A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing

Abstract

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor ␣2 (GlyR␣2) pre-mRNA, and this binding is implicated in Novadependent regulation of splicing. Cotransfection assays with a GlyR␣2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyR␣2 E3A. brPTB binds to a 90-nt GlyR␣2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyR␣2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissuerestricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.N eurons make extensive use of alternative splicing to regulate functional differences in proteins. A wide variety of neurotransmitter receptor activities are regulated by alternative splicing, including NR1 N-methyl-D-aspartate (NMDA) receptor subcellular localization (1) and interaction with neurofilaments (2), the physiology of the glutamate (3) and NMDA (4) receptors, and the ability of agrin to induce clustering of acetylcholine receptors (5). Moreover, several neurologic diseases such as spinal muscular atrophy, amyotrophic lateral sclerosis, and paraneoplastic opsoclonus-myoclonus ataxia (POMA) have been associated with defects in proteins involved in generating the splicing machinery or in the accurate splicing of target .Since the discovery of tissue-specific splicing of the calcitonin͞ calcitonin gene-related peptide (CGRP) transcript in neurons, there has been an extensive search for cis-acting RNA elements and trans-acting RNA binding proteins that mediate neuronspecific splicing. The first example of cis-acting regulatory elements in neuronal pre-mRNAs identified was in calcitonin͞ CGRP pre-mRNA (9), and a number of specific sequences have been identified that are responsible for calcitonin͞CGRP tissuespecific processing (10, 11). Subsequent work identified regulatory sequences near other neuron-specific exons such as the N1 exon of src (12) and a 24-nt exon of the ␥-aminobutyric acid type A receptor ␥2 subunit (13).The identification of trans-acting factors that regulate neuronal splicing has been a greater challenge. Two general mechanisms might account for the way such factors could mediate regulation of neuronal splicing. Brain-specific variants in splicing ...

Structure-function relationships of the polypyrimidine tract binding protein

“…Therefore, it will be meaningful to examine its functional role, as well as its structure, in more detail and look for differences between the splice variants. Beside U2AF, the repressive role of PTB has been antagonized by many different RNA binding proteins, namely the CELF [13,25] proteins ETR3 and CUG-BP, RBM4 [33], TIA-1 [27,34], Nova [40] and Fox-1 [102]. It will be valuable to gain a better understanding of the molecular mechanisms behind these antagonisms.…”

“…Raver1 recruitment is essential for effective repressor function of PTB in certain pre-mRNAs, for example for the splicing repression of a-tropomyosin exon 3, while in other systems PTB alone is sufficient. Furthermore, tissue-specific paralogs of PTB exist, such as neuronally enriched PTB [9,40] (nPTB), the smooth muscle-specific variant (smPTB) [41] or ROD1 the variant found in rat hematopoietic cell [42], which appear to have different effects on splicing regulation [9,41]. The second most studied function of PTB is its role in internal ribosomal entry site (IRES)-mediated translation initiation of both cellular and viral mRNAs.…”

“…and RRM4 are highly conserved across species, as well as in PTB paralogs [41,42] like nPTB [9,40] and hence it seems likely that this topology is functionally significant. The NMR structures of free RRM1 and RRM2 have been assessed in another study [82].…”

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

“…The expression levels of various SR proteins (Ayane et al, 1991;Mayeda and Krainer, 1992;Screaton et al, 1995;Zahler et al, 1993) and hnRNPs (Kamma et al, 1995) are variable among tissues and could therefore account for differences in splice site selection. Several examples of antagonistic splicing factors have been described (Cá ceres et al, 1994;Gallego et al, 1997;Mayeda et al, 1993;Polydorides et al, 2000), where one factor promotes inclusion of an exon and the other factor promotes its skipping. In the nucleus, factors involved in pre-mRNA processing are concentrated in speckles-dynamic structures (Misteli et al, 1997) that are sensitive to phosphorylation (Nayler et al, 1998b).…”

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].…”