Recent evidence supports the idea that pre-mRNA splicing and mRNA export are mechanistically coupled. In metazoans, this process appears to be mediated by a multicomponent complex, which associates with the spliced RNA upstream of the exon–exon junction. One of these components (Aly/REF) has a homolog in the budding yeast Saccharomyces cerevisiae known as Yra1p. The YRA1 gene is essential for growth and required for mRNA export. Notably, YRA1 is one of the only ∼5% intron-containing genes in yeast. Moreover, the YRA1 intron has several unusual features and is conserved in other budding yeast species. Previously, overexpression of intronless YRA1 was shown to be toxic. We show here that overexpression of the intron-containing gene results in increased levels of unspliced pre-mRNA but normal levels of Yra1 protein; conversely, expression of the cDNA results in increased levels of protein and accumulation of nuclear poly(A)+ RNA. Two additional lines of evidence suggest that expression of Yra1p is autoregulated: First, expression of excess Yra1p from a plasmid reduces the level of tagged, chromosomal Yra1p, and, second, this effect requires wild-type protein. Replacement of the YRA1 intron with that of other S. cerevisiae genes cannot rescue the dominant-negative growth defect of intronless YRA1. We conclude that the level of Yra1p is negatively autoregulated by a mechanism that involves splicing of its unusual intron. Tight control of the levels of Yra1p might be necessary to couple the rates of pre-mRNA splicing and mRNA export.

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a −1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a −1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5′ splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803–817]. In the 3′ intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3′ stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3′ stem-loop structure.

The 5′ and 3′ domains of yeast U6 snRNA contain sequences that are thought to be important for binding to Prp24 and Lsm proteins. By extensive mutational analysis of yeast U6 snRNA, we confirmed that the 3′ terminal uridine tract of U6 snRNA is important for U6 binding to Lsm proteins in yeast. Binding of Prp24 protein to U6 RNA is dependent on or is strongly enhanced by U6 binding of Lsm proteins. This supports a model for U6 snRNP assembly in which U6 RNA binds to the Lsm2–8 core prior to binding Prp24 protein. Using compensatory base-pairing analysis, we show that at least half of the recently identified U6 telestem as well as a nucleotide sequence in the other half of the telestem are important for binding of U6 RNA to Prp24 protein. Surprisingly, disruption of base pairing in the unconfirmed half of the telestem enhanced U6–Prp24 binding. Truncation of the entire 3′ terminal domain or nearly the entire 5′ terminal domain of yeast U6 allowed for detectable levels of splicing to proceed in vitro. In addition to gaining knowledge of the function of the 5′ and 3′ domains of yeast U6, our results help define the minimal set of requirements for yeast U6 RNA function in splicing. We present a revised secondary structural model of yeast U6 snRNA in free U6 snRNPs.

Target regions for posttranscriptional silencing of transgenes often reside in the 3′ region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic background, we have performed detailed analyses of target regions in three spontaneously β-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3′ half of the GUS coding region but upstream of the last 200 nt. The quantities of small (21–25 nt) RNAs homologous to 5′ or 3′ regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant genetic background are not major determinants of silencing target regions. We also show that virus-induced gene silencing (VIGS) of GUS in Nicotiana benthamiana is induced equally effectively with Potato virus X carrying either the 5′ or 3′ third of the GUS coding region. This indicates that both regions can act as efficient inducers as well as targets of posttranscriptional silencing, although the 3′ region is the predominant target region in the spontaneously silencing transgenic plant lines examined. Finally, we investigated spreading of the target region in the N. benthamiana plants undergoing VIGS. Surprisingly, only evidence for spreading of the target region in the 5′-3′ direction was obtained. This finding may help explain why the majority of target regions examined to date lie within the 3′ region of transgenes.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase “stuttering” (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3′ UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code.

Slu7 and Prp18 act in concert during the second step of yeast pre-mRNA splicing. Here we show that the 382-amino-acid Slu7 protein contains two functionally important domains: a zinc knuckle (122CRNCGEAGHKEKDC135) and a Prp18-interaction domain (215EIELMKLELY224). Alanine cluster mutations of 215EIE217 and 221LELY224 abrogated Slu7 binding to Prp18 in a two-hybrid assay and in vitro, and elicited temperature-sensitive growth phenotypes in vivo. Yet, the mutations had no impact on Slu7 function in pre-mRNA splicing in vitro. Single alanine mutations of zinc knuckle residues Cys122, His130, and Cys135 had no effect on cell growth, but caused Slu7 function during pre-mRNA splicing in vitro to become dependent on Prp18. Specifically, zinc knuckle mutants required Prp18 in order to bind to the spliceosome. Compound mutations in both Slu7 domains (e.g., C122A-EIE, H130A-EIE, and C135A-EIE) were lethal in vivo and abolished splicing in vitro, suggesting that the physical interaction between Slu7 and Prp18 is important for cooperation in splicing. Depletion/reconstitution studies coupled with immunoprecipitations suggest that second step factors are recruited to the spliceosome in the following order: Slu7 → Prp18 → Prp22. All three proteins are released from the spliceosome after step 2 concomitant with release of mature mRNA.

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5′ splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5′ splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5′ splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5′ splice site usage in an equivalent manner. Second, increasing the distance between the internal 5′ splice site and flanking A1 binding sites does not compromise activation of the distal 5′ splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5′ splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5′ splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5′ splice site and lead to distal 5′ splice site activation.

For D. Gandini Attardi and G.P. Tocchini-Valentini, Trans-acting RNA inhibits tRNA suppressor activity in vivo, RNA 8:904–912. Due to authors' oversight, the following statement was inadvertently omitted from the Acknowledgments:

“This work has been supported by Progetto Strategico Genetica Molecolare Legge 449/97; Programma Biomolecole per la Salute Umana MURST-CNR Legge 95/95 5%; Progetto Strategico Tecnologie di Base della Post Genomica (CNR).”