Members of the serine/arginine-rich (SR) protein family have multiple functions in the pre-mRNA splicing reaction. In addition to being required for the removal of constitutively spliced introns, SR proteins can function to regulate alternative splicing both in vitro and in vivo (Ge & Manley, 1990; Krainer et al., 1990a; Fu et al., 1992; Zahler et al., 1993a; Caceres et al., 1994; Wang & Manley, 1995). In the cell, SR proteins migrate from speckles—subnuclear domains that may function as storage sites for certain splicing factors—to sites of active transcription (Misteli et al., 1997; Misteli & Spector, 1999) and some SR proteins have been found to shuttle in and out of the nucleus (Caceres et al., 1998). The subcellular localization of SR proteins can be modulated by phosphorylation (Misteli & Spector, 1998; Misteli et al., 1998) and this undoubtedly underlies some regulated splicing events. However, once in the nucleus and localized to the nascent pre-mRNA, exactly how SR proteins engage the general splicing machinery to recognize specific splice sites is unclear and is an area of intense investigation.

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)63+) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)63+ is a useful spectroscopic probe for Mg(H2O)62+-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)63+. We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C[bull ]G base pair is replaced by U[bull ]G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3′ direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)63+ and RNA protons were used to locate the site of Co(NH3)63+ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5′ by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.

The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD). A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified. The HUpf1 has also been shown to play a role in NMD in mammalian cells. Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved. The yeast Upf1p demonstrates RNA-dependent ATPase and 5′ → 3′ helicase activities. In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein. We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5′ → 3′ helicase activity. Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP. Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.

Eukaryote ribosomal translation is terminated when release factor eRF1, in a complex with eRF3, binds to one of the three stop codons. The tertiary structure and dimensions of eRF1 are similar to that of a tRNA, supporting the hypothesis that release factors may act as molecular mimics of tRNAs. To identify the yeast eRF1 stop codon recognition domain (analogous to a tRNA anticodon), a genetic screen was performed to select for mutants with disabled recognition of only one of the three stop codons. Nine out of ten mutations isolated map to conserved residues within the eRF1 N-terminal domain 1. A subset of these mutants, although wild-type for ribosome and eRF3 interaction, differ in their respective abilities to recognize each of the three stop codons, indicating codon-specific discrimination defects. Five of six of these stop codon-specific mutants define yeast domain 1 residues (I32, M48, V68, L123, and H129) that locate at three pockets on the eRF1 domain 1 molecular surface into which a stop codon can be modeled. The genetic screen results and the mutant phenotypes are therefore consistent with a role for domain 1 in stop codon recognition; the topology of this eRF1 domain, together with eRF1-stop codon complex modeling further supports the proposal that this domain may represent the site of stop codon binding itself.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(−1) upstream of the 5′ splice site. P(−1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(−1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min−1 for the wild-type RNA to ∼4 min−1 at 30 °C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Loop–loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop–loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5′ noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop–loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.

It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 3′ ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 3′ ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 3′ ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 3′ ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 3′ ends were not uridylated in vitro, or when U6 snRNA with 3′ AOH was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 3′ end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 3′ uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 3′ end of some small RNAs and suggest that one of the functions of the 3′ adenylation may be to negatively affect the 3′ uridylation of small RNAs.

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160–455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the β-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between β strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

The selenocysteine (Sec) tRNA[Ser]Sec population in higher vertebrates consists of two major isoacceptors that differ from each other by a single nucleoside modification in the wobble position of the anticodon (position 34). One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this position, whereas the other contains 5-methylcarboxymethyluridine-2′-O-methylribose (mcmUm). The other modifications in these tRNAs are N6-isopentenyladenosine (i6A), pseudouridine (ψ), and 1-methyladenosine (m1A) at positions 37, 55, and 58, respectively. As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated the effect of the modifications at other positions as well as tertiary structure on its formation. Mutations were introduced within a synthetic gene encoded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases. The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an intact tertiary structure, whereas formation of modified bases at other positions, including mcmU, is not as stringently connected to precise primary and tertiary structure. These results, along with the observations that methylation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoacceptor must have a role in selenoprotein synthesis different from that of the mcmU isoacceptor.

The crystal structure of r(GCCACCCUG)[bull ]r(CAGGGUCGGC), helix II of the Xenopus laevis 5S rRNA with a cytosine bulge (underlined), has been determined in two forms at 2.2 Å (Form I, space group P42212, a = b = 57.15 Å and c = 43.54 Å) and 1.7 Å (Form II, space group P43212, a = b = 32.78 Å and c = 102.5 Å). The helical regions of the nonamers are found in the standard A-RNA conformations and the two forms have an RMS deviation of 0.75 Å. However, the cytosine bulge adopts two significantly different conformations with an RMS deviation of 3.9 Å. In Form I, the cytosine bulge forms an intermolecular C+∗G[bull ]C triple in the major groove of a symmetry-related duplex with intermolecular hydrogen bonds between N4C and O6G, and between protonated N3+C and N7G. In contrast, a minor groove C∗G[bull ]C triple is formed in Form II with intermolecular hydrogen bonds between O2C and N2G, and between N3C and N3G with a water bridge. A partial major groove opening was observed in Form I structure at the bulge site. Two Ca2+ ions were found in Form I helix whereas there were none in Form II. The structural comparison of these two forms indicates that bulged residues can adopt a variety of conformations with little perturbation to the global helix structure. This suggests that bulged residues could function as flexible latches in bridging double helical motifs and facilitate the folding of large RNA molecules.

Compelling in vivo studies suggest a tight functional linkage between RNA polymerase II transcription and pre-messenger RNA splicing. At present, the specific interactions involved in this coupling are poorly understood and deserve investigation. To this end, we developed an in vitro system that permits study of coupled transcription and splicing. Transcripts generated by RNA polymerase II were accurately and efficiently spliced under reaction conditions that permitted both transcription and splicing to occur simultaneously. The splicing of RNA-polymerase-II-driven transcripts was accelerated relative to that of the same transcripts driven by T7 RNA polymerase. Moreover, the product of exon ligation was found associated with the DNA template in reactions driven by RNA polymerase II. These two findings indicate that transcription and splicing were coupled in the in vitro system driven by RNA polymerase II, and suggest that this system will be useful for the biochemical study of this coupling.