The intracellular Ca(2+) signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca(2+)-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca(2+)-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca(2+) mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca(2+)-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca(2+) mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40-42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca(2+) signaling pathways.
Keywords: Alternative Splicing; Calcium; Calcium Signaling; Calcium-binding Proteins; Nuclear Speckles; Protein-Protein Interactions; RNA Polymerase II; SR Proteins; Subcellular Organelles.