The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3′ splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5′ pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an ∼62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of α phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5′-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid–base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.

Although ribozymes offer tremendous potential for posttranscriptionally controlling expression of targeted genes, their utility is often limited by the accessibility of the targeted regions within the RNA transcripts. Here we describe a method that identifies RNA regions that are accessible to oligonucleotides. Based on this selection protocol, we show that construction of hammerhead ribozymes targeted to the identified regions results in catalytic activities that are consistently and substantially greater than those of ribozymes designed on the basis of computer modeling. Identification of accessible sites should also be widely applicable to design of antisense oligonucleotides and DNAzymes.

The cap-binding complex eIF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: eIF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865–4874). Here we exploit this approach to assess which of the other subunits of eIF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the λ phage antiterminator protein N to generate the chimeric proteins λ4A, λ4E-102 (abolished cap binding), and λ4E-73–102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific λ-N binding site (boxB) in the intercistronic space. We show that λ4E-102, but neither the double mutant λ4E-73–102 nor λ4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all λ-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, λ4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.

The biosynthesis and function of eukaryotic mRNAs requires a series of events including nuclear polyadenylation, transport to the cytoplasm, translation, and ultimately mRNA degradation. To identify the interrelationships between these events, we examined the synthesis and fate of mRNAs in several strains defective in mRNA export. Strains carrying lesions in RAT7, GLE1, MEX67, and RAT8, produce nascent transcripts carrying poly(A) tails roughly 30 residues longer than the nascent poly(A) tails observed in wild type. In the rat7-1, rat8-2, and mex67-5 strains, the hyperadenylated transcripts undergo a novel form of deadenylation to chase into a population with normal poly(A) tail lengths, which cofractionate with polysomes, undergo nonsense-mediated decay, and are degraded by the normal cytoplasmic decay machinery. This suggests a relationship between the mechanism of processing to a normal poly(A) tail length and the ability of these transcripts to proceed in their metabolism. These observations provide further support for the view that mRNA 3′-end formation and mRNA export are mechanistically coupled events.

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells. Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3′-untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3′ UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3′ UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

We describe a new approach to elucidate the role of 3′-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 3′-end-processing machinery, is sufficient for the inhibition of not only 3′-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3′-end processing, thereby establishing that 3′-end processing is required for the splicing of a 3′ terminal intron in vivo. Because the NS1A protein causes a global suppression of 3′-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 3′-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3′-end formation. In contrast, the 3′-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

Members of the 70-kDa family of molecular chaperones assist in a number of molecular interactions that are essential under both normal and stress conditions. These functions require ATP and co-chaperone molecules and are associated with a cyclic transition of intramolecular conformational changes. As a new putative function, we have previously shown that mammalian Hsp/Hsc70 as well as a distant relative, Hsp110, selectively bind certain RNA sequences via their N-terminal ATP-binding domain. To investigate this phenomenon in more detail, here we examined RNA-binding affinity and specificity of various deletion mutants of human Hsp70. We demonstrate, that, although the N-terminal ATPase domain alone is sufficient for RNA binding, its binding affinity is considerably reduced when compared to that of the full-length protein. Additionally, we provide evidence that binding of RNA to a membrane-immobilized protein partner results in complete loss of RNA sequence specificity. Using various Hsp70 homologs, we show distinct RNA-binding properties of these proteins judged by sequence specificity, ribopolymer sensitivity, and northwestern analysis. Finally, we present data disclosing that RNA binding by DnaK, the Escherichia coli homolog, is influenced by the activity of its co-chaperones, DnaJ and GrpE. We conclude that the RNA-binding capability of this class of molecular chaperones is a conserved feature and it is strongly influenced by the structural and conformational properties. Furthermore, the notion that RNA binding of some Hsp70 family members is influenced by co-chaperones suggests an RNA-binding cycle resembling the protein-binding property of the chaperones.

Cricket paralysis-like viruses have a dicistronic positive-strand RNA genome. These viruses produce capsid proteins through internal ribosome entry site (IRES)-mediated translation. The IRES element of one of these viruses, Plautia stali intestine virus (PSIV), forms a pseudoknot immediately upstream from the capsid coding sequence, and initiates translation from other than methionine. Previously, we estimated that the IRES element of PSIV consists of seven stem-loops using the program MFOLD; however, experimental evidence of the predicted structures was not shown, except for stem-loop VI, which was responsible for formation of the pseudoknot. To determine the whole structure of the PSIV-IRES element, we introduced compensatory mutations into the upstream MFOLD-predicted helical segments. Mutation analysis showed that stem-loop V exists as predicted, but stem-loop IV is shorter than predicted. The structure of stem-loop III is different from predicted, and stem-loops I and II are not necessary for IRES activity. In addition, we identified two new pseudoknots in the IRES element of PSIV. The complementary sequence segments that are responsible for formation of the two pseudoknots are also observed in cricket paralysis virus (CrPV) and CrPV-like viruses such as Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), himetobi P virus (HiPV), Triatoma virus (TrV), and black queen-cell virus (BQCV), although each sequence is distinct in each virus. Considering the three pseudoknots, we constructed a tertiary structure model of the PSIV-IRES element. This structural model is applicable to other CrPV-like viruses, indicating that other CrPV-like viruses can also initiate translation from other than methionine.

