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A Comparative Profile of the MicroRNA Transcriptome in Immature and Mature Porcine Testes using Solexa Deep Sequencing

ABSTRACT: MicroRNAs (miRNAs) are small non-coding regulatory RNAs that play key roles in many diverse biological processes such as spermatogenesis. However, no study has been performed on the miRNA transcriptome of developing porcine testes. Here, we employed Solexa deep sequencing technology to extend the repertoire of porcine testis miRNAs and extensively compare the expression patterns of the sexually immature and mature porcine testes. Solexa sequencing of two small RNA libraries derived from immature (30 days) and mature (180 days) pig testis samples yielded over 25 million high-quality reads. Overall, the two developmental stages had significantly different small RNA compositions. A custom data analysis pipeline identified 398 known and/or homologous conserved porcine miRNAs, 15 novel pig-specific miRNAs, and 56 novel candidate miRNAs. We further observed multiple mature miRNA variants (isomiRs) and identified a new bidirectional transcribed miRNA locus, ssc-mir-181a. One hundred twenty-two miRNAs were differentially expressed in the immature and mature testes, and 10 were validated using quantitative RT-PCR. Furthermore, GO and KEGG pathway analyses of the predicted miRNA targets further illustrate the likely roles for these differentially expressed miRNAs in spermatogenesis. This study is the first comparative profile of the miRNA transcriptome in immature and mature porcine testes using a deep sequencing approach, and it provides a useful resource for future studies on the role of miRNAs in spermatogenesis and male infertility treatment.

ORGANISM(S): Sus scrofa

PROVIDER: GSE28169 | GEO | 2011/09/23

SECONDARY ACCESSION(S): PRJNA139519

REPOSITORIES: GEO

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Project description:Sus scrofa (pig, or swine) is one of the most important economic animals and a close biological model for complex human diseases such as obesity and diabetes. It is therefore utterly important to decode the porcine microRNAome (miRNAome) as in the literature only a small portion of it is known. In this work, a comprehensive search for porcine microRNAs (miRNAs) by Illumina sequencing was performed in ten small RNA libraries prepared from mixtures of assorted tissues, which included those collected from fetuses to adult pigs. The millions of the sequencing reads were analyzed with reference to 77 known porcine miRNA precursors (pre-miRNAs) and 3,443 distinct pre-miRNAs of other mammals listed in miRBase 13.0, and the most updated porcine genome (Sscrofa9, April 2009) and available EST sequences. Additionally, miRNA candidates specific to pig are predicated by genome & EST match and hairpin folding. Our search found 72 out of 78 (~92%) known porcine miRNAs and miRNA*s, and 36 previously unannotated miRNA*s are also indentified. Furthermore, we discovered 397 novel miRNAs by mapping to the sequencing transcripts to other mammalian pre-miRNAs and 493 candidate miRNAs which do not map to other mammalian miRNAomes and could be pig-specific. We constructed sequence- and genome-position clusters for the total of 998 miRNA candidates originating from 862 pre-miRNAs, which represent 777 unique miRNA sequences. Together with the six known porcine miRNAs that not been observed in our study, we report herein the sequence families of 783 unique miRNAs and genomic distribution patterns of 622 pre-miRNAs. We preformed q-PCR experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing data and the q-PCR data. We envision that our report will serve as a valuable resource for future studies aimed at understanding miRNAome of pig Ten small RNA libraries from samples of porcine fetuses (30 days after insemination) to mixed tissues (180 days after birth) at ten developmental stages were sequenced.

Project description:Purpose: To identify the correlation of the expression of testicular miRNAs and piRNAs with the infertility of Mtdh exon 3 deficient mice Methods: Total RNAs from individual testis of two WT mice and three homozygous Mtdh exon 3-depleted mice were used to prepare the miRNA sequencing library. After the completed libraries were quantified with an Agilent 2100 Bioanalyzer, the DNA fragments in the libraries were denatured with 0.1M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ and sequenced for 36 cycles on Illumina HiSeq 2000 according to the manufacturer’s instructions. Image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8.0). Subsequently, 3’ adapter sequences were trimmed from clean reads (reads that passed Solexa CHASTITY quality filter) and any reads shorter than 15nt were discarded. Next the 3’-adapter-trimmed-reads (? 15nt) were aligned to the latest known human reference miRNA precursor set (Sanger miRBase 19) using Novoalign (v2.07.11). Reads (counts < 2) were discarded when calculating miRNA expression. In order to characterize the isomiR variability, any sequences that matched the miRNA precursors in the mature miRNA region ±4nt (no more than one mismatch) were accepted as mature miRNA isomiRs, which were grouped according to the 5-prime (5p) or 3-prime (3p) arm of the precursor hairpin. For piRNAs, the 3’-adapter-trimmed reads (length ? 15nt) were aligned to the latest piRNA set in piRNAbank using Novoalign software (v2.07.11). Expression of each piRNA was defined as the mapped tag counts. Results: Reduced expression of miRNAs was detected in homozygous Mtdh exon 3-deficient testes (Table I). As shown in tables II and III, piRNA expression levels were dysregulated, with some being reduced and others increased in homozygous Mtdh exon 3-deficient testes compared to WT mice. Conclusions: Our study represents the first detailed analysis of Mtdh deficiency on the expression of small noncoding RNAs, generated by RNA-seq technology. We conclude that Mtdh is correlated to the expression of small non-coding RNAs including miRNAs and piRNAs at testis of mouse. Examine expression of miRNAs and piRNAs at testes of wild type and Mtdh deficent mice

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A Comparative Profile of the MicroRNA Transcriptome in Immature and Mature Porcine Testes using Solexa Deep Sequencing

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