Life today requires the transmission of genetic information in the form of DNA, and a large number of catalytic and structural functions carried out by proteins. Without DNA to specify useful sequences of amino acids, functional proteins would not exist. Without proteins to carry out high-fidelity DNA replication and gene expression, DNA could not serve as the genetic material. How could either arise without the other?

The availability of the complete sequence of the genome of Saccharomyces cerevisiae (Goffeau et al., 1996) and other organisms has provided a substantial amount of information on their chromosomal organization. We need now to understand the coordinated expression of the many genes present in a given genome and the function of the various encoded proteins. Microarrays (DNA chips) offer the opportunity to analyze nucleic acids at the genomic scale and have been used to perform global gene expression studies (Lander, 1999). However, these techniques also have the potential to give us a new perspective on posttranscriptional processes involved in the regulation of gene expression. We provide here a striking example through the investigation of yeast pre-mRNA splicing. Our analysis demonstrates that pre-mRNA splicing is quantitatively a much more important process in this species than previously thought and shows that splicing-signal conservation is correlated with transcription efficiency.

Two studies have provided separate pieces of information that bear on the functional and evolutionary significance of introns in the budding yeast Saccharomyces cerevisiae (Holstege et al., 1998; Spingola et al., 1999). By the measure of the number of introns within its genes, budding yeast seems a disappointing eukaryote. Fewer than 250 of the more than 6,200 annotated genes are known to have introns, and fewer than 10 are known have more than one intron (Spingola et al., 1999). In contrast, metazoan genes are estimated to average nearly 10 introns, and the intronless gene is the exception rather than the rule. Although many essential yeast genes have introns, it would appear that introns are on the way out of the yeast genome, perhaps by a cDNA-directed homologous recombination mechanism proposed by Fink (1987).

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3′,5′-phosphodiester linkage at the cleavage site; however, a 2′,5′-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2′,3′-cyclic phosphate and 5′-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3′,5′-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2′,5′-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2′,5′-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5′ to the cleavage site.

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 5′-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 3′-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 5′- and 3′-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5′ end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5′ end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 Å, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5′ displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells. These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif. Such loops are important in RNA–RNA and RNA–protein interactions. Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN. Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements. The sequence of individual selected IRES elements was determined. All functional IRES elements had a tetraloop with a 3′ terminal A residue. Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus. In contrast, IRES elements containing YCYA tetraloops were severely defective.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNASec and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3′ of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc Natl Acad Sci USA 94:6676–6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA–ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNAPhe anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNAPhe, ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNAPhe enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with Kps ≈ 50–60 nM, were afforded significantly greater protection by both yeast tRNAPhe and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (Kps ≈ 100–200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (Kps between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (Kps = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.

Subcellular localization signals for several mRNAs are positioned in their 3′ untranslated regions (UTR). We have utilized the human α- and β-actin 3′ UTRs as signals for colocalizing hammerhead ribozymes with a lacZ target mRNA. Ribozyme and target genes containing matched or unmatched 3′ UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. β-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl β-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched α- or β-actin 3′ UTR (α–α or β–β) was enhanced approximately threefold relative to unmatched 3′ UTRs. The increase in ribozyme-mediated inhibition of β-galactosidase activity observed when matched 3′ UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.

Eukaryotic translation initiation factor 4A (eIF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5′ untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5′-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to eIF4A and on stimulation of its ATPase activity once bound. Substitution of 2′-OH groups with 2′-H or 2′-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2′-OH groups participate in an eIF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to ∼15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2′-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2′-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and eIF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow eIF4A to operate within regions of 5′ UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5′ UTR influences translation efficiency.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5′ ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5′ and one 3′ purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a “kissing-loop” complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first ≈400 residues, whereas the C-terminus provides a nonspecific binding surface.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

DNA templates modified with C2′-methoxyls at the last two nucleotides of the 5′ termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single- and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.