Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs.
Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs.
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DOI：
10.1016/j.ydbio.2016.08.003
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发表时间：
2016-10-15
影响因子：
2.7
通讯作者：
Bennett KL
中科院分区：
文献类型：
--
作者：
McEwen TJ;Yao Q;Yun S;Lee CY;Bennett KL
在模式生物秀丽隐杆线虫的基因组中已经鉴定出超过400种不同的microRNAs （miRNAs）。由于生殖系致力于保存每个物种，并且成年线虫中几乎一半的细胞都是生殖系，因此调节性mirna可能对生殖系的发育和维持很重要。在秀丽隐杆线虫中，miR35家族具有很强的母性效应，有助于正常的胚胎发生和成虫的繁殖力。为了确定是否有任何特定的miRNA在秀丽隐杆线虫种系中大量富集，我们使用RNA-seq比较了几种成年秀丽隐杆线虫种系缺陷菌株的miRNA群体，包括glp-4（种系增殖-4），glh-1（种系解旋酶-1）和dcr-1（dicer-1）。RNA-seq比较的统计分析鉴定出13个种系富集的miRNA，包括七个研究得很好的miR35家族成员，在TaqMan qRT PCR miRNA分析中减少了多达1000倍。与mir35一起，其他六个：miR-56 （miR51家族的成员），- 70,- 244,- 260，- 788和- 4813，之前都没有被认为是这样，也被RNA-seq鉴定为种系富集候选者。我们继续为秀丽隐杆线虫开发了一种成功的miRNA原位杂交方案，揭示了mir35在卵子发生期间特异性地集中在性腺的粗线区，并持续整个早期胚胎发生，而在成年动物中，let-7和miR-228都没有种系偏向。
Over four hundred different microRNAs (miRNAs) have been identified in the genome of the model organism the nematode Caenorhabditis elegans. As the germline is dedicated to the preservation of each species, and almost half of all the cells in an adult nematode are germline, it is likely that regulatory miRNAs are important for germline development and maintenance. In C. elegans the miR35 family has strong maternal effects, contributing to normal embryogenesis and to adult fecundity. To determine whether any particular miRNAs are greatly enriched in the C. elegans germline we used RNA-seq to compare the miRNA populations in several germline-defective strains of adult C. elegans worms, including glp-4(germline proliferation-4), glh-1(germline helicase-1) and dcr-1(dicer-1). Statistical analyses of RNA-seq comparisons identified 13 miRNAs that are germline-enriched, including seven members of the well-studied miR35 family that were reduced as much as 1000-fold in TaqMan qRT PCR miRNA assays. Along with the miR35s, six others: miR-56 (a member of the miR51 family),−70, −244, −260 , −788 and −4813, none of which previously considered as such, were also identified by RNA-seq as germline-enriched candidates. We went on to develop a successful miRNA in situ hybridization protocol for C. elegans, revealing miR35s specifically concentrate during oogenesis in the pachytene region of the gonad, and persist throughout early embryogenesis, while in adult animals neither let-7 nor miR-228 has a germline-bias.