Quantification of RNAs’ Biochemical Compartmentalizations in Cardiac Myocytes and Cardiac Fibroblasts

RNA localization is a mechanism for establishing specific subcellular protein populations. Previously, we determined that each RNA has distinct spatial distributions using RNAScope. Here, we leveraged the biochemical properties of different detergents to assess the biochemical compartmentalization of several RNAs in primary cardiac myocytes and cardiac fibroblasts. Our fractionation technique partitioned the cells into four compartments: Cytosol, Membrane, Nuclear, and Insoluble. After optimization, we reliably recovered over 75% of the RNA on average from both cell types. Unexpectedly, two of the RNAs, Acta2 and Tnni3, appear to bind so tightly to proteins they require denaturing conditions for maximal recovery when using Trizol. Our analysis also revealed that the RNAs’ biochemical compartmentalization was largely independent of their spatial localization. For example, transcripts with distinct spatial distributions frequently shared similar biochemical compartmentalization profiles and vice versa. When we compared cardiac myocytes to cardiac fibroblasts, we found that compartmentalization is cell-type-dependent, with all but two transcripts (Ryr2 and Tnnt2) changing their compartmentalization profile across the cell types. These findings indicate that spatial localization and biochemical compartmentalization represent distinct layers of RNA organization and likely rely on different molecular mechanisms. Thus, our analysis provides new insight into the complexity of intracellular RNA regulation.