Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody
Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody
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DOI：
10.5582/bst.2012.v6.5.248
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发表时间：
2012-01-01
影响因子：
5.5
通讯作者：
Honda, Susumu
中科院分区：
文献类型：
--
作者：
Nishibu, Takahiro;Hayashida, Yukinobu;Honda, Susumu
MIWI 是主要在小鼠生殖细胞中表达的 PIWI 蛋白亚家族之一，与粗线期 piRNA 相关。 MIWI 被认为通过粗线期 piRNA 的生物发生和/或稳定性、逆转录转座子沉默和靶 mRNA 的转录后调节在精子发生和精子发生中发挥重要作用。然而，MIWI 的详细作用和功能尚不清楚。在这项研究中，我们生产了一种抗 MIWI 小鼠单克隆抗体，并通过成年小鼠睾丸裂解物的免疫沉淀鉴定了 MIWI 相关的 Poly(A) RNA。大约 70% 的 MIWI 相关 Poly(A) RNA 是已知的 mRNA，其中 30% 是未知的非编码 RNA。这些poly(A) RNA包含从piRNA簇区域转录的piRNA编码RNA和piRNA编码mRNA，例如Aym1 mRNA。这些 piRNA 编码 RNA 中特异编码的成熟 piRNA 在粗线期精母细胞中产生，但在 Miwi 缺陷 (Miwi(-/-)) 睾丸中未检测到。此外，MIWI与大量已知的mRNA相关，这些mRNA的表达水平在粗线期精母细胞中增加，而这些mRNA的表达在产后20天粗线期精母细胞最丰富的Miwi(-/-)睾丸中降低。这些结果强烈表明，MIWI 参与粗线期 piRNA 生物合成，并通过与粗线期 piRNA 前体和靶 mRNA 的关联，参与粗线期精母细胞中靶 mRNA 代谢的正向调节。
MIWI is one of the PIWI subfamily of proteins mainly expressed in mouse germ cells, and associates with pachytene piRNAs. MIWI has been thought to play an essential role in spermatogenesis and spermiogenesis via biogenesis and/or stability of pachytene piRNAs, retrotransposon silencing, and post-transcriptional regulation of target mRNAs. However, MIWI's detailed role and function are not well understood. In this study, we produced an anti-MIWI mouse monoclonal antibody and identified MIWI-associated poly(A) RNAs by immunoprecipitation from adult mouse testes lysates. Approximately 70% of the MIWI-associated poly(A) RNAs were known mRNAs and 30% of them were unknown non-coding RNAs. These poly(A) RNAs contained piRNA-encoding RNAs transcribed from piRNA cluster regions and piRNA-encoding mRNA, such as Aym1 mRNA. Mature piRNAs specifically encoded in these piRNA-encoding RNAs were generated in pachytene spermatocytes and not detected in Miwi-deficient (Miwi(-/-)) testes. Moreover, MIWI associated with a large number of known mRNAs whose expression levels were increased in pachytene spermatocytes, and the expression of these mRNAs was decreased in Miwi(-/-) testes at 20 days postpartum when pachytene spermatocytes were most abundant. These results strongly suggest that MIWI is involved in pachytene piRNA biogenesis and the positive regulation of target mRNA metabolism in pachytene spermatocytes via association with pachytene piRNA precursors and target mRNAs.