Ribosome synthesis is a highly complex process and constitutes a major cellular activity. The biogenesis of this ribonucleoprotein assembly requires a multitude of protein trans-acting factors including several putative ATP-dependent RNA helicases of the DEAD-box and related protein families. Here we show that the previously uncharacterized Saccharomyces cerevisiae open reading frame YLR276C, hereafter named DBP9 (DEAD-box protein 9), encodes an essential nucleolar protein involved in 60S-ribosomal-subunit biogenesis. Genetic depletion of Dbp9p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This terminal phenotype is likely due to the instability of early pre-ribosomal particles, as evidenced by the low steady-state levels and the decreased synthesis of the 27S precursors to mature 25S and 5.8S rRNAs. In agreement with a role of Dbp9p in 60S subunit synthesis, we find that increased Dbp9p dosage efficiently suppresses certain dbp6 alleles and that dbp6/dbp9 double mutants show synthetic lethality. Furthermore, Dbp6p and Dbp9p weakly interact in a yeast two-hybrid assay. Altogether, our findings indicate an intimate functional interaction between Dbp6p and Dbp9p during the process of 60S-ribosomal-subunit assembly.

RNA editing in Trypanosoma brucei produces mature mRNAs by posttranscriptional insertion and deletion of uridylates (Us) by a series of catalytic steps, which include endoribonucleolytic cleavage, 3′ terminal addition or removal of Us, and RNA ligation. Preedited mRNA (pre-mRNA) and guide RNA (gRNA) that are mutated at or near the editing site (ES) were used to examine the effects on the specificity of in vitro editing. Sequences that are not predicted to form a gRNA/pre-mRNA base pair immediately 5′ to the ES still supported accurate editing. Substitution of a non-U nucleotide at various positions within a stretch of Us that are normally removed from the ES resulted in deletion of only the Us that were 3′ to the substituted nucleotide. Overall, ES selection by the endoribonuclease, the specificity of the 3′ exoribonuclease for Us, and ligation appear to act in concert to ensure the production of accurately edited RNA.

Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (∼435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads and used to deplete rabbit reticulocyte lysate (RRL) of IRES-binding proteins. Translation initiation factors eIF4G, eIF4A, and eIF4B bound to the 3′ domain of the FMDV IRES. Depletion of eIF4G from RRL by this region of the FMDV IRES correlated with the loss of translational capacity of the RRL for capped, uncapped, and FMDV IRES-dependent mRNAs. However, this depleted RRL still supported hepatitis C virus IRES-directed translation. Poly (rC) binding protein-2 bound to the central domain of the FMDV IRES, but depletion of RRL with this IRES domain had no effect on FMDV IRES-directed translation initiation.

We previously reported a bifunctional ribozyme that catalyzes self-aminoacylation and subsequent acyl-transfer to a tRNA. The ribozyme selectively recognizes a biotinyl-glutamine substrate, and charges the tRNA molecule in trans. Structurally, there are two catalytic domains, referred to as glutamine-recognition (QR) and acyl-transferase (ATRib). We report here the essential catalytic core of the QR domain as determined by extensive biochemical probing, mutation, and structural minimization. The minimal core of the QR domain is a 29-nt helix-loop RNA, which is also able to glutaminylate ATRib in trans. Its amino acid binding site is embedded in an 11-nt cluster that is adjacent to the loop that interacts with the ATRib domain. Our study shows that a minihelix-loop RNA can act as a trans-aminoacylation catalyst, which lends support for the critical role of minihelix-loops in the early evolution of the aminoacylation system.

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codon[bull ]anticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

The well-studied interaction between the MS2 coat protein and its cognate hairpin was used to test the utility of the methylphosphonate linkage as a phosphate analog. A nitrocellulose filter binding assay was used to measure the change in binding affinity upon introduction of a single methylphosphonate stereoisomer at 13 different positions in the RNA hairpin. Comparing these data to the available crystal structure of the complex shows that all phosphates that are in proximity to the protein show a weaker binding affinity when substituted with a phosphorothioate and control positions show no change. However, in two cases, a methylphosphonate isomer either increased or decreased the binding affinity where no interaction can be detected in the crystal structure. It is possible that methylphosphonate substitutions at these positions affect the structure or flexibility of the hairpin. The utility of the methylphosphonate substitution is compared to phosphate ethylation and phosphorothioate substitution experiments previously performed on the same system.

A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons.

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs that are present in the recently isolated LEM125 strain [Thiemann et al., EMBO J, 1994, 13:5689–5700]. We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.

Nonsense-mediated decay (NMD), also called mRNA surveillance, is an evolutionarily conserved pathway that degrades mRNAs that prematurely terminate translation. To date, the pathway in mammalian cells has been shown to depend on the presence of a cis-acting destabilizing element that usually consists of an exon–exon junction generated by the process of pre-mRNA splicing. Whether or not mRNAs that derive from naturally intronless genes, that is, mRNAs not formed by the process of splicing, are also subject to NMD has yet to be investigated. The possibility of NMD is certainly reasonable considering that mRNAs of Saccharomyces cerevisiae are subject to NMD even though most derive from naturally intronless genes. In fact, mRNAs of S. cerevisiae generally harbor a loosely defined splicing-independent destabilizing element that has been proposed to function in NMD analogously to the spliced exon–exon junction of mammalian mRNAs. Here, we demonstrate that nonsense codons introduced into naturally intronless genes encoding mouse heat shock protein 70 or human histone H4 fail to elicit NMD. Failure is most likely because each mRNA lacks a cis-acting destabilizing element, because insertion of a spliceable intron a sufficient distance downstream of a nonsense codon within either gene is sufficient to elicit NMD.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of α phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5′-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid–base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.