Nova-1 Regulates Neuron-Specific Alternative Splicing and Is Essential for Neuronal Viability

Abstract

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.

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

“…It is interesting to note that some of the splicing factors like PSI and Mer1p, identified to be involved in recruiting U1 snRNP to pre-mRNA, contain KH RNAbinding motifs+ Studies of crystal structures of the KH RNA-binding domains revealed that it has a baabba topology and the three-stranded antiparallel b sheets oriented against the three a helices form a favorable surface for RNA recognition (Lewis et al+, 1999)+ The overall specificity of this motif for RNA may be achieved through the interaction of loops and other exposed surfaces of the domain with additional interaction sites on the target RNA+ Absence of a strong sequence specificity for this RNA-binding motif would explain how these different splicing factors consisting of KH motifs could recognize and bind to different or distinct regions near particular weak 59 splice sites+ Our studies reveal a short pyrimidine-rich consensus (RCYYCUURYRC; Fig+ 2A) for PSI RNA binding+ Detailed RNA-binding studies on two KH domain proteins, Nova-1 and Nova-2, showed a preference for tetranucleotide UCAY sequences in their target RNAs (Yang et al+, 1998;Jensen et al+, 2000)+ The KH domain splicing factors SF1/mBBP and yBBP both recognize the YNCURAY branch point sequence (Berglund et al+, 1998) and it was recently shown that the SF1/mBBP recognizes specific nucleotides in the branch point sequence (Peled-Zehavi et al+, 2001)+ Changing the consensus branchpoint sequence UAC UAAC at position 4 from U to G had a strong negative effect on SF1/mBBP binding (Peled-Zehavi et al+, 2001)+ These studies, in conjunction with our SELEX data for PSI indicate that KH domain-containing proteins generally recognize short pyrimidine-rich sequences in their target RNAs+ The different KH domain proteins may achieve their overall affinity and specificity for target RNAs by contacting additional sequences surrounding short pyrimidine-rich core motifs+ The context surrounding these core motifs therefore may play a major role in generating high affinity binding sites+ The extended PSI consensus sequence we have identified (RCYYCUURYRC; Fig+ 2A) shows the importance of nucleotides surrounding the CUU core in the PSI RNA-binding sites+ Interestingly, the resemblance of the consensus PSI SELEX sequence to the P element 59 exon F2 pseudo-59 splice site also 1250 A.K. Amarasinghe et al shows the preference of PSI for the pyrimidine-rich sequences in its natural target+ The recruitment of U1 snRNP to pseudo-59 splice sites, as seen for the P element IVS3 59 exon, or to weak 59 splice sites such as for MER2 and fas, could modulate the recognition of 59 splice sites to get either inhibition or activation, respectively+ Our data showed that a high affinity PSI SELEX RNA-binding sequence can function as a splicing inhibitor element and that the PSI protein is the major contributor to the inhibitory activity seen in the hybrid SELEX Up#6-P element IVS3 substrate splicing+ It is possible that similar sites are found near other 59 splice site-like sequences in PSI target cellular RNAs, in much the same way as the U-rich auxiliary regions are found near the msl-2 and fas 59 splice sites that function in TIA-1 binding (Forch et al+, 2000)+ Other examples of proteins with pyrimidinerich RNA-binding sites acting as splicing inhibitors include the Drosophila Sex-lethal protein (Singh et al+, ...…”

Control of alternative pre-mRNA splicing by Ca++ signals

“…The relative levels of positive and negative regulatory factors determine the generation of variant mRNA isoforms in specific cell types [73][74][75]. Brainenriched splicing factors include Nova-1 and -2 [62][63][64][65][66], FOX-1 and -2 [54-61], PTBP2 (nPTB) and neuronal Hu proteins [76][77][78][79][80][81][82][83].…”

Region-specific alternative splicing in the nervous system: Implications for regulation by the RNA-binding protein NAPOR

“…The spliceosome is the active arrangement of small nuclear ribonucleoprotein particles (snRNPs), which aligns the splice site boundaries and serves as the catalytic machinery for splicing+ The relative efficiency of spliceosome assembly on multiple introns of a complex pre-mRNA is instrumental in determining patterns of alternative splicing, as well as the accuracy of individual splicing events+ Whether an internal exon is constitutively spliced or subject to exon skipping is determined by the strength of the individual splice sites flanking the exon in combination with the effects of splicing enhancer and/or repressor elements (Burge et al+, 1999;Smith & Valcarcel, 2000)+ Serine/argininerich (SR) protein splicing factors and hnRNP proteins, which in specific combinations interact with such regulatory elements, have been shown to exhibit some tissue-specific variations (Kamma et al+, 1995;Hanamura et al+, 1998)+ However, these previous studies do not reveal a striking pattern of enrichment, or deficiency, of these factors in nervous system tissue, leaving open the possibility that subtle combinatorial differences in these proteins may contribute to splicing regulation in many tissues+ A number of RNA-binding proteins have been implicated in the regulation of neural-specific splicing events+ Nova-1 is a splicing activator with three hnRNP K homology (KH) domains, and its expression pattern is brain specific (Buckanovich et al+, 1996;Buckanovich & Darnell, 1997)+ Targets of Nova-1 regulation include the neural exons of the GlyRa2 and GABA A receptor g2 transcripts, which are positively regulated in transient coexpression assays (Jensen et al+, 2000a(Jensen et al+, , 2000b)+ In Nova-1 knockout mice, the ratio of the exon-included and exon-skipped mRNA products is disrupted in the corresponding midbrain and hindbrain regions where Nova-1 protein has been genetically depleted+…”