Abstract
Copper-zinc superoxide dismutase (SOD-1) is an enzyme that is widely expressed in eukaryotic cells and performs a vital role in protecting cells against free radical damage. In mouse testis, three different sizes of SOD-1 mRNAs of about 0.73, 0.80, and 0.93 kilobases (kb) are detected. The 0.73-kb mRNA is found in early stages of male germ cells and in all somatic tissues. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase H digestions and Northern blot analyses reveal that the three SOD-1 mRNAs are derived from two transcripts, a ubiquitously expressed transcript and a post-meiotic transcript, which differ by 114-120 nucleotides. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. Using a probe derived from the 5'-untranslated region of the 0.93-kb SOD-1 mRNA, we have established that it originates from an alternative upstream promoter contiguous with the somatic SOD-1 promoter. Polysomal gradient analysis of the three mouse testis SOD-1 mRNAs reveals that the 0.93-kb SOD-1 mRNA is primarily non-polysomal, while the 0.80- and 0.73-kb SOD-1 mRNAs are mostly polysome associated. A faster migrating form of the 0.93-kb SOD-1 mRNA is present on polysomes as a result of partial deadenylation. In a cell-free translation system, the 0.73-kb SOD-1 mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-1 mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-1 mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-1 mRNAs undergo adenylation changes, and one of the post-meiotic SOD-1 mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form.
Highlights
From the Wepartment of Biology, Tufts University, Medford, Massachusetts 02155 and the §Department of Anatomy, McGill University, Montreal, Quebec H3A 2B2, Canada
In a cell-free translation system, the 0.73-kb Superoxide dismutase (SOD)-! mRNA translates about 2-fold more efficiently than the 0.93-kb SOD-! mRNA. These data demonstrate that male germ cells transcribe two size classes of SOD-! mRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-! mRNAs undergo adenylation changes, and one of the post-meiotic SOD-! mRNAs is transcribed during mid-spermiogenesis and translated days later in a partially deadenylated form
Sin ce the 0 .80- a n d 0 .93-kb SOD-! mRNAs first a p pea r in the teste s of prepuberal mice a t th e tim e of differentiation when po st-mei otic spe rm a t ids are see n . we a na lyzed e n ri ched populations of cells t o better d efin e the cellul ar locat ions of the two large SOD·! tran scripts
Summary
236-243, 1995 Printed in U.S.A. In Male Mouse Germ Cells, Copper-Zinc Superoxide Dismutase Utilizes Alternative Promoters That Produce Multiple Transcripts with Different Translation Potential*. The mRNAs of 0.80 and 0.93 kb are exclusively detected in post-meiotic germ cells. RNase protection assays demonstrate that the additional nucleotides present in the post-meiotic mRNA are solely in the 5'-untranslated region. MRNAs with different translation potential by utilizing two different promoters, post-meiotic SOD-! We present evidence for three size classes ofSOD-1 mRNAs in the mouse testis; one, a post-meiotic transcript, is translationally controlled during mouse spermiogenesis. The extended 5'UTR of the longest post-meiotic SOD-1 mRNA may fine tune synthesis of SOD-1 in late stages of germ cell development since it is less efficiently translated in vitro and in vivo.
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