Playing an essential role in the central dogma of molecular biology – bringing amino acids to the elongating polypeptide chain during translation – transfer RNAs (tRNAs) were long considered to be solely part of the translation machinery. However, discoveries made in the last two decades have indicated that enzymatically cleaved tRNA-derived fragments (tRFs) are ubiquitous in cells. These findings group them with other small RNAs (such as piRNA, miRNA, snoRNA, etc.) as biologically active molecules that regulate diverse cellular processes. Accurate deep sequencing of tRNAs and their fragments has historically been constrained by their structural complexity and extensive posttranscriptional modifications. In this work, I first benchmark a novel cloning protocol (OTTR) for sequencing tRNAs to characterize intact tRNAs and tRFs in yeast and mouse tissues. I show that OTTR captures a wide variety of tRF species in the sample. And, unlike standard cloning methods, I also show that OTTR captures both 5’ and 3’ fragments at similar levels, giving us the most accurate picture of tRF levels to date.

Given our lab’s interest in transgenerational epigenetic inheritance, I also used OTTR on mature mouse spermatozoa. My data demonstrate that mammalian sperm carries a far more complex payload of tRFs than previously appreciated, including both 5’-tRFs and 3’-tRFs derived from the majority of tRNAs. These results force us to alter our understanding of the mature sperm RNA payload and the potential epigenetic functions it may carry.