Thomas C. Evans

Translational control of maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryo

Abstract In C. elegans, the glp-1 gene encodes a membrane receptor that is required for anterior cell fates in the early embryo. We report that GLP-1 protein is localized to anterior blastomeres in 2- to 28-cell embryos. By contrast, glp-1 mRNA is present in all blastomeres until the 8-cell stage. Furthermore, the glp-1 3′ untranslated region can restrict translation of a reporter mRNA to anterior blastomeres. Therefore, the translation of maternal glp-1 mRNA is temporally and spatially regulated in the C. elegans embryo. The regulation of maternal glp-1 mRNA has striking parallels to the regulation of maternal hunchback mRNA in the Drosophila embryo. Thus, the establishment of embryonic asymmetry in diverse organisms may involve conserved mechanisms of maternal mRNA regulation.

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3′untranslated region (3′ UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3′ UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.

Maternal mRNAs are regulated by diverse P body–related mRNP granules during early Caenorhabditis elegans development

Processing bodies (P bodies) are conserved mRNA–protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body–like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3′ untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis.

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.

Translation Repressors, an RNA Helicase, and Developmental Cues Control RNP Phase Transitions during Early Development

Like membranous organelles, large-scale coassembly of macromolecules can organize functions in cells. Ribonucleoproteins (RNPs) can form liquid or solid aggregates, but control and consequences of these RNP states in living, developing tissue are poorly understood. Here, we show that regulated RNP factor interactions drive transitions among diffuse, semiliquid, or solid states to modulate RNP sorting and exchange in the Caenorhabditis elegans oocyte cytoplasm. Translation repressors induce an intrinsic capacity of RNP components to coassemble into either large semiliquids or solid lattices, whereas a conserved RNA helicase prevents polymerization into nondynamic solids. Developmental cues dramatically alter both fluidity and sorting within large RNP assemblies, inducing a transition from RNP segregation in quiescent oocytes to dynamic exchange in the early embryo. Therefore, large-scale organization of gene expression extends to the cytoplasm, where regulation of supramolecular states imparts specific patterns of RNP dynamics.

Electrophysiological evidence suggests a defective Ca2+ control mechanism in a new Paramecium mutant

SummaryA new mutant ofParamecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca2+-regulated currents were altered in the mutant. (i) The voltage-dependent Ca2+ current recovered from Ca2+-dependent inactivation two- to 10-fold more slowly than wild type. Ca2+ current amplitudes were also reduced in the mutant, but could be restored by EGTA injection. (ii) The decay of the Ca2+-dependent K+ tail current was slower in the mutant. (iii) The decay of the Ca2+-dependent Na+ tail current was also slower in the mutant. All other membrane properties studied, including the resting membrane potential and resistance and the voltage-sensitive K+ currents, were normal in k-shyA. Considered together, these observations are consistent with a defect in the ability of k-shyA to reduce the free intracellular Ca2+ concentration following stimulation. The possible targets of the genetic lesion and alternative explanations are discussed. The k-shy mutants may provide a useful tool for molecular and physiological analyses of the regulation of Ca2+ metabolism inParamecium.

Modifiers of solid RNP granules control normal RNP dynamics and mRNA activity in early development

Modifiers of aberrant solid RNP granules suggest new insights into pathways that control dynamics of large-scale RNP bodies and mRNAs during C. elegans oogenesis.