Abstract
Subcellular localization of RNAs is critical for a number of specific cellular functions, but we still lack global maps and organizing principles for how RNAs are localized in cells and tissues. Here we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. As expected, proteomic analyses of fractions confirmed separation of subcellular compartments. Surprisingly, transcriptomic analyses revealed that RNAs sedimenting similarly across gradients encode proteins in similar protein complexes, cellular compartments, or biological functions. In most cases, RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These results provide new insights into establishment and maintenance of subcellular organization of the transcriptome.
Methods
Mouse liver was mechanically dissociated in the absence of detergent and nuclei were removed by spinning at 5Kxg for 10 minutes. The supernatant was loaded onto a 10-50% continuous sucrose gradient and spun at 30K for 3 hours in an SW41 rotor. Twenty-four fractions were collected and subjected to RNAseq and mass spectrometry analysis. The data presented here represent transcriptomes from fractions 4-20 (gradient 1) and fractions 3-24 (gradient 2), and peptide counts from fractions 3-19 (gradient 2).
