Novel Alternative Splicing and Nuclear Localization of HumanRGS12 Gene Products*

RGS proteins are GTPase-activating proteins for certain Gα 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- andtrans-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.

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