It has been known for almost a decade and a half that in trypanosomes all mRNAs are trans-spliced by addition to the 5′ end of the spliced leader (SL) sequence. During the same time period the conviction developed that classical cis-splicing introns are not present in the trypanosome genome and that the trypanosome gene arrangement is highly compact with small intergenic regions separating one gene from the next. We have now discovered that these tenets are no longer true. Poly(A) polymerase (PAP) genes in Trypanosoma brucei and Trypanosoma cruzi are split by intervening sequences of 653 and 302 nt, respectively. The intervening sequences occur at identical positions in both organisms and obey the GT/AG rule of cis-splicing introns. PAP mRNAs are trans-spliced at the very 5′ end as well as internally at the 3′ splice site of the intervening sequence. Interestingly, 11 nucleotide positions past the actual 5′ splice site are conserved between the T. brucei and T. cruzi introns. Point mutations in these conserved positions, as well as in the AG dinucleotide of the 3′ splice site, abolish intron removal in vivo. Our results, together with the recent discovery of cis-splicing introns in Euglena gracilis, suggest that both trans- and cis-splicing are ancient acquisitions of the eukaryotic cell.

The inactivity or occlusion of the HIV-1 poly(A) signal when in the 5′ long terminal repeat (LTR) has been mechanistically investigated. First we show that neither the homologous HIV-1 promoter nor the close proximity of this RNA processing signal to the transcript initiation site is required for the occlusion effect. Instead we demonstrate that the major splice donor (MSD) site positioned about 200 bp downstream maintains the poly(A) site in an inactive state. Although mutation of MSD results in activation of the 5′ LTR poly(A) signal, this effect can be suppressed by targeting U1 snRNAs near to the mutated MSD by base pairing. We show that hybrid U7-U1 snRNAs can also suppress the poly(A) signal and that this suppression is dependent on the U1 stem-loop 1. In particular the binding site for the U1 snRNP protein 70K that binds to the loop structure of stem-loop 1 is associated with poly(A) site occlusion. These experiments were carried out with an HIV-1 proviral construct and as such emphasize the physiological importance of this splice donor–poly(A) site interaction.

The 3′ ends of most eukaryotic pre-mRNAs are generated by 3′ endonucleolytic cleavage and subsequent polyadenylation. 3′-end formation can be influenced positively or negatively by various factors. In particular, U1 snRNP acts as an inhibitor when bound to a 5′ splice site located either upstream of the 3′-end formation signals of bovine papilloma virus (BPV) late transcripts or downstream of the 3′-end processing signals in the 5′ LTR of the HIV-1 provirus. Previous work showed that in BPV it is not the first step, 3′ cleavage, that is affected by U1 snRNP, but rather the second step, polyadenylation, that is inhibited. Since in HIV-1 the biological requirement is to produce transcripts that read through the 5′ LTR cleavage site rather than being cleaved there, this mechanism seemed unlikely to apply. The obvious difference between the two examples was the relative orientation of the 3′-end formation signals and the U1 snRNP-binding site. In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3′ cleavage reaction. U1 snRNP was found to inhibit cleavage when bound to a 5′ splice site downstream of the cleavage/polyadenylation site, as in the HIV-1 LTR. U1 snRNP binding at this location was shown not to affect the recruitment of multiple cleavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP on polyadenylation of other pre-mRNAs, were shown not to be required for cleavage inhibition. Therefore, U1 snRNP bound to a 5′ splice site can inhibit cleavage and polyadenylation in two mechanistically different ways depending on whether the 5′ splice site is located upstream or downstream of the cleavage site.

The hairpin ribozyme catalyzes RNA cleavage in partially hydrated RNA films in the absence of added divalent cations. This reaction exhibits the characteristics associated with the RNA cleavage reaction observed under standard conditions in solution. Catalysis is a site-specific intramolecular transesterification reaction, requires the 2′-hydroxyl group of substrate nucleotide A−1, and generates 2′,3′-cyclic phosphate and 5′-hydroxyl termini. Mutations in both ribozyme and substrate abolish catalysis in hydrated films. The reaction is accelerated by cations that may enhance binding, conformational stability, and catalytic activity, and is inhibited by Tb3+. The reaction has an apparent temperature optimum of 4 °C. At this temperature, cleavage is slow (kobs: 2 d−1) and progressive, with accumulation of cleavage products to an extent of 40%. The use of synthetic RNAs, chelators, and analysis of all reaction components by inductively coupled plasma-optical spectrophotometry (ICPOES) effectively rules out the possibility of contaminating divalent metals in the reactions. Catalysis is minimal under conditions of extreme dehydration, indicating that the reaction requires hydration of RNA by atmospheric water. Our results provide a further caution for those studying the biochemical activity of ribozymes in vitro and in cells, as unanticipated catalysis could occur during RNA manipulation and lead to misinterpretation of data.

Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing systems. We previously showed that 3′-sulfur substitution at the 3′ splice site of a nuclear pre-mRNA has no effect on splicing. We now report that 3′-sulfur substitution at the 3′ splice site of a nuclear pre-mRNA causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome uses a catalytic metal ion to stabilize the 3′-oxyanion leaving group during the second step of splicing, as shown previously for the first step. The lack of a metal-specificity switch under cis splicing conditions indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.

U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis. Genetic evidence suggests they form an extensively base-paired structure within the spliceosome that is required for catalysis. Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome. To this end, we synthesized and purified large segments of human U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (Tm = 50–60 °C in the presence of 20 mM Mg2+) complex. To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked species were identified and characterized. The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 5′ to U2-U6 helix Ia and opposite the invariant AGC in U6. Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast. Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5′ end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5′ end of deacylated tRNAPhe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45° between the respective planes of the molecules. The terminal phosphates of 3′CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

tRNA–guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TΨC arm of in vitro-transcribed yeast tRNAPhe. Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TΨC arm of the yeast tRNAPhe indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TΨC stem into the loop). Posttranscriptional base modifications in tRNAPhe block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNAAsp. Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 °C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, we performed a phylogenetic analysis of sequences required for editing at the R/G site. We observed highly conserved sequences that were shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it was the bases in paired, helical regions whose identity was conserved, whereas bases in nonhelical regions varied, but maintained their nonhelical state. We speculate this pattern in part reflects constraints imposed by ADAR's unique specificity and gained support for our hypotheses with mutagenesis studies. Unexpectedly, we observed that some of the gluR introns were conserved beyond the sequences required for editing. The ∼600-nt intron 13 of gluR-C was particularly remarkable, showing >94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago.

The structure of the viral RNA (vRNA) inside intact nucleocapsids of vesicular stomatitis virus was studied by chemical probing experiments. Most of the Watson–Crick positions of the nucleotide bases of vRNA in intact virus and in nucleoprotein (N)-RNA template were accessible to the chemical probes and the phosphates were protected. This suggests that the nucleoprotein binds to the sugar–phosphate backbone of the RNA and leaves the Watson–Crick positions free for the transcription and replication activities of the viral RNA-dependent RNA polymerase. The same architecture has been proposed for the influenza virus nucleocapsids. However, about 5% of the nucleotide bases were found to be relatively nonreactive towards the chemical probes and some bases were hyperreactive. The pattern of reactivities was the same for RNA inside virus and for RNA in N-RNA template that was purified over a CsCl gradient and which had more than 94% of the polymerase and phosphoprotein molecules removed. All reactivities were more or less equal on naked vRNA. This suggests that the variations in reactivity towards the chemical probes are caused by the presence of the nucleoprotein.

The untranslated leader of the RNA genome of the human immunodeficiency virus type 1 (HIV-1) encodes multiple signals that regulate distinct steps of the viral replication cycle. The RNA secondary structure of several replicative signals in the HIV-1 leader is critical for function. Well-known examples include the TAR hairpin that forms the binding site for the viral Tat trans-activator protein and the DIS hairpin that is important for dimerization and subsequent packaging of the viral RNA into virion particles. In this study, we present evidence for the formation of a tertiary structure by the complete HIV-1 leader RNA. This conformer was recognized as a fast-migrating band on nondenaturing polyacrylamide gels, and such a migration effect is generally attributed to differences in compactness. Both the 5′ and 3′ domains of the 335-nt HIV-1 leader RNA are required for the formation of the compact RNA structure, and the presence of several putative interaction domains was revealed by an extensive analysis of the denaturing effect of antisense DNA oligonucleotides. The buffer conditions and sequence requirements for conformer formation are strikingly different from that of the RNA-dimerization reaction. In particular, the conformer was destabilized in the presence of Mg2+ ions and by the viral nucleocapsid (NC) protein. The presence of a stable RNA structure in the HIV-1 leader was also apparent when this RNA was used as template for reverse transcription, which yielded massive stops ahead of the structured leader domain. Formation of the conformer is a reversible event, suggesting that the HIV-1 leader is a dynamic molecule. The putative biological function of this conformational polymorphism as molecular RNA switch in the HIV-1 replication cycle is discussed.

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5′ end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.