Functional interactions between yeast mitochondrial ribosomes and mRNA 5' untranslated leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5'-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5'-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.     

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.     

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.     

Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae

The genetic depletion of yeast Rrp5p results in a synthesis defect of both 18S and 5.8S ribosomal RNAs (Venema J, Tollervey D. 1996. EMBO J 15:5701-5714). We have isolated the RRP5gene in a genetic approach aimed to select for yeast factors interfering with protein import into mitochondria. We describe here a striking feature of Rrp5p amino acid sequence, namely the presence of twelve putative S1 RNA-binding motifs and seven tetratricopeptide repeats (TPR) motifs. We have constructed two conditional temperature-sensitive alleles of RRP5 gene and analyzed them for associated rRNA-processing defects. First, a functional "bipartite gene" was generated revealing that the S1 and TPR parts of the protein can act independently of each other. We also generated a two amino acid deletion in TPR unit 1 (rrp5delta6 allele). The two mutant forms of Rrp5p were shown to cause a defect in 18S rRNA synthesis with no detectable effects on 5.8S rRNA production. However, the rRNA processing pathway was differently affected in each case. Interestingly, the ROK1 gene which, like RRP5, was previously isolated in a screen for synthetic lethal mutations with snR10 deletion, was here identified as a high copy suppressor of the rrp5delta6 temperature-sensitive allele. ROK1 also acts as a low copy suppressor but cannot bypass the cellular requirement for RRP5. Furthermore, we show that suppression by the Rok1p putative RNA helicase rescues the 18S rRNA synthesis defect caused by the rrp5delta6 mutation.     

Mrs5p, an essential protein of the mitochondrial intermembrane space, affects protein import into yeast mitochondria

We have isolated a yeast nuclear gene that suppresses the previously described respiration-deficient mrs2-1 mutation when present on a multicopy plasmid. Elevated gene dosage of this new gene, termed MRS5, suppresses also the pet phenotype of a mitochondrial splicing-deficient group II intron mutation M1301. The MRS5 gene product, a 13-kDa protein of low abundance, shows no similarity to other known proteins and is associated with the inner mitochondrial membrane, protruding into the intermembrane space. MRS5 codes for an essential protein, as the disruption of this gene is lethal even during growth on fermentable carbon sources. Thus, the Mrs5 protein seems to be involved in mitochondrial key functions aside from oxidative energy conservation, which is dispensable in fermenting yeast cells. Depletion of Mrs5p in yeast cells causes accumulation of unprocessed precursors of the mitochondrial hsp60 protein and defects in all cytochrome complexes. These findings suggest an essential role of Mrs5p in mitochondrial biogenesis.     

Reduced dosage of genes encoding ribosomal protein S18 suppresses a mitochondrial initiation codon mutation in Saccharomyces cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18a delta and rps18b delta null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18a delta then rps18b delta. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.     

Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate

Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.     

Variable effects of the conserved RNA hairpin element upon 3' end processing of histone pre-mRNA in vitro

We have studied the requirements for efficient histone-specific RNA 3' processing in nuclear extract from mammalian tissue culture cells. Processing is strongly impaired by mutations in the pre-mRNA spacer element that reduce the base-pairing potential with U7 RNA. Moreover, by exchanging the hairpin and spacer elements of two differently processed H4 genes, we find that this difference is exclusively due to the spacer element. Finally, processing is inhibited by the addition of competitor RNAs, if these contain a wild-type spacer sequence, but not if their spacer element is mutated. Conversely, the importance of the hairpin for histone RNA 3' processing is highly variable: A hairpin mutant of the H4-12 gene is processed with almost wild-type efficiency in extract from K21 mouse mastocytoma cells but is strongly affected in HeLa cell extract, whereas an identical hairpin mutant of the H4-1 gene is affected in both extracts. The hairpin defect of H4-12-specific RNA in HeLa cells can be overcome by a compensatory mutation that increases the base complementarity to U7 snRNA. Very similar results were also obtained in RNA competition experiments: processing of H4-12-specific RNA can be competed by RNA carrying a wild-type hairpin element in extract from HeLa, but not K21 cells, whereas processing of H4-1-specific RNA can be competed in both extracts. With two additional histone genes we obtained results that were in one case intermediate and in the other similar to those obtained with H4-1. These results suggest that hairpin binding factor(s) can cooperatively support the ability of U7 snRNPs to form an active processing complex, but is(are) not directly involved in the processing mechanism.     

Mutational analysis of the coat protein gene of potato virus X: effects on virion morphology and viral pathogenicity

The role of the coat protein of potato virus X (PVX) was investigated by site-directed mutation of the coat protein gene. Mutant viruses with in-frame deletions in the 5' end of the coat protein gene were capable of systemically infecting plants, but produced virions with atypical morphology. Viruses with a frameshift mutation near the 5' end or with deletions in the central part of the coat protein gene failed to accumulate at detectable levels, even in the inoculated leaf. In protoplasts, mutants that infected systemically either had a wild-type phenotype or showed a small reduction in accumulation of genomic RNA. The other mutants, which did not accumulate in the inoculated leaf, were unaffected in genomic RNA accumulation 8 hr postinoculation, but at 16 hr and later they accumulated less genomic RNA than wild-type virus. None of the mutations had an effect on accumulation of negative-strand RNA. The data indicate that efficient accumulation and spread of PVX, even in the inoculated leaf, requires coat protein production and encapsidation of the viral RNA.     

Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm.     

Retransplantation of patients with severe posttransplant hepatitis B in the first allograft

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.