An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry.

The STIM1-ORAI1 pathway of store-operated Ca(2+) entry is an essential component of cellular Ca(2+) signalling. STIM1 senses depletion of intracellular Ca(2+) stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca(2+) channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca(2+) entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum-plasma membrane junctions before and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.

Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.