Two Arabidopsis late pollen transcripts are detected in cytoplasmic granules

Many of mRNAs synthesized during pollen development are translated after germination, and we hypothesize that they are stored in cytoplasmic granules. We analyzed the cellular localization of the SKS14 and AT59 Arabidopsis mRNAs, which are orthologues of the tobacco NTP303 and tomato LAT59 pollen mRNAs, respectively, by artificially labeling the transcripts with a MS2-GFP chimera. A MATLAB-automated image analysis helped to identify the presence of cytoplasmic SKS14 and AT59 mRNA granules in mature pollen grains. These mRNA granules partially colocalized with VCS and DCP1, two processing body (PB) proteins. Finally, we found a temporal correlation between SKS14 protein accumulation and the disappearance of SKS14 mRNA granules during pollen germination. These results contribute to unveil a mechanism for translational regulation in Arabidopsis thaliana pollen.Fil: Scarpin, Maria Regina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Sigaut, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Temprana, Silvio Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pietrasanta, Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Muschietti, Jorge Prometeo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentin

CGG Repeat RNAs Regulate Granule Translation

Fragile X tremor ataxia syndrome (FXTAS) and fragile X primary ovarian insufficiency (FXPOI) are associated with CGG repeat expansions in the 5’UTR of the FMR1 gene. RNAs containing an hnRNP A2 response element (A2RE) are localized and transported in hnRNP A2 granules that are organized by multivalent interactions. We show that RNAs containing CGG repeats are also localized in hnRNP A2 granules. Exogenous expanded CGG repeat RNA microinjected in mouse hippocampal neurons or oocytes inhibits granule RNA translation. Endogenous premutation CGG repeats in FMR1 in human fibroblast cells from females at risk for FXPOI also inhibit granule RNA translation. We suggest that CGG repeat RNAs encoding calcium regulatory proteins may be in RNA granules and show that CGG repeat RNAs in premutation human fibroblast cells affect calcium homeostasis which may contribute to FXTAS and FXPOI pathogenesis. Premutation CGG repeat RNA containing the 5’UTR and part of the FMR1 ORF and intron 1 in the transgenic TG296 mouse does not inhibit granule RNA translation. We suggest that FXTAS and FXPOI are granule based phenomena and RNA gain of function disorders. We suggest two mechanisms for RNA gain of function mechanisms, sequestration of translational machinery and activation of the double stranded binding protein, protein kinase R (PKR). Differences in the magnitude of translational inhibition by CGG repeat RNA in neurons, fibroblasts and oocytes suggests that activation of PKR is most likely the pathogenic mechanism for FXTAS and FXPOI

CGG Repeats in the 5'UTR of FMR1 RNA Regulate Translation of Other RNAs Localized in the Same RNA Granules.

CGG repeats in the 5'UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55-200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI