Evolution and Diversification of Components of Small RNA Pathways

Singh, Ravi Kumar (2016) Evolution and Diversification of Components of Small RNA Pathways. PhD thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Small RNAs (smRNAs) are the non-coding, regulatory molecules that modulate the expression of genes in a sequence dependent manner in the process often called as RNA interference. Plants harbours multiple types of non-coding smRNAs that play important roles during a wide range of biological processes such as growth and development, stress adaptations and combating diseases. Several types of smRNAs are present in plants, for instance the miRNAs, hcsiRNAs, tasiRNAs, phasiRNAs, and natsiRNAs. Seven families of proteins that participate in biogenesis and action of these smRNAs, include the RNA-directed RNA polymerases (RdRs), suppressor of gene silencing (SGS), silencing defective (SDE), ds-RNA binding protein (DRBs), Dicer-like proteins (DCLs), HUA enhancer (HEN) and the Argonautes (AGOs). Information on their biochemical and physiological functions has been extensively generated in the model plants. However, molecular details on how smRNA pathways have evolved to device functional diversity in plants remains little understood. The broad goal of this investigation is to understand the molecular basis of evolution of diverse smRNA pathways in plants. As the number of genes in the AGO family is large, and that they are crucial to the formation of RNA-induced silencing complex (RISC), we began our investigation with using the AGO as our model protein family. We gained insights into the molecular evolution of smRNA pathways in plants by asking three specific questions: (i) what is the basis of molecular evolution and diversification of AGO family of proteins; (ii) during the course of evolution, how did sequence variation effect the structural and functional diversity of AGOs, and (iii) how do the AGOs and other 6 families of proteins interact on an evolutionary scale to shape the current diversity of smRNA pathways. We have mined more than 1100 genes of 7 gene families in 53 plant genomes and performed comprehensive analysis on sequence diversity, phylogenetic classifications and evaluation of topological heterogeneity by creating maximum clade credibility (MCC) tree, analysis of gene structure, by estimating of evolutionary rates, and co-evolution within a family. Also, we estimated context-dependent changes in protein sequences, as well as gradient dependent changes in evolutionary rates. We further explored how the above mentioned parameters could help in evolution of protein structure and its function (in the context of interaction with the substrates and their possible outcome). Our findings suggest that the evolution of smRNA pathways has been a highly dynamic and complex process. After originating in unicellular life forms, the evolution of smRNA pathways highly concur with the evolution of multicellularity and vascular systems in the plants. miRNAs seem to have originated first in the unicellular organisms; the hcsiRNAs appeared along with the evolution of vasculature, while the secondary siRNA pathways (tasiRNA, phasiRNA, and natsiRNAs) evolved with the advent of angiosperms. Our analysis suggests that families of smRNA pathways such as the AGOs, have dynamically expanded by duplication in plant genomes. Complex set of molecular events have shaped the composition (as well as diversity), and function of the smRNA pathway in plants to help them adapt to complex habitats. Interestingly, bacteria harbours a structural and functional analog of the AGO, called YbeY. Therefore, we have further investigated the evolutionary diversification of this potential analog (objective 4), which is indeed a central component of RNA metabolism in bacteria.

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