Our knowledge of RNA structure and function has progressed at an astonishingly fast pace in the last five years. Since then, we have seen the X-ray structure of a full ribozyme, the awesome structure of a large domain of a group I intron, and recently the striking structure of the loop E of 5S rRNA. NMR studies have yielded structures, not only of small recurrent loops, but also of several RNA aptamers and of a fragment of the 16S rRNA complexed with an antibiotic. Both methods have also contributed enormously to our understanding of RNA–protein interactions; the richness of the tRNA synthetase interactions is now recognized, whereas the structures of complexes from the spliceosome and the ribosome point to new horizons. The 22 chapters of this new book compose a tribute to the fascinating and ubiquitous versatility of the RNA molecule.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor–substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor–cargo interaction and imparts directionality to the transport process.

Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.

A novel three-dimensional model of tertiary interactions in the core region of the eukaryotic selenocysteine tRNA is proposed based on the analysis of available nucleotide sequences. The model features the 7/5 tRNASec secondary structure characterized by seven and five base pairs in the acceptor and T-stems, respectively, and four nucleotides in the connector region between the acceptor and D-stems. The model suggests a unique system of tertiary interactions in the area between the major groove of the D-stem and the first base pair of the extra arm that provides a rigid orientation of the extra arm and contributes to the overall stability of the molecule. The model is consistent with available experimental data on serylation, selenylation, and phosphorylation of different tRNASec mutants. The important similarity between the proposed model and the structure of the tRNASer is shown. Based on this similarity, the ability of some tRNASer mutants to be serylated, selenylated, and phosphorylated was evaluated and found to be in a good agreement with experimental data.

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.

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.

At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.

The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids.

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5′ splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of β-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

While exons were originally defined as coding regions of split eukaryotic genes, introns have long been considered as mainly noncoding “genetic junk.” However, recognition that a large number of small nucleolar RNAs (snoRNAs) are processed from introns of pre-mRNAs demonstrated that introns may also code for functional RNAs. Moreover, recent characterization of the mammalian UHG gene that encodes eight box C/D intronic snoRNAs suggested that some genes generate functional RNA products exclusively from their intron regions. In this study, we show that the human U19 box H/ACA snoRNA, which is encoded within the second intron of the U19H gene, represents the only functional RNA product generated from the long U19H primary transcript. Splicing of the U19H transcript, instead of giving rise to a defined RNA, produces a population of diverse U19H RNA molecules. Although the first three exons of the U19H gene are preserved in each processed U19H RNA, the 3′ half of the RNA is generated by a series of apparently random splicing events. Because the U19H RNA possesses limited potential for protein coding and shows a predominant nucleoplasmic localization, we suggest that the sole function of the U19H gene is to express the U19 intronic snoRNA. This suggests that, in marked contrast to our previous dogmatic view, genes generating functionally important RNAs exclusively from their intron regions are probably more frequent than has been anticipated.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (−)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (−)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (−)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine–adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (−)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3–20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (−)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.

An RNA family that binds isoleucine with Kd = 200–500 μM was repetitively isolated from a ribonucleotide transcript pool containing 50 randomized positions. The RNA site is specific, discriminating against branched side chains of different size (valine—one methylene smaller than isoleucine) by at least 1.3 kcal/mol and against the shape (linear) of norleucine by 0.6 kcal/mol. The binding site was localized by sequence comparison, by synthesis of mutant and truncated active derivatives, and by chemical modification–interference. The binding site is small, no more than 10–12 nt, containing an asymmetric internal loop (6 over 2 nt) that includes the isoleucine codon AUU and a sequence of four G's, two of which are involved in G·U and G·C base pairs. Areas of U/G concentration like these may signal a hydrophobic RNA site.

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome “reads” the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.