The U1 snRNP is essential for recognition of the pre-mRNA 5′-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron Pl.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5′ noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5′-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5′ cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5′ cap-independent and 5′ cap-dependent translational initiation mechanisms.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5′ splice site selection requires base pairing of U6 snRNA with intron positions 4–6. However, no factor recognizing the highly conserved 5′ splice site GU has yet been identified. We have tested if the known U6 snRNA–pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5′ splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5′ splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5′ splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.

Escherichia coli tRNAVal with pyrimidine substitutions for the universally conserved 3′-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNAVal with a 3′-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3′-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNAVal:EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNAVal for EF-Tu:GTP, thus explaining the inability of these tRNAVal variants to function in polypeptide synthesis. Valyl-tRNAVal terminating in 3′-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3′ end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3′ cytosine. Similar observations were made in studies of the interaction of 3′ end mutants of E. coli tRNAAla and tRNAPhe with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3′ end of aminoacyl-tRNAs.

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 lcp1 to lcp5 (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.

Unlike most eukaryotic mRNAs studied to date, Xenopus serum albumin mRNA has a short (17-residue), discrete poly(A) tail. We recently reported that this short poly(A) tail results from regulation of the length of poly(A) on albumin pre-mRNA. The purpose of the present study was to locate the cis-acting element responsible for this, the poly(A)-limiting element or PLE. An albumin minigene consisting of albumin cDNA joined in exon 13 to the 3′ end of the albumin gene produced mRNA with <20 nt poly(A) when transfected into mouse fibroblasts. This result indicates both that cis-acting sequences that regulate poly(A) length are within this construct, and that nuclear regulation of poly(A) length is conserved between vertebrates. Poly(A) length regulation was retained after replacing the terminal 53 bp and 3′ flanking region of the albumin gene with a synthetic polyadenylation element (SPA). Conversely, fusing albumin gene sequence spanning the terminal 53 bp of the albumin gene and 3′ flanking sequence onto the human β-globin gene yielded globin mRNA with a 200-residue poly(A)tail. These data indicate that the PLE resides upstream of the sequence elements involved in albumin pre-mRNA 3′ processing. Poly(A) length regulation was restored upon fusing a segment bearing albumin intron 14, exon 15, and 3′ flanking sequence onto the β-globin gene. We demonstrate that exon 15 contains two PLEs that can act independently to regulate the length of poly(A).

A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing-ribozyme and a small internal group I-like ribozyme (GIR1), probably involved in protein expression. GIR1 was found to cleave at two internal sites in an obligate sequential order. Both sites are located 3′ of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160–190 nt, they are the smallest functional group I ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates a compactly folded ribozyme with the functionally essential P15 exposed in the cleft between the two principal domains P3–P8 and P4–P6.

Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNAArg and C36 and G37 in tRNAAsp (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069–5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNAArg and tRNAAsp, the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899–4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different kcat and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] catalyzes the addition and regeneration of the 3′-terminal CCA sequence of tRNAs. We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the “top half” of tRNA — the acceptor stem and TψC stem-loop of tRNA. CCA addition to the top half tDNA minihelices requires a 2′ as well as a 3′ OH at the 3′ terminus of the tDNA. Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TψC loop. These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5–5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.

