Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K_d 40 nM). This finding supports our model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. We narrowed down the best binding site to a 20 nt long sequence A(U₅)A(U₃)G(U₂)A(U₆) in an alternative 3′ exon. We propose and test a model in which the regulated use of this alternative 3′ exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne.     

Molecular Characterization of Barley 3H Semi-Dwarf Genes

The barley chromosome 3H accommodates many semi-dwarfing genes. To characterize these genes, the two-rowed semi-dwarf Chinese barley landrace ‘TX9425’ was crossed with the Australian barley variety ‘Franklin’ to generate a doubled haploid (DH) population, and major QTLs controlling plant height have been identified in our previous study. The major QTL derived from ‘TX9425’ was targeted to investigate the allelism of the semi-dwarf gene uzu in barley. Twelve sets of near-isogenic lines and a large NILF₂ fine mapping population segregating only for the dwarfing gene from ‘TX9425’ were developed. The semi-dwarfing gene in ‘TX9425’ was located within a 2.8 cM region close to the centromere on chromosome 3H by fine mapping. Molecular cloning and sequence analyses showed that the ‘TX9425’-derived allele contained a single nucleotide substitution from A to G at position 2612 of the HvBRI1 gene. This was apparently the same mutation as that reported in six-rowed uzu barley. Markers co-segregating with the QTL were developed from the sequence of the HvBRI1 gene and were validated in the ‘TX9425’/‘Franklin’ DH population. The other major dwarfing QTL derived from the Franklin variety was distally located on chromosome 3HL and co-segregated with the sdw1 diagnostic marker hv20ox2. A third dwarfing gene, expressed only in winter-sown trials, was identified and located on chromosome 3HS. The effects and interactions of these dwarfing genes under different growing conditions are discussed. These results improve our understanding of the genetic mechanisms controlling semi-dwarf stature in barley and provide diagnostic markers for the selection of semi-dwarfness in barley breeding programs.     

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV''s biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity.     

The C-terminal domain of Escherichia coli Hfq is required for regulation

The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the γ- and β-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq₆₅), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq₆₅ retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.