magnesium-dependent enzymes. Evaluation of the mechanistic role of the essential metal cofactor in Escherichia coli exonuclease III.

An investigation of the metal ion dependence of Escherichia coli exonuclease III, 3'-5'-exonuclease and exoribonuclease H activities is reported. Catalytic activation of E. coli exonuclease III has been examined for a series of inert chromium complexes Cr(NH3)6-x(H2O)x3+ (x = 0-6) that bear water and ammine ligands in well defined inner-sphere geometries. The importance of hydrogen bonding and electrostatic stabilization for catalysis of this reaction were quantitatively evaluated. Catalytic activation by the essential metal cofactor appears to be mediated through transition-state stabilization by outer-sphere complex formation with substrate. Hydrogen bonding to metal-bound water molecules is the dominant stabilizing interaction.

Catalysis↗