RESUMO
Translational control is a widespread mechanism that allows the cell to rapidly modulate gene expression in order to provide flexibility and adaptability to eukaryotic organisms. We applied translating ribosome affinity purification combined with RNA sequencing to characterize translational regulation of mRNAs at early stages of the nitrogen-fixing symbiosis established between Medicago truncatula and Sinorhizobium meliloti Our analysis revealed a poor correlation between transcriptional and translational changes and identified hundreds of regulated protein-coding and long noncoding RNAs (lncRNAs), some of which are regulated in specific cell types. We demonstrated that a short variant of the lncRNA Trans-acting small interference RNA3 (TAS3) increased its association to the translational machinery in response to rhizobia. Functional analysis revealed that this short variant of TAS3 might act as a target mimic that captures microRNA390, contributing to reduce trans acting small interference Auxin Response Factor production and modulating nodule formation and rhizobial infection. The analysis of alternative transcript variants identified a translationally upregulated mRNA encoding subunit 3 of the SUPERKILLER complex (SKI3), which participates in mRNA decay. Knockdown of SKI3 decreased nodule initiation and development, as well as the survival of bacteria within nodules. Our results highlight the importance of translational control and mRNA decay pathways for the successful establishment of the nitrogen-fixing symbiosis.
Assuntos
Reprogramação Celular/fisiologia , Fixação de Nitrogênio/fisiologia , Raízes de Plantas/metabolismo , Polirribossomos/metabolismo , RNA de Plantas/metabolismo , RNA não Traduzido/metabolismo , Simbiose/fisiologia , Reprogramação Celular/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Ácidos Indolacéticos/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , Nitrogênio/metabolismo , Fixação de Nitrogênio/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nodulação/genética , Nodulação/fisiologia , Raízes de Plantas/genética , RNA de Plantas/genética , RNA não Traduzido/genética , Nódulos Radiculares de Plantas , Sinorhizobium meliloti/metabolismo , Simbiose/genéticaRESUMO
Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.
