Small non-coding RNA profiling and the role of piRNA pathway genes in the protection of chicken primordial germ cells

Abstract

BackgroundGenes, RNAs, and proteins play important roles during germline development. However, the functions of non-coding RNAs (ncRNAs) on germline development remain unclear in avian species. Recent high-throughput techniques have identified several classes of ncRNAs, including micro RNAs (miRNAs), small-interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs). These ncRNAs are functionally important in the genome, however, the identification and annotation of ncRNAs in a genome is challenging. The aim of this study was to identify different types of small ncRNAs particularly piRNAs, and the role of piRNA pathway genes in the protection of chicken primordial germ cells (PGCs).ResultsAt first, we performed next-generation sequencing to identify ncRNAs in chicken PGCs, and we performed ab initio predictive analysis to identify putative piRNAs in PGCs. Then, we examined the expression of three repetitive sequence-linked piRNAs and 14 genic-transcript-linked piRNAs along with their linked genes using real-time PCR. All piRNAs and their linked genes were highly expressed in PGCs. Subsequently, we knocked down two known piRNA pathway genes of chicken, PIWI-like protein 1 (CIWI) and 2 (CILI), in PGCs using siRNAs. After knockdown of CIWI and CILI, we examined their effects on the expression of six putative piRNA-linked genes and DNA double-strand breakage in PGCs. The knockdown of CIWI and CILI upregulated chicken repetitive 1 (CR1) element and RAP2B, a member of RAS oncogene family, and increased DNA double-strand breakage in PGCs.ConclusionsOur results increase the understanding of PGC-expressed piRNAs and the role of piRNA pathway genes in the protection of germ cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-757) contains supplementary material, which is available to authorized users.

The role of non-coding RNAs in male sex determination and differentiation

“…At the amino acid level, CIWI shows 78, 76 and 64% similarity with human, mouse and zebrafish PIWIs respectively, with especially highly conserved PAZ and PIWI domains (80%). RNAi-induced knockdown of CIWI and CILI results in upregulated expression of the repetitive element CR1 and an increase in DNA double-strand breakage in isolated chicken PGCs (Rengaraj et al 2014). These data indicate that, similar to zebrafish and mice, the PIWI/piRNA pathway is operational in avian gametes.…”

“…In addition, other piRNAs are derived from the 3 0 UTRs of proteincoding genes, which has been shown to be conserved from Xenopus to mammals (Robine et al 2009, Ha et al 2014. Using shotgun cloning or high-throughput nextgeneration sequencing, piRNAs have been identified in embryonic and adult chicken testis , Rengaraj et al 2014. Despite some challenges associated with the low sequence conservation of small ncRNAs, chicken piRNAs have been annotated and mapped in clusters across the chicken genome.…”

Transcriptional and translational dynamics during maternal‐to‐zygotic transition in early chicken development

“…Each miRNA forward primer and chicken small nucleolar RNA primer (Supplemental Table 1) was designed following the guidelines from Agilent Technologies (Santa Clara, CA, USA). miRNA expression was normalized to that of a chicken small nucleolar RNA, which has been validated as an internal control (25, 26), and was calculated by using the 2 ‐ΔΔ Ct method (27).…”

Expression profiles of long and short RNA s in the cytoplasm and nuclei of growing chicken (Gallus gallus domesticus) oocytes

“…piРНК экспресси-руются в половых клетках, участвуя в инактивации мобильных элементов генома и регуляции экспрессии спе-цифичных для клеток яичника и/или семенников генов (Aravin, Hannon, 2008;Lau et al, 2009;Malone et al, 2009). У курицы piРНК и piРНК-по-добные РНК были недавно охарак-теризованы с помощью технологии секвенирования следующего поколе-ния (NGS) в семенниках Zhang et al, 2013) и в ранних эмбрионах (Shao et al, 2012), в том числе в клетках зародышевого пути (Rengaraj et al, 2014), но не в расту-щих ооцитах.…”