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ANALYSES of Trna and Rrna DERIVED FRAGMENTS ACROSS ANIMAL

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ANALYSES of Trna and Rrna DERIVED FRAGMENTS ACROSS ANIMAL ANALYSES OF tRNA AND rRNA DERIVED FRAGMENTS ACROSS ANIMAL KINGDOM by LINGYU GUAN A dissertation submitted to the Graduate School-Camden Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Computational and Integrative Biology Written under the direction of Dr. Andrey Grigoriev And approved by ______________________________ Dr. Andrey Grigoriev ______________________________ Dr. Shantanu Bhatt ______________________________ Dr. Kwangwon Lee ______________________________ Dr. Sunil Shende Camden, New Jersey May 2021 ABSTRACT OF THE DISSERTATION Analyses of tRNA and rRNA derived fragments across animal kingdom by LINGYU GUAN Dissertation Director: Dr. Andrey Grigoriev Transfer RNA (tRNA) and ribosomal RNA (rRNA) are known for their textbook functions in the translation machinery. With further advances in the high-throughput sequencing, accumulating evidence has revealed their novel functionality as sources of short RNA fragments which may act as post-transcriptional regulators in various organisms. Such tRNA-derived fragments (tRFs) and rRNA-derived fragments (rRFs) have been shown to be implicated in many biological pathways, e.g., ageing, neuronal disorders and cancers, etc. Using large-scale computational analyses of various types of sequencing data, we characterized tRFs and rRFs in flies, mouse and human. We revealed the loading patterns of age-associated rRFs to different Argonaute proteins and suggested their roles in the ageing process of flies. We inferred the potential biogenesis pathway of human rRFs. By comprehensively analyzing the experimentally crosslinked target RNAs of tRFs and rRFs, we investigated the binding mechanisms of these molecules to their targets in the Argonaute proteins and suggested potential regulatory functions. ii Table of Contents Abstract ............................................................................................................................... ii Table of Contents ............................................................................................................... iii List of Tables .......................................................................................................................v List of Figures .................................................................................................................... vi Chapter 1 Introduction .........................................................................................................1 1.1 RNAi pathway ............................................................................................................1 1.2 tRNA and tRNA-derived fragments (tRFs) ...............................................................1 1.3 rRNA and rRNA-derived fragments (rRFs) ...............................................................4 Chapter 2: Inferring Targeting Modes of Argonaute-loaded tRNA Fragments ..................6 2.1 Introduction ................................................................................................................7 2.2 Results ......................................................................................................................11 2.3 Discussion ................................................................................................................24 2.4 Materials and Method...............................................................................................30 2.5 References ................................................................................................................33 Chapter 3: Large-scale Computational Discovery of Binding Motifs in tRNA fragments ............................................................................................................................................36 3.1 Introduction ..............................................................................................................38 3.2 Result ........................................................................................................................39 3.3 Discussion ................................................................................................................49 iii 3.4 Materials and Method...............................................................................................53 3.5 Reference ..................................................................................................................55 Chapter 4: Age-related Argonaute Loading of Ribosomal RNA Fragments .....................57 4.1 Introduction ..............................................................................................................59 4.2 Results ......................................................................................................................62 4.3 Discussion ................................................................................................................76 4.4 Materials and Method...............................................................................................81 4.5 Reference ..................................................................................................................83 Chapter 5: Computational Meta-Analysis of Ribosomal RNA Fragments: Potential Targets and Interaction Mechanisms ..............................................................................................85 5.1 Introduction ..............................................................................................................86 5.2 Results and Discussion .............................................................................................91 5.3 Materials and Method.............................................................................................127 5.4 Reference ................................................................................................................131 Chapter 6 Discussion and Future Directions ...................................................................137 Chapter 7 Legends for Supplementary Tables and Figures .............................................142 7.1 Supplementary Tables ............................................................................................142 7.2 Supplementary Figures ...........................................................................................143 iv List of Tables Table 3.1. Numbers of reads supporting hybrids with specific types of targets in forward and reverse pairs of tRFs. Table 4.1. Number of reads mapped to rRNAs, tRNAs and miRNAs. Table 5.1. Boundaries of rRFs in double-stranded (DS) or single-stranded (SS) regions of rRNA. Table 5.2. Numbers of interactions between rRFs and different target types. Table 5.3. Nucleotide contents of analyzed RNAs. v List of Figures Figure 1.1. Six types of tRFs. Figure 2.1. Ago-1 loaded tRFs. Figure 2.2. Abundance heatmap for tRFs generated from mature nuclear tRNAs. Figure 2.3. tRFs guide Ago1 to a variety of RNA targets. Figure 2.4. Base-pairing patterns for unique tRF-3/target chimeras. Figure 2.5. Computationally and experimentally identified interaction sites for tRF-3 type tRFs. Figure 3.1 Length distribution of rRFs. Figure 3.2. rRF generation profile across 45S pre-rRNA. Figure 3.3. Alignments of the rRFs isoforms in the two most abundant groups. Figure 3.4. rRFs aligned to different rRNAs change with age. Figure 3.5. Differential and preferential loadings. Figure 3.6. Candidate seed regions for the top two rRFs 18S-1597-1618 and 28S-1863- 1885. Figure 3.7. Candidate seed regions for two Ago1-guided rRFs 28S-2293-2312 and 28S- 2327-2347. Figure 3.8. Seed matches of 28S-2293-2312 in the conserved CDS of a putative targeted gene Brat. Figure 4.1 Comparison of the tRFs identified in forward and reverse pairs. Figure 4.2. PCA plots of dinucleotide composition. Figure 4.3. Analyses of Ago-crosslinking and target binding sites. vi Figure 5.1. Analysis and properties of rRFs and other sRNA guides paired with targets in CLASH hybrids. Figure 5.2. CLASH rRFs originated from multiple regions across 45S pre-rRNA. Figure 5.3. Distribution of putative rRF-targeted mRNA regions. Figure 5.4. Clustering of 20-nt rRFs from 18S rRNAs reveals potential binding modes. Figure 5.5. Positions of T→C conversion sites are compatible with the predicted interaction motifs show conservation between species. Figure 5.6. The same Ago-bound rRFs are found in both the cytoplasm and nucleus of mouse cells. Figure 5.7. Secondary structure of putative rRFs-target interaction sites. vii 1 CHAPTER 1 INTRODUCTION 1.1 RNAi pathway RNA interference (RNAi) describes a mechanism whereby small regulatory RNAs bind to target RNAs via forming Watson-Crick base pairings and modulate various biological processes. It was initially found for the exogenous small-interfering RNA (siRNA) silence the gene expression in RNA-induced silencing complex (RISC). Other better-known small RNAs functioning in RNAi pathway involve endogenous microRNA (miRNA) (Bartel 2009), Piwi protein interacting RNA (piRNA) (Hamilton and Baulcombe 1999), etc. The involvement of small RNAs in the post-transcriptional regulation of gene expression is not limited to repressing translation, it may degrade the target RNAs or even promote translation (Fabian, Sonenberg et al. 2010). RISC is a multi-protein complex playing essential role in RNAi, of which the key protein is Argonaute. The Argonaute superfamily contains numerous proteins and are mainly divided into Piwi proteins (Piwi clade),
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