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Biomedical subjects

R E Braun

Publications and source records attributed to R E Braun.

At least 19 recordsLinked to original sources
PubMed2001

Absence of germline infection in male mice following intraventricular injection of adenovirus.

The possibility of inadvertent exposure of gonadal tissue to gene therapy vectors has raised safety concerns about germline infection. We show here that the receptor for coxsackie B viruses and adenoviruses 2 and 5 (CXADR) is expressed in mouse germ cells, suggesting the possibility that these viruses could infect germ cells. To directly assess the risk of germline infection in vivo, we injected an adenovirus carrying the germ-cell-specific protamine promoter fused to the bacterial lacZ reporter gene into the left ventricular cavity of mice and then monitored expression of the reporter gene in germ cells. To differentiate between infection of stem cells and differentiating spermatogenic cells, we analyzed expression of the reporter cassette at different times after viral delivery. Under all conditions tested, mice did not express the Escherichia coli beta-galactosidase protein in developing spermatids or in mature epididymal spermatozoa. Primary germ cells cultured in vitro were also refractory to adenoviral infection. Our data suggest that the chance of vertical germline transmission and insertional mutagenesis is highly unlikely following intracoronary adenoviral delivery.

Adenoviridae↗
PubMed2001

A highly conserved sequence essential for translational repression of the protamine 1 messenger rna in murine spermatids.

Translational regulation of the protamine 1 mRNA is mediated by sequences in its 3' untranslated region. In this study, we demonstrate that a highly conserved sequence, the translational control element, is solely responsible for protamine 1 translational regulation. Mutation of the conserved sequence causes premature translation of a transgene containing a fusion between the human growth hormone coding sequence and the protamine 1 3' untranslated region. Temporal expression of the transgene was monitored in prepubertal animals by Northern and Western blotting and in adult animals by immunocytochemistry. Messenger RNAs lacking the translational control element sediment in the messenger ribonucleoprotein particle and ribosomal fractions of polysome gradients, suggesting that the translational control element is required for translational repression but not for incorporation of mRNAs into ribonucleoprotein particles.

Acrosome↗
PubMed2001

MSY2 and MSY4 bind a conserved sequence in the 3' untranslated region of protamine 1 mRNA in vitro and in vivo.

Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5'-UCCAUCA-3', present in the 3' untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of U(AC)C(A)CAU(C)CA(CU) (subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

3' Untranslated Regions↗
PubMed2001

Requirement for the murine zinc finger protein ZFR in perigastrulation growth and survival.

The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function.

Animals↗
PubMed2000

A sequence-specific RNA binding complex expressed in murine germ cells contains MSY2 and MSY4.

The protamine mRNAs are stored for up to 8 days as translationally repressed ribonucleoprotein particles during murine spermatogenesis. Translational repression of the protamine 1, Prm1, mRNA is controlled by sequences in its 3'-untranslated region (UTR). In this study we used the yeast three-hybrid system to clone Msy4, which encodes a novel member of the Y box family of nucleic acid binding proteins. MSY4 specifically binds to a site within the 5' most 37 nucleotides in the Prm1 3' UTR. Msy4 is highly expressed in the testis, and the protein is detected in the cytoplasm of germ cells in both the testis and the ovary, where repressed messages are stored. Analysis of a previously described 48/50-kDa binding activity in testis extracts by electrophoretic mobility shift assays and immunoprecipitation indicates the activity is composed of MSY4 and MSY2, another mouse Y box protein. Polysome analysis demonstrates MSY4 is associated with mRNPs, consistent with MSY4 having a role in storing repressed messages.

3' Untranslated Regions↗
PubMed2000

Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells.

Fanconi anemia is a polygenic trait hypothesized to be a DNA damage repair disease. We show that all three Fanconi anemia loci that have been cloned are expressed in the embryonic gonad during the period of primordial germ cell proliferation. Mice mutant for the Fanconi anemia complementation group C locus (Fancc) have reduced germ cell numbers as early as embryonic day E12.5, suggesting the Fancc protein functions prior to meiosis in both sexes. Depletion in the mutant occurs at a time when all three loci would be expressed in a wild-type gonad, implying a function in the early germline. Determination of the mitotic index of primordial germ cells by BrdU incorporation shows that germ cells in Fancc(-/-) mice proliferate significantly more slowly than littermate controls. This study demonstrates Fancc is required for mitotic proliferation of primordial germ cells.

Animals↗
PubMed2000

Male contraception: views to the 21st century, Bethesda, MD, USA, 9-10 September 1999.

For men who still wish to father children, the contraceptive options currently available are withdrawal and the condom. Although significant progress has been made on hormonal and vaccine-related approaches to male contraception, a marketed product is, at best, several years away. Therefore, the National Institute of Child Health and Human Development convened a workshop to discuss novel strategies for development of male contraceptives that focused on the testis and epididymis. Participants recognized that exploration of these new approaches will necessitate considerable investment of funds and research efforts.

Contraception↗
PubMed2000

Temporal control of protein synthesis during spermatogenesis.

