Small RNA species in the mammalian meiotic nucleus

MiRNAs and possibly the recently discovered piRNAs can function in reversible gene silencing. In meiosis, transcriptional silencing of the XY bivalent and the late pairing regions of autosomes is a necessary event and defects in these processes lead to infertility. The exact mechanisms of meiotic gene silencing are not well understood but it is thought that some RNA component might be involved at least in the XY transcriptional silencing, similar to Xist silencing of the X chromosome in females. We wanted to investigate if small RNA species are present in the meiotic nucleus and, if so, to determine their localization pattern. We found both miRNAs and piRNAs in the nucleus of spermatogenic cells. In Sertoli cells, miRNAs and piRNAs are localized to the fibrillogranular compartment of the nucleolus, while in meiotic cells they are found mainly in the XY-associated dense body but also are associated with chromosome cores, telomerers and the sex chromatin. Interestingly, in MIWI null male mice, the nucleolar localization of miRNAs is decreased while the localization of piRNAs is diminished. However, the meiotic localization of these two components is not affected. These data suggest that small RNA species might have different roles during different stages of male gametogenesis and undergo differential regulation. Based on the localization of miRNAs and piRNAs in the male-specific, XY-associated dense body, it is probable that at least some are involved in the silencing of the XY bivalent. Others might be involved in translational repression of transcripts used in later stages of spermiogenesis when transcription is halted. Keywords: total RNA direct hybridization Small RNAs were profiled by custom Agilent arrays with complementary probes for 257 mature mouse miRNAs. Total RNA was directly labelled and hybridized to these arrays. 5 probes were designated for each miRNA from which a median intensity was derived and compared to 4000 intensities arising from random probe sequences.微RNA（miRNAs）以及近年新发现的Piwi互作RNA（piRNAs）均可参与可逆性基因沉默过程。在减数分裂过程中，XY二价体与常染色体迟配对区域的转录沉默是必需事件，该过程出现缺陷会引发不育。目前减数分裂基因沉默的确切机制尚未完全阐明，但研究认为至少XY染色体的转录沉默过程可能涉及某种RNA组分，这与雌性体细胞中X失活特异性转录本（Xist）介导的X染色体沉默机制类似。 本研究旨在探究减数分裂细胞核内是否存在小RNA分子，并明确其定位模式。 本研究在生精细胞的细胞核中同时检测到miRNAs与piRNAs。在支持细胞（Sertoli细胞）中，miRNAs与piRNAs定位于核仁的纤维颗粒区；而在减数分裂细胞中，二者主要定位于XY相关致密体，同时也与染色体轴、端粒及性染色质存在关联。值得注意的是，在MIWI基因敲除雄性小鼠中，miRNAs的核仁定位水平显著降低，而piRNAs的核仁定位则明显减弱。但二者在减数分裂期的定位模式并未受到影响。 上述结果表明，小RNA分子在雄性配子发生的不同阶段可能发挥不同功能，并受到差异化调控。基于miRNAs与piRNAs定位于雄性特异性XY相关致密体这一发现，推测其中至少部分分子参与了XY二价体的沉默过程。其余小RNA则可能在转录活动停止后的精子形成后期，参与靶转录本的翻译抑制过程。 关键词：总RNA直接杂交；本研究通过定制安捷伦（Agilent）芯片对257种成熟小鼠miRNAs进行小RNA谱分析，该芯片配备针对上述靶标的互补探针。实验中对总RNA进行直接标记后，与上述芯片进行杂交。每个miRNA对应设计5条探针，通过计算得到其中位信号强度，并与4000条随机探针序列产生的信号强度进行比对。