Small non-coding RNAs (sRNAs) of about 20~30 nucleotides (nt) play an essential role in a variety of animal developmental processes, such as embryonic, neuronal, muscle, and germline development (Moazed D. 2009; Stefani G, et al., 2008; Pauli A, et al., 2011). MicroRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), different from biogenesis and biological function, are two predominant types of sRNAs ( Bartel DP, 2004; Houwing S, et al., 2007). Mature single-strand miRNAs are 21~25 nt and derived from longer primary miRNA molecules (pri-miRNAs), which are double-stranded RNAs (dsRNAs) and are successively processed by two RNase III endonucleases, namely Drosha and Dicer (Moazed D. 2009; Bartel DP, 2004). It was estimated that miRNAs regulate the gene expression of at least one third of all human genes and are involved in a broad spectrum of biological processes, such as development, metabolism, and tumorigenesis by either translational repression, RNA degradation or both through an RNA-induced silencing complex (RISC) (Moazed D. 2009; Stefani G, et al., 2008; Lewis BP, et al., 2005; Lu J, et al., 2005). In contrast to miRNAs, piRNAs are slightly longer with a peak size distribution of 26~28nt and mainly necessary for germ cell maintenance and genome protection by silencing transposable elements (Malone CD, et al., 2009). The primary piRNAs are maternally deposited or generated through an unclear mechanism, while more secondary piRNAs are originated from discrete genomic loci termed piRNA clusters, which produce piRNAs from both genomic strands and then reciprocally promote the generation of complementary piRNAs following a Ping-Pong model (Brennecke J, et al., 2007; Aravin AA, et al., 2007).
In this work, the sRNA-seq technology was first used to determine the expression profiles of sRNAs during eight stages of early zebrafish embryonic development. Based on known zebrafish pre-miRNAs, we designed a zebrafish-specific algorithm of ZmirP (zebrafish miRNA prediction), with 8 new and 57 previously reported sequence and structure features. These features were combined together to construct an SVM model for validating the miRNAs predicted from MIREAP and miRDeep2 (Friedländer MR, et al., 2012). The performance and robustness of ZmirP were extensively evaluated by 4-, 6-, 8-, 10-fold and leave-one-out validation (LOO). Compared with other existing approaches, ZmirP exhibits greater sensitivity of 95.64% and specificity of 98.84%. Then we greatly improved the CPSS (Zhang Y, et al., 2012) and developed a more specific platform as CSZ (characterization of small RNAome for zebrafish) for the analysis of the high-through sequencing data. From the results, we observed that the expression levels of piRNAs are gradually decreased, while miRNA expressions are gradually increased during early embryonic stages. Thus, the sRNA class transition from piRNA to miRNA was confirmed in early zebrafish embryonic development. Furthermore, we observed that the diverse and complex of expression patterns and levels of 129 known miRNA families are dramatically increased as development proceeds. Moreover, 25 novel miRNA candidates were predicted by CSZ with high confidence. We randomly selected three predicted miRNAs for further experimental investigation, and two of them, m0027-5p and chr6_7844-5p, were confirmed through Northern blots. In addition, widespread expression of piRNAs before MZT suggested piRNAs may play a potential role during early development. Taken together, our studies contributed valuable clues for further investigating the sRNA regulation of embryonic development, and provided useful techniques for small RNAome analysis. The CSZ package was implemented in Perl and freely downloadable at: http://csz.biocuckoo.org/download/csz_1.0.zip.
※ FLOWCHART OF CSZ:
Systematic characterization of small RNAome during zebrafish early developmental stages
Yuangen Yao, Lili Ma, Qiong Jia, Wankun Deng, Zexian Liu, Yuanwei Zhang, Jian Ren, Yu Xue, Haibo Jia and Qing Yang.
BMC Genomics. 2014, 15(1):117.