👤 J C Richardson

Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis [PABP1, embryonic PABP (ePABP), and PABP4] is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein-protein interactions. Show less

Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis. Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L). Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required. Show less

The basic helix-loop-helix transcriptional repressor Hairy-related transcription factor 2 (Hrt2) is expressed in ventricular, but not atrial, cardiomyocytes, and in endothelial and vascular smooth muscle cells. Mice homozygous for a null mutation of Hrt2 die perinatally from a spectrum of cardiac abnormalities, raising questions about the specific functions of this transcriptional regulator in individual cardiac cell lineages. Using a conditional Hrt2 null allele, we show that cardiomyocyte-specific deletion of Hrt2 in mice results in ectopic activation of atrial genes in ventricular myocardium with an associated impairment of cardiac contractility and a unique distortion in morphology of the right ventricular chamber. Consistent with the atrialization of ventricular gene expression in Hrt2 mutant mice, forced expression of Hrt2 in atrial cardiomyocytes is sufficient to repress atrial cardiac genes. These findings reveal a ventricular myocardial cell-autonomous function for Hrt2 in the suppression of atrial cell identity and the maintenance of postnatal cardiac function. Show less

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression. Show less

Mitogen-activated protein kinase (MAPK) pathways couple intrinsic and extrinsic signals to hypertrophic growth of cardiomyocytes. The MAPK kinase MEK5 activates the MAPK ERK5. To investigate the potential involvement of MEK5-ERK5 in cardiac hypertrophy, we expressed constitutively active and dominant-negative forms of MEK5 in cardiomyocytes in vitro. MEK5 induced a form of hypertrophy in which cardiomyocytes acquired an elongated morphology and sarcomeres were assembled in a serial manner. The cytokine leukemia inhibitory factor (LIF), which stimulates MEK5 activity, evoked a similar response. Moreover, a dominant-negative MEK5 mutant specifically blocked LIF-induced elongation of cardiomyocytes and reduced expression of fetal cardiac genes without blocking other aspects of LIF-induced hypertrophy. Consistent with the ability of MEK5 to induce serial assembly of sarcomeres in vitro, cardiac-specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death. These findings reveal a specific role for MEK5-ERK5 in the induction of eccentric cardiac hypertrophy and in transduction of cytokine signals that regulate serial sarcomere assembly. Show less

HP1-like chromobox genes comprise an evolutionarily conserved family of genes that encode components of centromeric heterochromatin. In order to investigate the role of the murine HP1-like gene, M31, in heterochromatin formation we have isolated its gene and characterised its transcripts and protein products. PCR products that represent M31 transcripts were detected at the one-cell stage and were maternal in origin. Maternal provision of M31 transcripts may reflect a need for M31 in the formation of a functional centromere in order that there is proper segregation of chromosomes during the early cleavage divisions; studies in fission yeast and Drosophila have suggested a crucial role for HP1-like genes in centromere function. There are three protein products encoded by the M31 gene. Surprisingly, the two smaller products are found almost exclusively in the cytoplasm. Show less

The basement membrane plays an important role in maintaining the structural and functional integrity of tissues. Altered basement membrane structure has been associated with severe functional impairment of the testis in several conditions, including vasectomy, autoimmune orchitis, cryptorchidism, and following x-irradiation. We have used efferent duct ligation as a model to examine seminiferous tubular basement membrane morphology, synthesis, and gene expression to determine whether altered basement membrane synthesis is responsible for the aberrant structures noted after tissue injury. On days 2 and 3 after ligation, both the seminiferous epithelium and the basement membrane appeared normal, but 7 days after ligation, the seminiferous epithelium began to degenerate. The basement membrane appeared detached from the epithelium, and redundant patches of basement membrane were observed adjacent to the Sertoli cells at 14 and 21 days postligation. Immunoprecipitation indicated an increase in laminin protein synthesis in the ligated tubules at the same time. Northern blot analysis showed increases in transcript levels for laminin as well as collagen IV and heparan sulfate proteoglycan. These data show that new protein synthesis is responsible, at least in part, for the duplication of the basement membrane coincident with the tissue damage caused by efferent duct ligation. Show less

Age-related increases in basement membrane thickness have been noted in many tissues, including the testis. The current investigation examined the morphology of the basement membrane in the aged Brown Norway rat and sought to determine whether the accumulation of basement membrane was the result of an increase in the expression of the basement membrane genes. The aged testis was characterized by atrophy of the seminiferous tubules. Closer examination of the degenerated tubules revealed that the seminiferous epithelium was completely devoid of germ cells and that the basement membrane of these tubules was thickened and highly convoluted. In some animals, there was a measurable increase in basement membrane thickness in tubules of normal diameter together with an apparently normal epithelium, suggesting that the thickening is not solely due to a shrinkage of the tubules. To determine whether an increase in basement membrane synthesis was responsible for the thickening, the expression of the genes for laminin, collagen IV, heparan sulfate proteoglycan, and fibronectin was analyzed by Northern blot. There were no changes in the expression of the genes for the laminin B1 and B2 chains, heparan sulfate proteoglycan, or fibronectin that could be correlated with increasing age. Surprisingly, however, the levels of mRNA for the laminin A chain and collagen IV decreased with age.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Both Sertoli and myoid cells have been shown to be required for the appropriate deposition of basement membrane in the testis. We sought to define the pattern of basement membrane gene expression in Sertoli and peritubular myoid cells in vitro in order to begin to understand the regulatory mechanisms involved in basement membrane synthesis. Sertoli and myoid cells cultured alone or together were examined for synthesis of basement membrane components. Immunocytochemical localization demonstrated that Sertoli cells alone produced laminin and collagen IV, but not fibronectin, while myoid cells produced all three proteins. In Sertoli:myoid cocultures, a sequential deposition of the components into extracellular fibers was noted during 5 days of culture. Northern blot analysis revealed that mRNA levels for the laminin B1 chain and collagen IV increased from Days 3 to 5 in Sertoli cell monocultures. By contrast, the levels of laminin B1, collagen IV, heparan sulfate proteoglycan, and fibronectin decreased in the cocultures. Transcripts for the laminin A chain were not detected in the myoid cells; instead these cells produced the mRNA for the laminin homologue, merosin. This observation was confirmed by immunolocalization of merosin to the tunica propria of the testis and in cultured myoid cells. These data describe the expression of the basement membrane genes by Sertoli and peritubular myoid cells and provide the basis for future studies to determine the mechanisms that regulate the expression of the basement membrane genes in the testis. Show less

Human pregnancy-specific beta 1-glycoprotein (PSG) is found in high concentrations in the serum of pregnant women, but also has been found in the serum of males and nonpregnant females. Northern slot-blot analysis has demonstrated the presence of PSG mRNA in a variety of tissues in the rat, with the highest levels being found in the testis. Therefore, we have investigated further the expression of PSG in the rat male reproductive tract using in situ hybridization. In testes from immature and adult rats, PSG mRNA was localized in Leydig and peritubular cells, and in the walls of the interstitial blood vessels. PSG transcripts were noted also in the tunica albuginea and in the stromal tissue of the caput and cauda epididymis, prostate, and seminal vesicle from adult rats. The function of PSG is unknown, but it has been speculated that PSG may have immunosuppressive properties or that it may serve as a paracrine regulator of growth and differentiation. It is possible, then, that PSG could contribute to the immunological privilege of the testis or that it plays a role in the cellular interactions which increasingly are being shown to be important in the regulation of male reproductive tract tissues. Show less

Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3. Show less