In Male Mouse Germ Cells, Copper-Zinc Superoxide Dismutase Utilizes Alternative Promoters That Produce Multiple Transcripts with Different Translation Potential

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.

Characterization of the mouse Men1 gene and its expression during development

“…It would be interesting to investigate whether the two major Men1 transcripts have di erent translation potential, like in the case of the copper-zinc superoxide dismutase gene (SOD1). 5' variants of SOD1 mRNAs are di erentially expressed in various stages of male germ cells and have been shown to di er by their ability to associate to polysomes, and thus to be translated e ciently (Gu et al, 1995).…”

An Alternative Splicing Event Which Occurs in Mouse Pachytene Spermatocytes Generates a Form of DNA Ligase III with Distinct Biochemical Properties That May Function in Meiotic Recombination

“…Several examples of differences in gene expression between somatic and germs cells, including different transcription initiation sites (16), differences in the length of the poly(A) tract (13,18), alternative splicing (15), and the existence of homologous genes, one expressed only in somatic cells and the other expressed only in germ cells (19,27), have been described. In this report, we have characterized a testis-specific alternative splicing mechanism that generates a 96-kDa DNA ligase III polypeptide, DNA ligase III-␤, which differs with respect to carboxy terminus and molecular mass from the ubiquitously expressed 103-kDa DNA ligase III-␣.…”

Feedback Regulation between Zipcode Binding Protein 1 and β-Catenin mRNAs in Breast Cancer Cells

“…The assays were performed as described previously (11). Briefly, RNA aliquots (15 g) were denatured in 80% formamide at 65°C for 10 min and annealed with 0.5 g of specific oligonucleotide(s) complementary to the ZBP1 mRNA for 90 min at 50°C in a buffer containing 10 mM PIPES (pH 6.4), 40% formamide, 0.4 M NaCl, and 1 mM EDTA.…”