Single-nucleotide Differences and Cell Type Decide the Subcellular Localization of miRNA Isoforms (isomiRs), tRNA-derived Fragments (tRFs) and rRNA-derived Fragments (rRFs)

BackgroundMicroRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent ~95% of all short RNAs found in cells. All three types modulate mRNA and protein abundance and are dysregulated in diseases. Experimental studies to date assumed that the subcellular localization of these molecules is well understood and constant across cell types. ResultsWe investigated the localization of isomiRs, tRFs, and rRFs in biological replicates from three frequently-used model cell lines. In each case, we analyzed the contents of the nucleus, cytoplasm, whole mitochondrion, mitoplast, and the whole cell. We used a rigorous mathematical model to account for cross-fraction contamination and technical errors and adjusted abundances accordingly. We found that isomiRs, tRFs, and rRFs exhibit complex and unexpected patterns of subcellular localization. These patterns depend on the type of the RNA molecule, its exact sequence, and the cell type. Even for "sibling" RNAs from the same parental RNA whose sequences differ by only a few nucleotides, their subcellular localization depends on each siblings exact sequence and the cell type. ConclusionsPrevious studies of isomiRs, tRFs, and rRFs that used ectopic expression without accounting for isoforms may need to be re-evaluated. Future experiments with these molecules will need to distinguish among the multiple isoforms and account for the fact that each isoforms abundance and destination depend on its exact sequence and cell type. The findings additionally suggest the existence of an intracellular trafficking program for isomiRs, tRFs, and rRFs and, by extension, expanded roles for these molecules - both dimensions await characterization. To help design future experiments, we compiled a first-of-its-kind Atlas to catalogue the subcellular localization and abundance of 5,898 isomiRs, tRFs, and rRFs across three model cell lines. Results SummaryO_LIWe analyzed the distribution of microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) in the{whitebullet} nucleus {whitebullet}cytoplasm {whitebullet}mitochondrion, and {whitebullet}mitoplast of biological replicates from three cell lines from the same tissue. C_LIO_LIWe corrected the measured abundances by accounting for cross-fraction contamination and technical errors through a rigorous mathematical model. C_LIO_LIOur analysis revealed complex localization patterns involving numerous isomiRs, tRFs, and rRFs. C_LIO_LIThe subcellular localization of these RNAs depends on their exact sequence and differs even for molecules that arise from the same parental miRNA, tRNA, or rRNA. C_LIO_LIFor a given RNA, its subcellular localization additionally depends on cell type. C_LIO_LIThe findings have implications for previous and future molecular studies of the function of isomiRs, tRFs, and rRFs. C_LIO_LIThe findings suggest the existence of a complex subcellular trafficking program, and hint at expanded functions for these RNA molecules that differ by compartment. C_LIO_LITo assist with the design of future experiments, we created a first-of-its-kind Atlas that catalogues the subcellular distribution and abundance of 5,898 isomiRs, tRFs, and rRFs across three cell lines. C_LI