Novel Alternative Splicing and Nuclear Localization of HumanRGS12 Gene Products

Abstract

RGS proteins are GTPase-activating proteins for certain G␣ subunits, accelerating the shutoff mechanism of G protein signaling, and also may interact with receptors and effectors to modulate G protein signaling. Here, we report identification of 12 distinct transcripts of human RGS12 that arise by unusually complex splicing of the RGS12 gene, which spans 70 kilobase pairs of genomic DNA and contains 16 exons. These transcripts arise by both cis-and trans-splicing mechanisms, are expressed in a tissue-specific manner, and encode proteins ranging in size from 356 to 1447 amino acids. Both 5-and 3-splicing of two primary RGS12 transcripts occur to generate RGS12 mRNAs encoding proteins with four distinct N-terminal domains, three distinct C-terminal domains, and a common internal region where the semiconserved RGS domain is located. Confocal microscopy and subcellular fractionation of COS-7 cells expressing RGS12 proteins with three different N termini (brain (B), peripheral (P), and trans-spliced (TS)) and a shared short (S) C-terminal domain demonstrated exclusive nuclear localization of these proteins and an influence of the N-terminal region on the pattern of intranuclear distribution. Both native RGS12TS-S in HEK-293T cells and ectopically expressed RGS12TS-S localized to discrete nuclear foci (dots), a characteristic of various tumor suppressor proteins. Subnuclear localization of RGS12TS-S into nuclear dots was cell cycle-dependent. Native RGS12TS-S associated with the metaphase chromosome during mitosis, and ectopically expressed RGS12TS-S induced formation of abnormally shaped and multiple nuclei in COS-7 cells. Expression of RGS12 proteins with long and intermediate C-terminal domains was not observed in COS-7 cells, suggesting that 3-splicing of RGS12 transcripts may influence the expression or stability of the encoded proteins. These results document extraordinary structural complexity in the RGS12 family and the role of alternative splicing and cell cycle-dependent mechanisms in expression and subnuclear targeting of RGS12 proteins.Heterotrimeric G proteins are components of many major signaling systems used by cells to transduce a variety of signals (neurotransmitters, hormones, light, and olfactory and taste signals) from specific cell-surface receptors to effector proteins (see Refs. 1 and 2 for review). These receptors activate G proteins by stimulating exchange of GTP for GDP on the ␣ subunit (G␣) of the inactive G protein heterotrimer (G␣␤␥) to promote dissociation into G␣-GTP and G␤␥ subunits. Both of these G protein subunits function as signal-transducing molecules by regulating activities of various effector proteins including enzymes and ion channels. The intensity and duration of signaling by G proteins are highly regulated. The key element that controls the lifetime of active G␣ and G␤␥ is the intrinsic GTPase activity of the G␣ subunits, producing G␣-GDP and its reassembly with G␤␥.RGS (regulators of G protein signaling) proteins constitute a family of proteins originally defined by the pr...

Human RGS6 Gene Structure, Complex Alternative Splicing, and Role of N Terminus and G Protein γ-Subunit-like (GGL) Domain in Subcellular Localization of RGS6 Splice Variants

“…Yet, the physiological function of RGS proteins has been shown only for pheromone signaling in yeast (4), neuronal signaling in C. elegans (5), and, in mammals, phototransduction in the eye where RGS9 is required for the rapid inactivation of transducin (9,10). Moreover, recent evidence from this and other laboratories has shown that several RGS proteins are localized predominantly at intracellular sites other than the plasma membrane including the nucleus (11)(12)(13)(14)(15)(16), where G proteins as well as their effectors and activating receptors are not thought to localize. These findings raise the fascinating possibility that some members of the RGS protein family may have functions apart from, or in addition to, regulatory control of heterotrimeric G protein signaling.…”

“…Recent evidence suggests that this complexity may arise not only from the more than 20 mammalian genes encoding RGS protein members, but also by the existence of multiple forms of a given RGS gene product. We identified two major transcripts for human RGS3 (27) and subsequently demonstrated the existence of twelve alternatively spliced forms of human RGS12 (12). RGS9 has also been found to exist in two splice variant forms (28).…”

“…PCR Amplification of RGS6 cDNAs-Full-length cDNAs encoding various forms of RGS6 were amplified using a PCR-based strategy we described previously (12). We utilized a 505-bp expressed sequence tag identified as RGS6 (GenBank TM accession number H09621) to design primers for use in 5Ј-and 3Ј-RACE to amplify overlapping segments of RGS6 cDNAs essentially as we described previously (12).…”

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

“…AGS3-5 were subsequently named G-protein signaling modulator (GPSM) 1-3, respectively by the HUGO Gene Nomenclature Committee. AGS6 encodes RGS12 (Chatterjee and Fisher, 2000). AGS5 is widely expressed, whereas expression of AGS3 and AGS4 are more restricted (Blumer et al, 2002;Cao et al, 2004;Pizzinat et al, 2001;Takesono et al, 1999).…”

Pre-mRNA trans-splicing: from kinetoplastids to mammals, an easy language for life diversity

“…-Another possibility in the generation of hybrid mRNA molecules is trans-splicing of transcripts coded by a cluster of genes. Hybrid mRNAs were detected between members of the immunoglobulin locus (Shimizu et al 1989, Fujieda et al 1996, the human GTPase RSG12 gene and a sequence localized 170 kb downstream from the RGS12 gene (Chatterjee & Fisher 2000), transcripts coded by genes of the cytochrome P450 3A cluster (Finta & Zaphiropoulos 2002), and members of the mouse protocadherin locus .The four cytochrome P450 3A genes (CYP3A4, CYP3A5, CYP3A7, and CYPA43) are located in a cluster of human chromosome 7 (Finta & Zaphiropoulos 2002). CYPA43 is in a head-to-head orientation to the CYP3A4 and CYP3A5 genes, i.e.…”