Characterization of Sus scrofa small non-coding RNAs present in both female and male gonads.

Christophe Antoniewski,Dorota Kowalczykiewicz,Jan Wrzesinski,Aleksandra Świercz,Luiza Handschuh,Marek Figlerowicz,Katarzyna Leśniak

doi:10.1371/journal.pone.0113249

Abstract

Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30–36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5′ uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.

Highlights

Three major families of small non-coding RNAs have been identified in eukaryotic cells: microRNAs, short interfering RNAs, and Piwi-interacting RNAs [1, 2]. miRNAs, which are the best-described family, are RNA molecules 21–23 nucleotides in length that are encoded in the nuclear genome
In the RNA-induced silencing complex (RISC) complex, the miRNA serves as a probe that recognizes the complementary mRNA and inhibits its expression [7]. miRNAs play a significant role in gametogenesis
In the testis of an adult male the functional spermatozoa are produced in each cycle of spermatogenesis, lasting approximately 40 days, and males reach their sexual maturity at 7 months of age, provided, that they must be at least 10–12 months old before being used for reproduction [34]

Summary

Introduction

Three major families of small non-coding RNAs (sncRNAs) have been identified in eukaryotic cells: microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs) [1, 2]. miRNAs, which are the best-described family, are RNA molecules 21–23 nucleotides (nt) in length that are encoded in the nuclear genome. Similar to miRNAs, siRNAs associate with the Ago proteins family to form RISC complexes, which bind to the target mRNAs destroying them. The piRNAs are a different class of small non-coding RNAs and, in contrast to miRNAs and siRNAs, are found exclusively in the gonads. They have been detected in many organisms (from Drosophila to human) with diverse maternal and paternal reproductive systems [21]. Disturbances of the piRNA biogenesis pathway and the loss of Piwi protein expression in males typically result in infertility. We report the characteristics of the small RNA molecules (piRNAs, miRNAs and tRFs) detected in porcine gonads by deep sequencing. The main goal of our analysis was to determine if there is a pool of small RNAs that are present in both female and male gonads or if the small RNAs that occur in testes and ovaries significantly differ from each other

Materials and Methods

Results and Discussion

10. Sus scrofa