核糖核酸酶C29F5.3於秀麗隱桿線蟲中調控精子生成機制

Abstract

小核糖核酸在精子生成的過程中扮演著重要的角色。先前的研究揭示了一組小核糖核酸與其相關的argonautes蛋白和秀麗隱桿線蟲的精子生成有關。我們發現當特定的小核糖核酸-argonautes複合物的缺失後，會導致精子在高溫的環境下（25℃）發生缺陷。而ALG-3/4即是這一組argonautes，而與其結合的26G小核糖核酸對高溫下精子的功能非常重要。過去的研究表明，小核糖核酸-ALG-3/4複合物能夠藉由觸發另一種argonaute，CSR-1，所介導的小核糖核酸路徑，確保在25°C時能大量生成精子相關的特異性蛋白。而除了ALG-3/4外，ERI/DICER蛋白複合物是另一群參與在ALG-3/4 26G小核糖核酸生成的成員。因此，這些複合物的成員一旦缺失，它們也會導致線蟲精子在25°C下有所缺陷。然而，這些ALG-3/4 26G小核糖核酸的產生機制為何尚不清楚。 在我的研究中，我們研究一個尚未被報導的基因，c29f5.3。並使用兩個c29f5.3的突變種：核糖核酸酶缺陷型突變種c29f5.3（hyt044）和一個假定的胞苷脫氨酶缺失突變種c29f5.3（tm2499）。發現兩者皆會在高溫下（25° C）不孕。進一步研究發現更這個不孕的現象是精子方面出現了問題，並且可能是發生在精子活化的過程中。相似的情況同樣發生在alg-3/4突變種中。此外，我們也發現c29f5.3突變體會導致ALG-3/4的26G小核糖核酸減少。最後，使用免疫沉澱法後能得到HA-C29F5.3的蛋白質相互作用物。在進行後續的質譜分析後發現，ALG-3確實是C29F5.3的蛋白質相互作用物之一。這些資訊表明C29F5.3參與在ALG-3/4調控的小RNA路徑中，並藉此去調控精子生成的機制。

Small RNAs participate in spermatogenesis is a conserved regulatory pathway from C. elegans to human. Previous studies have revealed a sub-group of small RNAs, 26G small RNA, and their associated argonautes, ALG-3 and ALG-4 (abbreviate as ALG-3/4), are essential for sperm function at elevated temperature (25°C) in C. elegans. The ALG-3/4 26G small RNA protein complex is known triggers RNA dependent RNA polymerase (RdRP), EGO-1, to synthesize a subset of 22G small RNAs and these 22G small RNAs specifically associate with CSR-1, which is another argonaute. The CSR-1 mediated gene regulatory pathway is known results in up-regulation in the transcription level of the targeted genes, which has been known as licensing processes. Previous studies have also shown that the ALG-3/4 26G small RNA targeted genes are majority sperm specific genes. Though several proteins have been known participates in biogenesis of ALG-3/4 26G small RNAs, such as, the class I ERIs in the ERI/DICER complex, the detail mechanism of the biogenesis of ALG-3/4 26G small RNAs are still unknown. Here, we have identified a novel ZC3H12A-like NYN ribonuclease domain protein, C29F5.3. Our data show that the two c29f5.3 mutant strains, including one ribonuclease defective mutant, c29f5.3(hyt044), and a putative cytidine deaminase deletion mutant, c29f5.3(tm2499), result in sterile phenomenon at elevated temperature (25°C). Our further characterization shows that the sterility is solely contributed from sperm failure and likely during sperm activation processes, similar to what is reported in alg-3/4 mutant strain. Furthermore, we have also shown that c29f5.3 mutants not only result in decreases in one of the ALG-3/4 related small RNA but also alg-3/4 targeted mRNAs. Moreover, our mass spectrometry analysis of the protein interactors of immunoprecipitated HA-C29F5.3 has revealed that ALG-3 is one of the protein interactors of HA-C29F5.3. Our data suggest that C29F5.3 participates in ALG-3/4 26G small RNA regulatory pathway and is likely via interaction between C29F5.3 and ALG-3.