Drosophila ELAV promotes the generation of the neuron-specific isoform of Neuroglian by the regulation of pre-mRNA splicing. The findings reported in this paper demonstrate that ELAV is necessary, and the ectopic expression of ELAV in imaginal disc cells is sufficient, to mediate neuron-specific alternative splicing.

The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3 splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-)(1) and K(M) = 0.83 +/- 0.22 microM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 microM). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

The Drosophila gene elav encodes a 483-amino-acid-long nuclear RNA binding protein required for normal neuronal differentiation and maintenance. We molecularly analyzed the three known viable alleles of the gene, namely elavts1, elavFliJ1, and elavFliJ2, which manifest temperature-sensitive phenotypes. The modification of the elavFliJ1 allele corresponds to the change of glycine426 (GGA) into a glutamic acid (GAA). Surprisingly, elavts1 and elavFliJ2 were both found to have tryptophan419 (TGG) changed into two different stop codons, TAG and TGA, respectively. Unexpectedly, protein analysis from elavts1 and elavFliJ2 reveals not only the predicted 45-kD truncated ELAV protein due to translational truncation, but also a predominant full-size 50-kD ELAV protein, both at permissive and nonpermissive temperatures. The full-length protein present in elavts1 and elavFliJ2 can a priori be explained by one of several mechanisms leading to functional suppression of the nonsense mutation or by detection of a previously unrecognized ELAV isoform of similar size resulting from alternative splicing and unaffected by the stop codon. Experiments described in this article support the functional suppression of the nonsense mutation as the mechanism responsible for the full-length protein.