The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA

Abstract

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.

Emc, a negative HLH regulator with multiple functions in Drosophila development

“…Hel-N1 binding sequences are also present in the 3' UTR of the Drosophila emc mRNA (King et al, 1994). Elav is an RNA binding protein known to regulate splicing of several neural speci®c genes (Lisbin et al, 2001). It would thus be interesting to determine whether Elav, which accumulates in the neurons (Robinow and White, 1988), plays any role in the downregulation of Emc which appears necessary for neural development to proceed.…”

Coactivator-associated Arginine Methyltransferase 1, CARM1, Affects Pre-mRNA Splicing in an Isoform-specific Manner*♦

“…In Drosophila melanogaster, sex-lethal functions as a regulator of alternative splicing in sex determination (2), and the embryonic lethal abnormal visual system is a gene-specific regulator of alternative pre-mRNA processing in neurons (3). However, in mammals, only a limited number of splicing regulators have been identified to date, despite the large diversity of the mammalian gene transcripts, and little is known about the mechanisms by which alternative splicing is regulated.…”

“…The mechanism of alternative splicing permits diversity of translatable mRNAs, thereby increasing the proteome diversity encoded by a limited number of genes. Genetic switches based on alternative splicing are known to be important in many cellular and developmental processes, including sex determination, apoptosis, axon guidance, and tissue-specific differentiation (2)(3)(4).…”

The C2H2 zinc-finger protein SYD-9 is a putative posttranscriptional regulator for synaptic transmission

“…UNC-75 is the C. elegans ortholog of the CELF͞BrunoL family of proteins that control pre-mRNA splicing in mammals (35,36) and mediate translation repression in Drosophila oocytes (37,38). EXC-7 is the C. elegans ELAV (embryonic lethal abnormal vision)͞Hu family protein, a neuron-specific alternative splicing factor in Drosophila (39)(40)(41)(42) and neuron-specific RNA-processing regulator in mammals (43)(44)(45). We identified a previously uncharacterized nuclear protein, syd-9 (syd, synapse defective), in C. elegans that specifically regulates synaptic transmission: in particular, endocytosis.…”