To identify the bona fide RNA targets of an individual RBP in vivo, the crosslinking immunoprecipitation (CLIP) method was developed 4, 5, based on but beyond the regular RNA immunoprecipitation (RIP) 6, 7, by incorporation of key steps including ultraviolet (UV) crosslinking, stringent wash and gel transfer to improve signal specificity. These inconsecutive transcriptional events necessitate post-transcriptional gene regulation, in which RNA-binding proteins (RBPs) play a crucial role, shaping transcriptome and maintaining male fertility. An unique value of this model lies in the emergence of transcriptional inactivation at certain stages of spermatogenesis, typically when meiotic sex chromosome inactivation (MSCI) occurs 1, 2 and when round spermatids undergo drastic nuclear compaction during spermiogenesis 3. Mammalian testis represents an excellent developmental model wherein an intricate cell differentiation program runs cyclically to yield numerous spermatozoa. This study establishes an applicable basis for eCLIP in mammalian testis. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals.

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