Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3′ end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification–interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3′ end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p. U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events. A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle. Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S. Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits. The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo. The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus.

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid–base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.

The mammalian thyroid hormone receptor gene c-erbAα gives rise to two mRNAs that code for distinct isoforms, TRα1 and TRα2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRα1-specific polyadenylation site or TRα2-specific 5′ splice site. A previous investigation of TRα minigene expression defined a critical role for the TRα2 5′ splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEα2, enhance splicing of TRα2 in vitro as well as in vivo. Although SEα2 is located within the intron of TRα2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEα2 functions by binding trans-acting factors in HeLa nuclear extract. Protein–RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEα2. SEα2 also includes an element resembling a 5′ splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5′ splice site sequence have a dramatic effect on splicing and protein binding. Thus SEα2 and its associated factors are required for splicing of TRα2 pre-mRNA.

Several snoRNAs are essential for the sequence of cleavage events required to produce the mature forms of 18S, 5.8S, and 28S rRNA from the large precursor molecule. In the absence of U22, mature 18S rRNA fails to accumulate; U8 snoRNA is essential for accumulation of both 5.8S and 28S rRNA. The mechanisms by which snoRNAs facilitate these cleavage events is not known and might include direct cleavage or assisting the rate or efficiency of ribosome assembly. To learn more about the mechanisms of snoRNA-mediated pre-rRNA processing, an examination of the kinetics of pre-rRNA processing in Xenopus oocytes was undertaken. Correct pre-rRNA processing can be restored in snoRNA-depleted oocytes following cytoplasmic injection of the corresponding in vitro-synthesized snoRNA. Analysis of the kinetics of pre-rRNA processing in these snoRNA-rescue experiments demonstrated that the rate of accumulation of mature rRNAs was slower than that seen in untreated oocytes. The snoRNAs were imported into the nucleus at a rate and overall efficiency less than that of U1 snRNA, used as a control for import. However, sufficient levels of snoRNA were present in the nucleus to yield a functional phenotype (rescue of rRNA processing) several hours before the snoRNAs were directly detectable in the nucleus via autoradiography. This indicated that very low amounts of the snoRNA in the nucleus were sufficient for rescue. Finally, transcriptional inhibitors were used to separate transcription and processing. Failure to rescue snoRNA-mediated processing of pre-accumulated precursors is consistent with a scenario in which U8 and U22 must be present during transcription of pre-rRNA.

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5′-bulged promoter structure, as caused by the central unpaired residue A10 in its 5′ branch. Upon insertion of two uridine residues in the 3′ branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3′-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5′-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5′A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5′ bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3′ UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD → DQAD and HRIGR → HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA–protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3′ end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5′ or 3′ side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPαS (R = A, G, I) or Rp-c7-deaza-RMPαS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.

mRNA lacking a 3′ polyA tail is not translated efficiently in wild-type eukaryotic cells, but is translated efficiently in yeast ski mutants. This enhanced expression could be due to altered translational specificity. However, as the SKI genes are required for 3′ mRNA degradation, it could be a consequence of inhibition of 3′ mRNA decay. Therefore, we asked if inhibition of 3′ decay of a polyA-minus mRNA in cis would allow its efficient expression in wild-type cells. Capped in vitro reporter transcripts were prepared with or without a 3′ cis-acting element known to inhibit 3′ degradation (oligoG) and electroporated into yeast cells. The addition of oligoG to a polyA-minus mRNA enhanced expression 30-fold in wild-type cells. This level of expression was the same as that for an oligoG-minus, polyA-minus transcript in a ski mutant. The addition of oligoG did not significantly enhance the expression of polyA-minus mRNA in a ski mutant. The oligoG-dependent increase in expression was due to an increase in initial rate of translation and an increase in the functional half-life of the mRNA, similar to the effects observed in a ski mutant. The enhanced expression of the oligoG-containing RNA did not require Pab1p. We conclude that the enhanced translation of polyA-minus RNA in a ski mutant is due to inhibition of 3′ mRNA degradation. Furthermore, a polyA-minus mRNA is expressed in wild-type cells when terminated in an element known to inhibit 3′ decay in cis.

In Xenopus oocytes, the deadenylation of a specific class of maternal mRNAs results in their translational repression. Here we report the purification, characterization, and molecular cloning of the Xenopus poly(A) ribonuclease (xPARN). xPARN copurifies with two polypeptides of 62 kDa and 74 kDa, and we provide evidence that the 62-kDa protein is a proteolytic product of the 74-kDa protein. We have isolated the full-length xPARN cDNA, which contains the tripartite exonuclease domain conserved among RNase D family members, a putative RNA recognition motif, and a domain found in minichromosome maintenance proteins. Characterization of the xPARN enzyme shows that it is a poly(A)-specific 3′ exonuclease but does not require an A residue at the 3′ end. However, the addition of 25 nonadenylate residues at the 3′ terminus, or a 3′ terminal phosphate is inhibitory. Western analysis shows that xPARN is expressed throughout early development, suggesting that it may participate in the translational silencing and destabilization of maternal mRNAs during both oocyte maturation and embryogenesis. In addition, microinjection experiments demonstrate that xPARN can be activated in the oocyte nucleus in the absence of cytoplasmic components and that nuclear export of deadenylated RNA is impeded. Based on the poly(A) binding activity of xPARN in the absence of catalysis, a model for substrate specificity is proposed.

