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.     

Nova autoregulation reveals dual functions in neuronal splicing

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of pre-mRNAs encoding the inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2 and GlyRalpha2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA(A)Rgamma2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.     

ELAV multimerizes on conserved AU4-6 motifs important for ewg splicing regulation

ELAV is a gene-specific regulator of alternative pre-mRNA processing in Drosophila neurons. Since ELAV/Hu proteins preferentially bind to AU-rich regions that are generally abundant in introns and untranslated regions, it has not been clear how gene specificity is achieved. Here we used a combination of in vitro biochemical experiments together with phylogenetic comparisons and in vivo analysis of Drosophila transgenes to study ELAV binding to the last ewg intron and splicing regulation. In vitro binding studies of ELAV show that ELAV multimerizes on the ewg binding site and forms a defined and saturable complex. Further, sizing of the ELAV-RNA complex and a series of titration experiments indicate that ELAV forms a dodecameric complex on 135 nucleotides in the last ewg intron. Analysis of the substrate RNA requirements for ELAV binding and complex formation indicates that a series of AU(4-6) motifs spread over the entire binding site are important, but not a strictly defined sequence element. The importance of AU(4-6) motifs, but not spacing between them, is further supported by evolutionary conservation in several melanogaster species subgroups. Finally, using transgenes we demonstrate in fly neurons that ELAV-mediated regulation of ewg intron 6 splicing requires several AU(4-6) motifs and that introduction of spacer sequence between conserved AU(4-6) motifs has a minimal effect on splicing. Collectively, our results suggest that ELAV multimerization and binding to multiple AU(4-6) motifs contribute to target RNA recognition and processing in a complex cellular environment.     

Two neuronal,nuclear-localized RNA binding proteins involved in synaptic transmission

While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.     

UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation

Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3'-UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs.     

Intronic binding sites for hnRNP A/B and hnRNP F/H proteins stimulate pre-mRNA splicing

hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B–binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5′ splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.     

Pre-mRNA secondary structures influence exon recognition

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.     

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.     

RBP45 and RBP47, two oligouridylate-specific hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of plant cells

Introns in plant nuclear pre-mRNAs are highly enriched in U or U + A residues and this property is essential for efficient splicing. Moreover, 3'-untranslated regions (3'-UTRs) in plant pre-mRNAs are generally UA-rich and contain sequences that are important for the polyadenylation reaction. Here, we characterize two structurally related RNA-binding proteins (RBPs) from Nicotiana plumbaginifolia, referred to as RBP45 and RBP47, having specificity for oligouridylates. Both proteins contain three RBD-type RNA-binding domains and a glutamine-rich N-terminus, and share similarity with Nam8p, a protein associated with U1 snRNP in the yeast Saccharomyces cerevisiae. Deletion analysis of RBP45 and RBP47 indicated that the presence of at least two RBD are required for interaction with RNA and that domains other than RBD do not significantly contribute to binding. mRNAs for RBP45 and RBP47 and mRNAs encoding six related proteins in Arabidopsis thaliana are constitutively expressed in different plant organs. Indirect immunofluorescence and fractionation of cell extracts showed that RBP45 and RBP47 are localized in the nucleus. In vivo UV crosslinking experiments demonstrated their association with the nuclear poly(A)+ RNA. In contrast to UBP1, another oligouridylate-binding nuclear three-RBD protein of N. plumbaginifolia (Lambermon et al., EMBO J, 2000, 19:1638-1649), RBP45 and RBP47 do not stimulate mRNA splicing and accumulation when transiently overexpressed in protoplasts. Properties of RBP45 and RBP47 suggest they represent hnRNP-proteins participating in still undefined steps of pre-mRNA maturation in plant cell nuclei.     

Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability

We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.     

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation.