[Show abstract][Hide abstract] ABSTRACT: Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties,
such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in
response to membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However,
proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation
of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved
CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition
motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated
exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished
by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly
step to regulate the variant subunit composition of potassium channels.
[Show abstract][Hide abstract] ABSTRACT: The regulation of gene expression through alternative pre-mRNA splicing is common in metazoans and is often controlled by intracellular signaling pathways that are important in cell physiology. We have shown that the alternative splicing of a number of genes is controlled by membrane depolarization and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) through CaMKIV-responsive RNA elements (CaRRE1 and CaRRE2); however, the trans-acting factors remain unknown. Here we show that the heterogeneous nuclear ribonucleoprotein (hnRNP) L is a CaRRE1 binding factor in nuclear extracts. An hnRNP L high affinity CA (cytidine-adenosine) repeat element is sufficient to mediate CaMKIV and hnRNP L repression of splicing in a location (3'-splice site proximity)-dependent way. Depletion of hnRNP L by RNA interference followed by rescue with coexpressed exogenous hnRNP L demonstrates that hnRNP L mediates the CaMKIV-regulated splicing through CA repeats in heterologous contexts. Depletion of hnRNP L also led to increased inclusion of the stress axis-regulated exon and a CA repeat-harboring exon under depolarization or with activated CaMKIV. Moreover, hnRNP L binding to CaRRE1 was increased by CaMKIV and, conversely, was reduced by pretreatments with protein phosphatases. Therefore, hnRNP L is an essential component of CaMKIV-regulated alternative splicing through CA repeats, with its phosphorylation likely playing a critical role.
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing of a single pre-mRNA transcript can produce protein isoforms that promote either cell growth or death. Here we show that Ro-31-8220 (Ro), an apoptotic agent that inhibits protein kinase C and activates the c-Jun N terminal kinase, decreased the proportion of the cell growth-promoting Bcl-xL splice variant. Targeted mutagenesis analyses narrowed down a critical sequence to a 16-nt G-tract element (Gt16). Transferring this element to a heterologous gene conferred Ro response on an otherwise constitutive exon. The Ro effect was reduced by okadaic acid, an inhibitor of protein phosphatases PP1 and PP2A, in a concentration-dependent manner. Search in the human genome followed by RT-PCR identified a group of genes that contain similar exonic G-tract elements and are responsive to Ro. Moreover, the Gt16 element also mediates the regulation of alternative splicing by other cell apoptosis-inducers particularly retinoic acid. Therefore, the G-tract element likely plays a role in the apoptotic agents-induced alternative splicing of a group of genes. The functions of these genes imply that this regulation will have impact on cell growth/death.
Nucleic Acids Research 07/2008; 36(10):3320-31. DOI:10.1093/nar/gkn207 · 9.11 Impact Factor