Germ Cell-Specific Proteins Interact with the 3′ Untranslated Regions of Prm-1 and Prm-2 mRNA

Abstract

The testis-specific mouse protamine genes (Prm-1 and Prm-2) are transcribed in haploid round spermatids, their mRNAs stored as cytoplasmic ribonucleoprotein particles and translated about 1 week later in elongating spermatids. We have compared the in vitro translational efficiencies of deproteinized Prm-1 mRNA isolated from purified populations of germ cells and found that Prm-1 mRNA from round spermatids translates as efficiently as Prm-1 mRNA from elongating spermatids, suggesting that translation of Prm-1 mRNA is normally repressed in round spermatids. Previous studies in transgenic mice have shown that the 3' UTR of Prm-1 mRNA is necessary and sufficient for its translational control (Braun et al., 1989). In this manuscript, we have used an RNA band shift assay to identify an activity, present in cytoplasmic fractions of meiotic spermatocytes and postmeiotic round spermatids, that binds the 3'UTRs of both Prm-1 and Prm-2 mRNA. We have used 3' UTR deletion variants to map the binding site to a 22-nt region within the Prm-1 3' UTR and to a 20-nt region within the Prm-2 3' UTR. uv cross-linking of the RNA band shift activities detected with the Prm-1 and Prm-2 3' UTRs generated the same two RNA/protein complexes of 53 and 55 kDa. The presence of the binding activity in the cell type and subcellular compartment associated with Prm-1 and Prm-2 mRNA storage suggest that the activity may be actively engaged in translational repression of these mRNAs.

Post–transcriptional control of gene expression during spermatogenesis

“…39 Competition assays reveal that the complex is specific for Prm-1 and Prm-2 mRNAs and mutational analysis and RNAse footprinting studies have been used to map the binding site to a specific region in the 5Ј-end of the 3Ј Ž . UTRs Figure 1 .…”

“…To elucidate the mechanisms of translational regulation it is important to identify all of the factors involved. Despite significant progress in identifying protamine RNA-binding proteins, none of the proteins thus far described, PRBP, 41 TB-RBP, 23,24 or the 48r50 kDa Y box proteins, 39 have been shown to mediate the translational repression of the protamine mRNAs in vivo. Certain properties of these proteins, for example, the sequence-specific binding properties of the 48r50-kDa Y box proteins and TB-RBP, the ability of the binding site for the 48r50-kDa proteins to repress translation in vivo and the presence of the PRBP-binding site in a region of the Prm-1 3Ј UTR shown to be sufficient for translational repression in transgenic mice, are desirable for putative translational repressors.…”

Translation of a Testis-Specific Cu/Zn Superoxide Dismutase (SOD-1) mRNA Is Regulated by a 65-Kilodalton Protein Which Binds to Its 5′ Untranslated Region

“…A phosphoprotein which binds to highly conserved sequences (the Y and H element) of about 35 nucleotides located within the 3Ј UTR of mouse protamine 2 has been shown to repress translation in vitro (40,41). Recently, Fajardo and coworkers (18) have identified two testicular proteins that bind to a 22-nucleotide sequence within the 3Ј UTR of protamine 1 and a 20-nucleotide region within the 3Ј UTR of protamine 2. In contrast, SOD-RBP appears to require most of the 114 nucleotides of the 5Ј UTR of T SOD-1 for optimal RNA-protein complex formation in vitro, since deletion transcripts of either the 5Ј end or the 3Ј end of the 5Ј UTR dramatically decrease or abolish the binding activity.…”

“…In addition to the testicular RNA-binding proteins that interact with the 3Ј UTRs of mRNAs (18,40,41), p48 and p52, homologs of the Xenopus FRG Y2, p54 and p56 which function both as transcription factors and mRNA-masking proteins (48,49), are present in the testis (42). The testicular 48-and 52-kDa proteins not only bind to all RNA sequences including the 3Ј UTR of mP2 mRNA (42,64) but also bind to a conserved element in the promoter of testis-specific genes including protamine 2 (51).…”

Les gènes de la spermatogènese et leur régulation

“…La comparaison des r6gions 3'UTR des ARN de PRM1 et PRM2 interagissant avec des prot6ines r6v61e la pr6sence d'une courte homologie de s6quence, pouvant constituer un 616-merit important de liaison [54]. Par, ailleurs, des exp6riences de comp6tition indiquent que les m~mes prot6ines se lient ~ la r6gion 3'UTR de PRM1 PRM2.…”