A novel HIV-1 genome that stably utilizes tRNAHis rather than tRNALys,3 to initiate reverse transcription was used to study features for the interaction between the tRNA and viral RNA genome. In addition to a primer binding site (PBS) complementary to tRNAHis, this virus contains a six-nucleotide sequence in U5 complementary to the anticodon-loop of tRNAHis and three additional substitutions: U174-to-G, G181-to-A, and U200-to-C [HXB2(His-AC-GAC)]. Mutations in these three nucleotides resulted in viruses with three different genotypes: one group maintained a PBS complementary to tRNAHis with restored G174A181C200 or G174A181U200 configurations, one group reverted to a PBS complementary to tRNALys,3, and one group contained two or more PBSs complementary to different tRNAs on the same viral genome. Characterization of a previously identified virus with additional C152-to-A and C160-to-U substitutions [HXB2(His-AC-A152U160-GAC)] revealed that this virus maintained a PBS complementary to tRNAHis, whereas a mutant HXB2(His-AC-U152A160-GAC) reverted after culture to contain dual PBS complementary to tRNALys,3 and tRNAHis, respectively. Our results demonstrate that regions in U5 act in concert with the PBS to promote use of the tRNA primer for initiation of reverse transcription. These results are discussed with respect to structural models for the U5-PBS interactions with tRNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the “binding pocket” for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA–protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA–protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling contraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA–protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.

Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3·GDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3·GDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNAAlaGGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.

A 22-codon upstream open reading frame (uORF2) in the human cytomegalovirus UL4 transcript leader inhibits downstream translation in cis. Previous studies revealed that the peptide product of uORF2 mediates this inhibitory effect by interfering with translation termination at its own stop codon. The block at termination results both in accumulation of the nascent uORF2 peptide linked to tRNAPro, the tRNA that decodes the final codon of uORF2, and in stalling of ribosomes at the end of uORF2. The stalled ribosomes create a barrier that obstructs ribosomal transit to the downstream cistron. In the current studies, we further investigated the mechanism of uORF2-mediated translational inhibition by assessing the kinetics of uORF2 peptidyl tRNAPro hydrolysis and ribosomal release from the uORF2 termination site. Whereas hydrolysis of a mutant, noninhibitory uORF2 peptidyl tRNA is nearly complete in less than 1 min, hydrolysis of the wild-type peptidyl tRNA is negligible even after 30 min. In spite of this remarkably prolonged block to hydrolysis of the uORF2 peptidyl tRNAPro, most ribosomes are released from the uORF2 termination site within 15 min. Thus, peptidyl tRNA hydrolysis is not absolutely required for ribosomal release in this system. These results suggest that a eukaryotic cellular mechanism exists for removing stalled ribosomes from mRNAs in the absence of peptidyl tRNA hydrolysis.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 Å side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 Å distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 Å from the 4′ position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

We present a novel missense suppression system for the selection of tRNA2Gln mutants that can efficiently translate the CGA (arginine) codon as glutamine. tRNA2Gln mutants were cloned from a partially randomized synthetic gene pool using a plasmid vector that simultaneously expresses the tRNA gene and, to ensure efficient aminoacylation, the glutamine aminoacyl-tRNA synthetase gene (glnS). tRNA mutants that insert glutamine at CGA were selected as missense suppressors of a lacZ mutant (lacZ625(CGA)) that contains CGA substituted for an essential glutamine codon. Preliminary characterizations of four suppressors is presented. All of them contain two anticodon mutations: C → U at position 34 and U → C at position 35, which allow for cognate translation of CGA. U35 was previously shown to be an important determinant for glutaminylation of tRNA2Gln in vitro; suppression in vivo requires overexpression of the glutaminyl-tRNA synthetase gene (glnS). One tRNA variant contains no further mutations and has the highest missense suppression activity (8%). Three other isolates each contain an additional point mutation that alters suppression efficiency. This system will be useful for further studies of tRNA structure and function. In addition, because relatively efficient translation of the rare CGA codon as glutamine is not toxic for Escherichia coli, it may be possible to translate this sense codon with other alternate meanings, a property which could greatly facilitate protein engineering.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5′ side of internal loop 1, whereas Prp8p interacts with five different regions of the 5′ stem-loop, but not with the Sm site or 3′ stem-loop. Both internal loops in the 5′ domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5′ stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1–exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5′ splice site (5′SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5′SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5′SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5′SS RNA when the APA is placed within the 5′ exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5′SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5′SS junction (positions −2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5′ end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5′SS interaction. Unexpectedly, thio-modifications within the region of the 5′SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.