Mechanisms of translational repression of the Smcp mRNA in round spermatids

Abstract

The protamine 1 (Prm1) and sperm mitochondria-associated, cysteine-rich protein (Smcp) mRNAs exemplify a widespread pattern of mRNA-specific regulation of mRNA translation in post-meiotic spermatogenic cells, spermatids. Both mRNAs are transcribed and initially stored in free-mRNPs in early spermatids, and translated on polysomes in late spermatids. In this study, we demonstrate that the 5 0 and 3 0 -UTRs and the 3 0 terminus of the Smcp 3 0 -UTR are required for normal repression of the Smcp mRNA in transgenic mice. RNA affinity chromatography and mass spectrometry sequencing identified Y-box protein 2 (YBX2/MSY2) as the major protein that interacts with the 3 0 terminus of the Smcp 3 0 -UTR and a Y-box recognition sequence, GCCACCU, in the translation control element that is necessary for Prm1 mRNA repression. Depletion of YBX2 in Ybx2-null mice prematurely activates Prm1 and Smcp mRNA translation in early spermatids. Fluorescent in situ hybridization reveals that the Smcp intron, the Smcp mRNA, and both Smcp-Gfp transgenic mRNAs are strongly concentrated in the chromatoid body, and that theYbx2-null mutation does not eliminate the Smcp mRNA from the chromatoid body. This and previous findings suggest that the Smcp pre-mRNA is spliced and associates with YBX2 in the chromatoid body, and that repressed free-mRNPs are stored in the general cytoplasm. As YBX2 is the predominant protein in testis free-mRNPs, it likely represses many mRNAs in early spermatids. The mechanisms by which YBX2 represses the Smcp and Prm1 mRNAs are relevant to reproductive medicine because mutations in the human YBX2 gene correlate with abnormal protamine expression and male infertility.

Regulatory processes that control haploid expression of salmon sperm mRNAs

“…During this period, translation of many mRNAs may be delayed for functions required during later stages of sperm differentiation. Several different processes are employed that link transcription of these genes with subsequent mRNA processing and delayed translation [3–6]. Notable among these are the groups of mRNAs that are transcriptionally upregulated by the Y-box [25] and CREM [18] proteins before transcriptional arrest.…”

“…Interestingly, there is evidence that signal motifs residing within the untranslated regions (utrs) of some of these mRNAs may also serve as recognition binding elements for the CREM and Y-box proteins [26], as well as for many other regulatory proteins and RNAs [3–6, 27–29]. Once transcribed, the subsets of mRNAs important for late-stage sperm development are bound within RNA–protein complexes, stored, transported and localized to await disassembly and translation.…”

“…RNA-binding protein (RBP)–mRNA interactions can specify functional and subcellular localization units as part of a larger regulatory network [2]. In late-stage, transcriptionally quiescent sperm cells, recognition elements within various mRNAs provide specific signals for interactions with RBPs and RNA cofactors necessary for stability, storage, transport, localization and subsequent translation [3–6]. …”

Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay

“…These YRSs are described by the 7‐nt consensus sequence NNCAYCN, in which most, but possibly not all, permutations of bases at degenerate sites are bound avidly (Chowdhury and Kleene, ). Notably, NNCAYCN includes three YRSs implicated in in vivo translation repression by YBX2 in Xenopus oocytes and mouse spermatids: AACAUCU, UCCAUCA, and GCCACCU (Matsumoto et al, ; Giorgini et al, ; Bagarova et al, ; Cullinane et al, ).…”

“…The Prm1 3′UTR contains two imperfect copies of a translation control element (TCE): a TCE (GAACAAUGCCACCUGUC) immediately upstream of the poly(A) signal, and TCE‐prime (GAAAAAUGCCACCGUC) 41 nt upstream of the poly(A) signal (Zhong et al, ). Both TCEs contain YRSs (underlined), and YBX2 binds GCCACCU (Cullinane et al, ). Mutation of all the bases in both TCEs relieves the strong YBX2‐dependent repression in round spermatids (Zhong et al, ).…”