In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability. Recent results suggest a model with three main points. First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events. Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains. Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p. If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation. Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA. This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3′ cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G[bull ]U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.

Highly efficient cap-independent translation initiation at the 5′-proximal AUG is facilitated by the 3′ translation enhancer sequence (3′TE) located near the 3′ end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3′TE in regulating viral translation was examined. The 3′TE is required for translation and thus replication of the genomic RNA that lacks a 5′ cap (Allen et al., 1999, Virology 253:139–144). Here we show that the 3′TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3′TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5′ extremity of the sequence required in the 3′ untranslated region (UTR) for cap-independent translation in vivo coincides with the 5′ end of sgRNA2. Thus, sgRNA2 has the 3′TE in its 5′ UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3′TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5′ UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.

Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1α-like catalytic domain. By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S. pombe as well as of S. cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al. (1997, Mol Cell Biol 17:2798–2805). The activity of S. pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1α. These single mutational defects in the eRF1–eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction. Given that eRF3 is an EF-Tu/EF-1α homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1α for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1α share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively. These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1α (i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.

The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA− MG1655 either in rich or minimal medium at either 24, 37, or 42 °C. Plasmid rescue of the BL21/DE3 rsuA− strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.

The internal ribosome entry segment (IRES) of picornaviruses consists of ∼450 nt of 5′-untranslated region, terminating at the 3′ end with an ∼25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and finally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in encephalomyocarditis virus (EMCV) virtually all ribosomes initiate translation at this site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3′ end of the IRES (at nt 586 in PV type 1) is considered to be a silent entry site, with all ribosomes initiating translation at the next AUG downstream (nt 743). To investigate what determines this different behavior, chimeras were constructed with a crossover at the conserved UUUC motif: the body of the IRES, the sequences upstream of this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of the FMDV or PV IRESes was replaced by that of EMCV, there was a marked increase in the absolute and relative frequency of initiation at the upstream AUG, the Lab site of FMDV and 586AUG of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, initiation occurred with no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the context of the AUG at the 3′ end of the IRES may influence initiation frequency at this site, as was shown by improving the context of 586AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-competent manner is particularly efficient and accurate with the EMCV IRES.

The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8SS) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8SS rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

Recent studies have demonstrated that cells have evolved elaborate mechanisms to rid themselves of aberrant proteins and transcripts. The nonsense-mediated mRNA decay pathway (NMD) is an example of a pathway that eliminates aberrant mRNAs. In yeast, a transcript is recognized as aberrant and is rapidly degraded if a specific sequence, called the DSE, is present 3′ of a premature termination codon. Results presented here show that strains harboring the mof2-1, mof4-1, mof5-1, and mof8-1 alleles, previously demonstrated to increase the efficiency of programmed −1 ribosomal frameshifting, decrease the activity of the NMD pathway. The effect of the mof2-1 allele on NMD was characterized in more detail. Previous results demonstrated that the wild-type MOF2 gene is identical to the SUI1 gene. Studies on the mof2-1 allele of the SUI1 gene indicate that in addition to its role in recognition of the AUG codon during translation initiation and maintenance of the appropriate reading frame during translation elongation, the Mof2 protein plays a role in the NMD pathway. The Mof2p/Sui1p is conserved throughout nature and the human homolog of the Mof2p/Sui1p functions in yeast cells to activate NMD. These results suggest that factors involved in NMD are general modulators that act in several aspects of translation and mRNA turnover.

Translocation of DNA or RNA is a ubiquitous phenomenon. One intricate translocation process is viral DNA packaging. During maturation, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into a limited space within the procapsid. We have revealed that φ29 DNA packaging is accomplished by a mechanism similar to driving a bolt with a hex nut, which consists of six DNA-packaging pRNAs. Four bases in each of the two pRNA loops are involved in RNA/RNA interactions to form a hexagonal complex that gears the DNA translocating machine. Without considering the tertiary interaction, in some cases only two G/C pairs between the interacting loops could provide certain pRNAs with activity. When all four bases were paired, at least one G/C pair was required for DNA packaging. The maximum number of base pairings between the two loops to allow pRNA to retain wild-type activity was five, whereas the minimum number was five for one loop and three for the other. The findings were supported by phylogenetic analysis of seven pRNAs from different phages. A 75-base RNA segment, bases 23–97, was able to form dimer, to interlock into the hexamer, to compete with full-length pRNA for procapsid binding, and therefore to inhibit φ29 assembly in vitro. Our result suggests that segment 23–97 is a self-folded, independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, whereas bases 1–22 and 98–120 are involved in DNA translocation but dispensable for RNA/RNA interaction. Therefore, this 75-base RNA could be a model for structural studies in RNA dimerization.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2–S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214–1218; Held et al., 1974, J Biol Chem 249:3103–3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J Mol Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, S11, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 °C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II′ (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics.

Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.