The effect of higher order RNA processes on changing patterns of protein domain selection: a developmentally...

The domain structure of proteins synthesized from a single gene can be remodeled during tissue development by activities at the RNA level of gene expression. The impact of higher order RNA processing on changing patterns of protein domain selection may be explored by systematically profiling single-gene transcriptomes. itpr1 is one of three mammalian genes encoding receptors for the second messenger inositol 1,4,5-trisphosphate (InsP3). Some phenotypic variations of InsP3 receptors have been attributed to hetero-oligomers of subunit isoforms from itpr1, itpr2, and itpr3. However, itpr1 itself is subject to alternative RNA splicing, with 7 sites of transcript variation, 6 within the ORF. We have identified 17 itpr1 subunit species expressed in mammalian brain in ensembles that change with tissue differentiation. Statistical analyses of populations comprising >1,300 full-length clones suggest that subunit variation arises from a variably biased stochastic splicing mechanism. Surprisingly, the protein domains of this highly allosteric receptor appear to be assembled in a partially randomized way, yielding stochastic arrays of subunit species that form tetrameric complexes in single cells. Nevertheless, functional expression studies of selected subunits confirm that splicing regulation is connected to phenotypic variation. The potential for itpr1 subunits to form hetero-tetramers in single cells suggests the expression of a developmentally regulated continuum of molecular forms that could display diverse properties, including incremental sensitivities to agonist activation and varying patterns of Ca2+ mobilization. These studies illuminate the extent to which itpr1 molecular phenotype is induced by higher order RNA processing.