Identification of Potential Regulatory Elements in the 5′ and 3′ UTRs of 12 Translationally Regulated mRNAs in Mammalian Spermatids by Comparative Genomics

Abstract

To facilitate identifying translational control elements by studies of mutations in transgenic mice, a database of orthologous 5' and 3' ends of 12 messenger RNA (mRNA) species from 13 to 23 mammals that undergo delayed translational activation in spermatids was constructed for the Acev2, Akap3, Akap4v2, Gapdhs, Odf1, Prm1, Prm2, Prm3, Smcp, Spata18, Tnp1, and Tnp2 mRNAs. This database, available here, was searched for conserved sequences in conserved positions and known translational control elements. Numerous potential mRNA-specific elements were identified, including upstream open reading frames, conserved sequences upstream and downstream of the poly(A) signal, and noncanonical and multiple poly(A) signals. RNA electrophoresis mobility shift assays demonstrate that Y-box proteins bind 30 of the 36 permutations of the degenerate Y-box recognition sequence (YRS), [UAC][CA]CA[UC]C[ACU], and this information was used to identify hundreds of YRSs in the untranslated region (UTR) database. Collectively, these findings suggest that the distal ends of both UTRs are particularly well conserved, implying that translation of each mRNA is regulated by mechanisms involving the poly(A) binding protein and the closed loop. In addition, the 5' flanking regions of all 12 genes have conserved, gene-specific sequences and configurations of elements that resemble the binding site of the testis-specific isoform of cyclic AMP response element modulator, and all 12 genes lack retrogene paralogues, demonstrating the efficacy of mechanisms that limit the proliferation of retroposons in the male germ line. This study illustrates the power of comparative genomics in identifying novel hypothetical regulatory elements for analysis with biochemical and in vivo genetic approaches.