Small RNA deep sequencing of cellular subcompartments

RNA deep sequencing efforts have revealed abundant expression of small RNAs derived from small nucleolar (sno) RNAs. We employ here spatial RNA deep sequencing to assess the expression of 10-40 nt small RNAs in subcellular compartments of HeLa cells. Total cellular, cytoplasmic, nuclear and nucleolar fractions were isolated, RNA was purified and size-fractionated in a two-step process to yield 10-40 nt RNA fractions. cDNA libraries were constructed and sequenced on a Ion Torrent platform. Vast majority of cellular, cytoplasmic and nuclear small RNA reads were identified as miRNAs. We found the expression of eleven ten miRNAs in the nucleolar preparations using a cut-off rate of 10 reads. Several miRNAs had a greater relative abundance in the nucleolus compared to the other compartments. The nucleolar small RNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped both 5’ and 3’ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads from SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as 20 and 25 nt RNAs. This study reveals a rich representation of cell-compartment specific expression of small RNAs and the distinctive unique composition of the nucleolar small RNAs. Examination of small RNAs in cellular subcompartments