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SMALL NONCODING RNA CONTENTS OF RAT EPIDIDYMAL EXTRACELLULAR VESICLES INCLUDING A PUTATIVE NOVEL SMALL RNA

Extracellular vesicles (EVs) released from the epididymal epithelium (epididymosomes) impart functional competence on sperm as they transit the epididymis by merging with sperm and releasing a complex repertoire of molecules. The cargo of epididymosomes includes small noncoding RNAs (sncRNAs) that are modified by external factors such as stress, nutrition, and drug use. If incorporated into sperm, the EV sncRNA cargo can affect offspring and lead to heritable phenotypes. In the current study we characterized the RNA contents of EVs collected from the caput epididymis of adult male rats in order to fill a gap in knowledge in this species and to establish a sncRNA profile. Small RNAs of EVs were isolated from the caput portion of the epididymis of adult male rats, and sequenced on a NovaSeq 6000 on a SP flow cell in a single-end 50 bp configuration. The resulting reads were checked for quality, trimmed for adapter sequences, aligned to the unmasked rat genome (Rnor 6), and assigned an annotation designation. The majority of RNA reads aligned to either tRNA fragments (79.1%) or piRNA (18.1%) loci. Micro RNAs (miRNAs) accounted for a surprisingly small proportion of reads (0.18%). The third largest category of aligned reads (1.5%) was in intergenic space and not strictly associated with canonical sncRNA loci. In-depth investigation determined these latter reads (∼19 nt) aligned strictly within the boundaries of known CpG islands (CpGi), which have not previously been reported to express any form of sncRNA. These newly described “CpGi sRNAs” could not consistently be accounted for by overlaying features of any other annotation type (including rRNA and piRNA). The CpGi sRNAs have characteristics of RNA fragments that can associate with the Argonaute/PIWI family of proteins and therefore could have regulatory function via RNA induced silencing or de novo DNA methylation. We propose that CpGi sRNAs constitute a new family of sncRNA that may represent an important and unreported class of regulatory RNA in gametes.