This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

The pathway of mRNA degradation has been extensively studied in the yeast, Saccharomyces cerevisiae, and it is now clear that many mRNAs decay by a deadenylation-dependent mechanism. Although several of the factors required for mRNA decay have been identified, the regulation and precise roles of many of the proteins involved remains unclear. We have developed an in vitro system that recapitulates both the deadenylation and the decapping steps of mRNA decay. Furthermore, both deadenylation and decapping are inhibited by poly(A) binding proteins in our assay. Our system has allowed us to separate the decay process from translation and we have shown that the poly(A) tail is capable of inhibiting decapping in an eIF4E-independent manner. Our in vitro system should prove invaluable in dissecting the mechanisms of mRNA turnover.

Eukaryotic initiation factor (eIF) 4A functions as a subunit of the initiation factor complex eIF4F, which mediates the binding of mRNA to the ribosome. eIF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of eIF4A during translation initiation is to unwind the mRNA secondary structure in the 5′ UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that eIF4A is also required for the translation of mRNAs possessing minimal 5′ UTR secondary structure. Were this hypothesis correct, the requirement for eIF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian eIF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5′ untranslated region are more susceptible to inhibition by the eIF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of eIF4F subunits and eIF4B to the mRNA cap structure is dramatically reduced by the eIF4A mutant and RNA secondary structure. Finally, the eIF4A mutant forms a more stable complex with eIF4G, as compared to the wild-type eIF4A, thus explaining the mechanism by which substoichiometric amounts of mutant eIF4A inhibit translation.

The 5′-terminal 88 nt of poliovirus RNA fold into a cloverleaf RNA structure and form ribonucleoprotein complexes with poly(rC) binding proteins (PCBPs; AV Gamarnik, R Andino, RNA, 1997, 3:882–892; TB Parsley, JS Towner, LB Blyn, E Ehrenfeld, BL Semler, RNA, 1997, 3:1124–1134). To determine the functional role of these ribonucleoprotein complexes in poliovirus replication, HeLa S10 translation-replication reactions were used to quantitatively assay poliovirus mRNA stability, poliovirus mRNA translation, and poliovirus negative-strand RNA synthesis. Ribohomopoly(C) RNA competitor rendered wild-type poliovirus mRNA unstable in these reactions. A 5′-terminal 7-methylguanosine cap prevented the degradation of wild-type poliovirus mRNA in the presence of ribohomopoly(C) competitor. Ribohomopoly(A), -(G), and -(U) did not adversely affect poliovirus mRNA stability. Ribohomopoly(C) competitor RNA inhibited the translation of poliovirus mRNA but did not inhibit poliovirus negative-strand RNA synthesis when poliovirus replication proteins were provided in trans using a chimeric helper mRNA possessing the hepatitis C virus IRES. A C24A mutation prevented UV crosslinking of PCBPs to 5′ cloverleaf RNA and rendered poliovirus mRNA unstable. A 5′-terminal 7-methylguanosine cap blocked the degradation of C24A mutant poliovirus mRNA. The C24A mutation did not inhibit the translation of poliovirus mRNA nor diminish viral negative-strand RNA synthesis relative to wild-type RNA. These data support the conclusion that poly(rC) binding protein(s) mediate the stability of poliovirus mRNA by binding to the 5′-terminal cloverleaf structure of poliovirus mRNA. Because of the general conservation of 5′ cloverleaf RNA sequences among picornaviruses, including C24 in loop b of the cloverleaf, we suggest that viral mRNA stability of polioviruses, coxsackieviruses, echoviruses, and rhinoviruses is mediated by interactions between PCBPs and 5′ cloverleaf RNA.

To functionally classify AU-rich elements (AREs) from six different cytokine mRNAs, we made use of two previously described HT1080-derived cellular mutants (slowA, slowC) that lack a function required for the rapid degradation of interleukin-3 (IL-3) mRNA. Here we show that the defect is specific for ARE-containing mRNAs, whereas nonsense-mediated decay is intact. Degradation of β-globin reporter transcripts mediated by the AREs of IL-3, GM-CSF, and TNFα, as well as by the structurally different and less potent AREs of IL-2 and IL-6, is impaired in both mutants. All these reporter transcripts are also sensitive to decay induced by ectopic expression of the RNA-binding protein tristetraprolin in the slowC background. Thus, we concluded that the mutants slowA and slowC define a common mRNA degradation pathway that targets cytokine AREs. In NIH3T3 cells, this decay pathway becomes incapacitated by upstream signaling from p38 MAP- or PI3-kinases, which independently stabilize cytokine ARE-containing transcripts. In contrast, c-fos ARE-directed mRNA degradation proceeds through a different pathway not affected by these kinases.

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by eIF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, eIF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses. eIF4G is likely to mediate this translational stimulation through its direct interaction with the IRES. Here, we support this hypothesis by cleaving eIF4G to separate the PABP-binding site from the portion that promotes internal initiation. eIF4G cleavage abrogates the stimulatory effect of poly(A) tail on translation. In addition, translation in extracts in which eIF4G is cleaved is resistant to inhibition by the PABP-binding protein 2 (Paip2). The eIF4G cleavage-induced loss of the stimulatory effect of poly(A) on translation was mimicked by the addition of the C-terminal portion of eIF4G. Thus, PABP stimulates picornavirus translation through its interaction with eIF4G.