The abundance of a transfer rna-derived rna fragment small rna subpopulation is enriched in cauda spermatozoa

Abstract

The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis; a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.