Xenopus Vg1 mRNA is localized to the vegetal cortex during oogenesis in a process involving microtubules and microfilaments and proteins that specifically recognize the vegetal localization element (VLE) within the 3′ untranslated region. One of the best characterized VLE-binding proteins is Vg1RBP or Vera. Primary sequence analysis of Vg1RBP and its homologs suggests that most of its open reading frame is occupied by RNA-binding modules, including two RRMs and four KH domains, arranged as three pairs of didomains. In the first detailed domain analysis of Vg1RBP, we show that the interaction of Vg1RBP with the VLE requires both KH didomains, but not the RRM didomain, and moreover that the KH didomains contribute cooperatively to RNA binding. In the full-length protein, individual KH domains display significant redundancy, and their relative importance appears to vary with the RNA target. We also demonstrate that the KH34 didomain mediates Vg1RBP self-association, which is stabilized by RNA, and occurs in vivo as well as in vitro. Altogether, our findings highlight the importance of multiple KH domains in mediating RNA–protein and protein–protein interactions in the formation of a stable complex of Vg1RBP and Vg1 mRNA.

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3′ untranslated region (3′ UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3′ UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.

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 of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3′ untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson–Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements.