Identification of elements in the Smcp 5' and 3' UTR that repress translation and promote the formation of heavy inactive mRNPs in spermatids by analysis of mutations in transgenic mice

Abstract

The sperm mitochondria-associated cysteine-rich protein (Smcp) mRNA is transcribed in step 3 spermatids, and is stored in free mRNPs until translation begins ∼6 days later in step 11. To identify sequences that control the timing of Smcp mRNA translation, mutations in both UTRs were analyzed in transgenic mice using green fluorescent protein (GFP), squashes of seminiferous tubules, and quantification of polysomal loading in adult and 21 dpp testes in sucrose and Nycodenz gradients. GFP fluorescence is first detected in step 9 spermatids in lines harboring a transgene containing the Gfp 5' UTR and Smcp 3' UTR. Unexpectedly, this mRNA is stored in large, inactive mRNPs in early spermatids that sediment with polysomes in sucrose gradients, but equilibrate with the density of free mRNPs in Nycodenz gradients. Randomization of the segment 6-38 nt upstream of the first Smcp poly(A) signal results in early detection of GFP, a small increase in polysomal loading in 21 dpp testis, inactivation of the formation of heavy mRNPs, and loss of binding of a Y-box protein. GFP is first detected in step 5 spermatids in a transgene containing the Smcp 5' UTR and Gfp 3' UTR. Mutations in the start codons in the upstream reading frames eliminate translational delay by the Smcp 5' UTR. Collectively, these findings demonstrate that Smcp mRNA translation is regulated by multiple elements in the 5' UTR and 3' UTR. In addition, differences in regulation between Smcp-Gfp mRNAs containing one Smcp UTR and the natural Smcp mRNA suggest that interactions between the Smcp 5' UTR and 3' UTR may be required for regulation of the Smcp mRNA.