|Author:||Hung, Wing Hong|
|Title:||RNA interactome identification by next-generation sequencing (RIINGS) : a novel method for identifying microRNA-RNA interactome in exact correspondence|
|Advisors:||Yip, Shea-ping (HTI)|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
Small interfering RNA
|Department:||Department of Industrial and Systems Engineering|
|Pages:||xxxvi, 399 pages : color illustrations|
|Abstract:||MicroRNAs are important in epigenetic regulation of gene expression. They are short non-coding RNA molecules, and are able to regulate gene expression at the post-transcriptional level via binding to the target sites of target RNAs. Since target recognition does not require full complementarity between microRNAs and their target sites, a single microRNA can potentially regulate many targets. In addition, the regulation may show conditional dependence as evidenced by the fact that when luciferase reporter assays are used to confirm the inhibition effect of some microRNAs on their targets, the results can be positive or negative in different cell lines. Therefore, microRNA target identification is a very challenging task in microRNA studies. There was no reliable experimental method to identify microRNA targets in a massive way in the past and hence computational prediction has played an important role in and contributed a lot to this area for a long time. However, the accuracy of computation-based methods is far from desire in identifying the genuine targets. Recently, several high-throughput screening methods have been developed for identifying microRNA targets. These methods can be divided into two major categories. The first category can identify microRNA targets, but cannot reveal the exact correspondence between microRNAs and their targets; methods like High-Throughput Sequencing of RNAs Isolated by Crosslinking Immunoprecipitation (HITS-CLIP), Photoactivatable Ribonucleoside-enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), and Individual-nucleotide Resolution Cross-linking and Immunoprecipitation (iCLIP) fall into this category. The second category can additionally identify the exact correspondence between microRNAs and their targets, and is exemplified by the methods of Crosslinking, Ligation and Sequencing of Hybrids (CLASH), Mapping RNA interactome in vivo (MARIO), and Psoralen Analysis of RNA Interactions and Structures (PARIS). However, these methods are still not capable of detecting all possible interacting pairs because the ligation approaches used in all these methods for generating chimeras are not efficient and only interacting pairs with particular properties can be ligated (i.e. many interacting pairs cannot be ligated) during library preparation. Accordingly, the representation of the library for the entire population of the microRNA-target RNA interacting pairs is poor and may be biased.|
With regard to the restrictions and limitations of current experiment-based methods for identifying microRNA target, this study primarily aims at inventing a comprehensive method for identifying miRNA interactome. Based on a different principle, the current method potentially enhances the generation of microRNA-target RNA chimeras from all interacting pairs during library preparation. In order to develop the current method, several critical techniques have been developed and evaluated. First, a novel approach to conjugating the interacting miRNA-target RNA via a stem-loop adaptor has been developed and evaluated in a set of mock experiments. Second, a pair of "repairers" with enhanced ability to repair the mismatches at 5' and 3' ends of miRNA in the miRNA-interacting duplexes has been invented and evaluated in another set of mock experiments. Third, an approach to enhancing the ligation of adaptor to the 3' end of target RNA has been evaluated. This approach not only is suitable for the "Stem-Loop Adaptor (RNA)" ligation in this method, but also can be used in other RNA interactome identification methods, e.g. HITS-CLIP and PAR-CLIP. However, the invention of "Library Enrichment Beads", which are capable of both capturing the interacting microRNA-target RNA duplexes and eluting the captured sequences under an extremely mild and non-denaturing condition, is still in progress. Currently, the capturing ability of the beads has been solved in general whereas the elution part is still under testing. In fact, these beads can also be applied to other applications that require on-bead manipulations of nucleic acids if they are finally developed. On the other hand, mass spectrometry analysis has revealed that the purity of immunoprecipitated Argonaute complexes is good enough for evaluating the current method directly. Furthermore, melting curve analysis has found that the melting peak of miRNA-target RNA duplexes is about 40℃ and re-annealing of the melted duplexes is probably impossible once they are melted. This evidence supports the feasibility of the current method. The conditions for immunoprecipitation, "Stem-Loop Adaptor (RNA)" ligation, "Re-attaching Adaptor" ligation, RNA fragmentation and UV crosslinking have been well optimized. This is a method potentially capable of identifying the entire interactome of microRNA in a specific cell type or tissue, and revealing the interactome changes in different physiological states of cells. Furthermore, the method may also be used to identify other protein-mediated nucleic acid interactions. This newly developed method is called RNA Interactome Identification by Next Generation Sequencing (RIINGS).
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