Abstract
Post-transcriptional mechanisms including differential splicing expand the protein repertoire beyond that provided by the one gene-one protein model. Trans-splicing has been observed in mammalian systems but is low level (sometimes referred to as noise), and a contribution to hybrid protein expression is unclear. In the study of rat sperm tail proteins a cDNA, called 1038, was isolated representing a hybrid mRNA derived in part from the ornithine decarboxylase antizyme 3 (Oaz3) gene located on rat chromosome 2 fused to sequences encoded by a novel gene on chromosome 4. Cytoplasmic Oaz3 mRNA is completely testis specific. However, in several tissues Oaz3 is transcribed and contributes to hybrid 1038 mRNA synthesis, without concurrent Oaz3 mRNA synthesis. 1038 mRNA directs synthesis of a hybrid 14-kDa protein, part chromosome 2- and part chromosome 4-derived as shown in vitro and in transfected cells. Antisera that recognize a chromosome 4-encoded C-terminal peptide confirm the hybrid character of endogenous 14-kDa protein and its presence in sperm tail structures and 1038-positive tissue. Our data suggest that the testis-specific OAZ3 gene may be an example of a mammalian gene that in several tissues is transcribed to contribute to a hybrid mRNA and protein. This finding expands the repertoire of known mechanisms available to cells to generate proteome diversity.
Highlights
The mammalian sperm tail contains unique structures not present in cilia, viz. the outer dense fibers (ODF)2 and the fibrous sheath (FS)
This indicates that in these latter tissues, the ornithine decarboxylase antizyme 3 (Oaz3) gene is transcribed for the purpose of contributing to a hybrid mRNA, rather
Than expression of normal spliced Oaz3 mRNA. To our knowledge this is the first demonstration that a mammalian gene can be transcribed in one tissue to give rise to both normal and hybrid mRNA species but in other tissues only contributes to the hybrid mRNA
Summary
Male Germ Cell and Component Isolation—Rat male germ cells, including pachytene spermatocytes, round spermatids, and elongating spermatids, were isolated by centrifugal elutriation as described previously (31). Polyclonal antibodies were raised against total rat ODF and FS proteins, and anti-14 antibodies were isolated by affinity purification using filters containing immobilized 14-kDa protein as described previously (12). Genomic location of the 3Ј-end of 1038 cDNA was mapped using primers 5Ј-GGAGAGAATGTGGGGAATACCAG-3Ј and 5Ј-GCCTCTAGCCATGTTCCTCAAGC-3Ј, which generate a 346-bp PCR fragment. The pellet was washed three times in 1 ml of homogenization buffer and resuspended in 1 ml of TRIzol (Invitrogen) to isolate nuclear RNA. PCR fragments were analyzed at the University of Calgary Core DNA Services. Protein Analysis—Western blot analysis was carried out essentially as described previously (12). Immune complexes were precipitated using Protein A-Sepharose CL-4B beads (Pharmacia Biotech), washed three times with wash buffer (50 mM Tris, pH 8), and analyzed by SDS-PAGE and autoradiography as described (12). Immunoprecipitates were analyzed by Western blotting using the indicated antibodies as described above
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