During oogenesis and spermatogenesis transcription ceases prior to the differentiation of the mature cells. To complete germ cell differentiation and initiate early embryogenesis, proteins are synthesized from pre-existing mRNAs that are stored for several days. It is well established that important regulatory elements functioning in spatial localization, temporal translation or messenger RNA stability are located in the 3' untranslated region (3' UTR) of mRNAs. During mammalian spermatogenesis temporal translational regulation of the protamine 1 (Prm1) mRNA is dependent on a highly conserved sequence located in the distal region of its 3' UTR. The 17-nucleotide translational control element (TCE) mediates translational repression of the Prm1 mRNA. Mutation of the TCE causes premature synthesis of protamine protein and sterility. The Prm1 mRNA is stored as a cytoplasmic ribonucleoprotein (mRNP) particle in spermatids. Contained within the particle are several members of the Y box family of nucleic acid binding proteins. In the yeast three-hybrid system the murine Y box proteins MSY1, MSY2 and MSY4 bind in a sequence-dependent manner to a conserved region in the proximal portion of the Prm1 3' UTR. Sequence-specific binding by MSY4 to the Y box recognition sequence (YRS) is dependent on the highly conserved cold shock domain, possibly through the RNP1 and RNP2 motifs present within it. The Y box proteins may function as translational repressors in vivo. Alternatively, their primary function may be to protect mRNAs from degradation during their extended period of storage. Translational activation of stored mRNAs is essential for the completion of gametogenesis. Proper translational activation of the Prm1 mRNA in elongated spermatids requires the cytoplasmic double-stranded RNA binding protein TARBP2. Tarbp2 is expressed at low levels in many cells but is expressed at robust levels in late stage meiotic cells and in postmeiotic spermatids. Mice mutant for Tarbp2 are defective in proper translational activation of the Prm1 and Prm2 mRNAs and are sterile. Current studies are designed to determine the mechanism by which proteins bound to the 3' UTR communicate with the 5' end of the message to control translational silencing and activation.

Animals↗
PubMed1999

Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein.

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of the mouse interleukin enhancer binding factor 3 (Ilf3) homolog in a screen for RNA binding proteins.

In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells.

3' Untranslated Regions↗
PubMed1999

A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells.

Chromatin packaging in mammalian spermatozoa requires an ordered replacement of the somatic histones by two classes of spermatid-specific basic proteins, the transition proteins and the protamines. Temporal expression of transition proteins and protamines during spermatid differentiation is under translational control, and premature translation of protamine 1 leads to precocious nuclear condensation and sterility. We have previously suggested that the double-stranded (ds) RNA binding protein Prbp (encoded by the gene Tarbp2) functions as a translational regulator during mouse spermatogenesis. Here we show that Prbp is required for proper translational activation of the mRNAs encoding the protamines. We generated mice that carry a targeted disruption of Tarbp2 and determined that they were sterile and severely oligospermic. Using immunohistological analysis, we determined that the endogenous Prm2 mRNA and a reporter mRNA carrying protamine 1 translational-control elements were translated in a mosaic pattern. We showed that failure to synthesize the protamines resulted in delayed replacement of the transition proteins and subsequent failure of spermiation. The timing of Prbp expression suggests that it may function as a chaperone in the assembly of specific translationally regulated ribonucleoprotein particles.

Animals↗
PubMed1998

Post-transcriptional control of gene expression during spermatogenesis.

The synthesis and storage of mRNAs prior to their translation is a necessity during spermatogenesis as global transcription ceases several days prior to the completion of spermatid differentiation. Post-transcriptional control can be mediated by sequences in the 5' and 3' untranslated regions of mRNAs, and in some cases separate elements may regulate translational repression and translational activation. Translational repression is essential for spermatid differentiation as premature translation can lead to an arrest in spermatid differentiation and cause dominant male sterility.

Animals↗
PubMed1998

Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.

Animals↗
PubMed1997

Separate elements in the 3' untranslated region of the mouse protamine 1 mRNA regulate translational repression and activation during murine spermatogenesis.

The mouse protamine mRNAs, Prm-1 and Prm-2, are translationally repressed for several days during male germ cell differentiation. The translational delay of mouse Prm-1 mRNA has previously been shown to be dependent upon cis-acting elements that reside in the last 62 nucleotides of the Prm-1 3' untranslated region (3' UTR). We have previously identified a 48/50-kDa protein that binds the 3' UTRs of both Prm-1 and Prm-2 mRNAs in a sequence-specific manner, is present in cytoplasmic fractions of postmeiotic round spermatids where the protamine mRNAs are translationally silent, and is markedly reduced in elongated spermatids where the protamine mRNAs become activated for translation. Surprisingly, the binding site for this activity maps to a region of the Prm-1 3' UTR not contained within the functional 62 nucleotides described above. In this report we show that the binding site for the 48/50-kDa protein can also delay translation of a reporter RNA in vivo, suggesting that the 48/50-kDa protein can repress the translation of Prm-1 mRNA during murine spermatogenesis. This observation proves that two separate regions of the Prm-1 3' UTR are sufficient to repress Prm-1 translation. In addition, immunocytochemistry and polysome analysis have revealed that this transgenic reporter mRNA fails to undergo proper translational activation. These results suggest that an additional region of the Prm-1 3' UTR is required for proper translational activation and that Prm-1 translational repression elements can be separated from those involved in translational activation.

Animals↗