Methylation of -adenosine (mA) in mRNA is an important posttranscriptional gene regulatory mechanism in eukaryotes. mA provides a binding site for effector proteins ("readers") that influence pre-mRNA splicing, mRNA degradation, or translational efficiency. YT521-B homology (YTH) domain proteins are important mA readers with established functions in animals. Plants contain more YTH domain proteins than other eukaryotes, but their biological importance remains unknown. Here, we show that the cytoplasmic YTH domain proteins EVOLUTIONARILY CONSERVED C-TERMINAL REGION2/3 (ECT2/3) are required for the correct timing of leaf formation and for normal leaf morphology. These functions depend fully on intact mA binding sites of ECT2 and ECT3, indicating that they function as mA readers. Mutation of the close homolog,, enhances the delayed leaf emergence and leaf morphology defects of mutants, and all three ECT proteins are expressed at leaf formation sites in the shoot apex of young seedlings and in the division zone of developing leaves. ECT2 and ECT3 are also highly expressed at early stages of trichome development and are required for trichome morphology, as previously reported for mA itself. Overall, our study establishes the relevance of a cytoplasmic mA-YTH regulatory module in the timing and execution of plant organogenesis.
Constitutive photomorphogenic 1 (COP1) is a RING-finger E3 ligase that plays a central role in photomorphogenesis by destabilizing many light-regulated transcription factors and photoreceptors. Here, we reveal a novel function for COP1 E3 ligase in controlling global miRNA biogenesis in Arabidopsis thaliana. In cop1 mutants, the level of miRNAs is dramatically reduced because of the diminution of HYPONASTIC LEAVES 1 (HYL1), an RNA-binding protein required for precise miRNA processing. HYL1 is destabilized by an unidentified protease, which we tentatively call protease X, that specifically cleaves the N-terminal region from HYL1, thus neutralizing its function. Our results further show that the cytoplasmic partitioning of COP1 under light is essential to protect HYL1 against protease X. Taken together, we suggest a novel regulatory network involving HYL1, protease X, COP1 and light signalling that is indispensable for miRNA biogenesis in Arabidopsis thaliana.
These authors contributed equally to this work. SUMMARYWe have characterized a b-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to b-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to b-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the b-1,6-and b-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-b-1,6-Gal and b-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6-and 3,6-linked galactose (Gal) and reduced levels of 3-, 2-and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched b-1,3-and b-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.
RNA silencing refers to a collection of gene regulatory mechanisms that use small RNAs for sequence specific repression. These mechanisms rely on ARGONAUTE (AGO) proteins that directly bind small RNAs and thereby constitute the central component of the RNA-induced silencing complex (RISC). AGO protein function has been probed extensively by mutational analyses, particularly in plants where large allelic series of several AGO proteins have been isolated. Structures of entire human and yeast AGO proteins have only very recently been obtained, and they allow more precise analyses of functional consequences of mutations obtained by forward genetics. To a large extent, these analyses support current models of regions of particular functional importance of AGO proteins. Interestingly, they also identify previously unrecognized parts of AGO proteins with profound structural and functional importance and provide the first hints at structural elements that have important functions specific to individual AGO family members. A particularly important outcome of the analysis concerns the evidence for existence of Gly-Trp (GW) repeat interactors of AGO proteins acting in the plant microRNA pathway. The parallel analysis of AGO structures and plant AGO mutations also suggests that such interactions with GW proteins may be a determinant of whether an endonucleolytically competent RISC is formed.
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