Recent studies point to the existence of poorly characterized small regulatory RNAs generated from mRNAs, rRNAs and tRNAs. To explore the subcellular location of tRNA-derived small RNAs, 0–1 and 7–8 h Drosophila embryos were fractionated on sucrose density gradients. Analysis of 12,553,921 deep-sequencing reads from unfractionated and fractionated Drosophila embryos has revealed that tRFs, which are detected mainly from the 5’ends of tRNAs, co-sediment with the non-polysomal fractions. Interestingly, the expression levels of a subset of tRFs change temporally following the maternal-to-zygotic transition in embryos. We detected non-polysomal association of tRFs in S2 cells as well. Differential tRF expression pattern points to developmental significance at the organismal level. These results suggest that tRFs are associated primarily with the non-polysomal complexes in Drosophila embryos and S2 cells.

Bioinformatics programs have helped tremendously in identifying the targets of microRNAs, which are small noncoding RNAs that regulate gene expression posttranscriptionally. However, the partial complementarity between miRNAs and their targets hinders the accuracy of target prediction, necessitating the use of experimental validation procedures. Here, we describe a gene reporter assay typically used in our lab to validate putative miRNA-mRNA interactions in Drosophila S2 cells.