Absolute quantification of specific tRNA-derived small RNAs
Mammalian spermatocytes harbor a variety of RNAs that are mostly degradation products of abundant non-coding RNAs, including ribosomal RNA-derived small RNAs (rsRNAs) and tRNA-derived RNAs (tDRs). Notably, tDRs have been implicated in the inheritance of paternally acquired traits, primarily in rodents. Direct experimental proof for this notion comes from the manipulation of fertilized murine oocytes through microinjection of small RNA preparations resulting in an impact on specific metabolic pathways that were measurable in the offspring. How exactly these paternally transmitted small RNAs could function mechanistically in the fertilized oocyte remains to be understood. Since nothing is known about how many small RNA molecules would be required for functional impact in the developing zygote, we aimed at determining absolute copy numbers of specific small RNAs contained in a single murine spermatocyte. Using hybridization-based methods that avoid amplification-induced biases, we report here average copy numbers for specific tDRs and rsRNAs in single mouse spermatocytes. These results should allow an approximation of how many rRNA- and tRNA-derived RNAs will enter an oocyte in the physiological context of fertilization. The reported numbers underscore the need for a better understanding of the quantitative nature of the biological system that is being manipulated using small RNAs, and to be cautious when arriving at conclusions as to the effect of introducing unphysiological numbers of molecules into a biological system.