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The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells.

ABSTRACT: During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting approximately 0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage/translational delay in meiotic and postmeiotic male germ cells of the mouse.

SUBMITTER: Yang J

PROVIDER: S-EPMC547816 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells.

Yang Juxiang J Medvedev Sergey S Reddi P Prabhakara PP Schultz Richard M RM Hecht Norman B NB

Proceedings of the National Academy of Sciences of the United States of America 20050121 5

During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting approximately 0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former ...[more]

PMID: 15665108

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Project description:BackgroundSpermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library.MethodsPolyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea.ResultsThe authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2).ConclusionWe analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.

Project description:Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-? mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.

Dataset Information

The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells.

Publications

The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells.

Similar Datasets

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