Development and Application of Ribose Oxidation Sequencing (Riboxi-Seq) Yinzhou Zhu University of Connecticut, [email protected]
Total Page：16
File Type：pdf, Size：1020Kb
University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 6-7-2018 Development and Application of Ribose Oxidation Sequencing (RibOxi-seq) Yinzhou Zhu University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Zhu, Yinzhou, "Development and Application of Ribose Oxidation Sequencing (RibOxi-seq)" (2018). Doctoral Dissertations. 1881. https://opencommons.uconn.edu/dissertations/1881 Development and Application of Ribose Oxidation Sequencing (RibOxi-seq) Yinzhou Zhu, PhD University of Connecticut, 2018 In eukaryotes, a number of RNA sites are modified by 2’-O methylation (2’-OMe). Such editing is mostly guided by BoxC/D class small nucleolar RNAs (snoRNAs). These snoRNAs direct methylation via complementary RNA-RNA interactions. 2’-OMe has so far been shown to be present in rRNAs, tRNAs and some small RNAs and has been implicated in ribosome maturation and translational circuitries. A substantial portion of known methylated sites in rRNA lie in close proximity to ribosome functional sites such as regions around the peptidyl transferase center. It is not yet clear whether many mRNAs might possess internal 2’-OMe sites. It is therefore important to characterize 2’-OMe landscapes. We have developed a novel method for the highly accurate and transcriptome-wide detection of 2’-OMe sites. The core principle of this method is to randomly digest RNAs to expose 2’-OMe sites at the 3’-ends of digested RNA fragments. Next, an oxidation step using sodium periodate destroys all fragment-3’-ends except those that are 2’-O methylated. Only these oxidation-resistant fragments are available for linker ligation and subsequent sequencing library preparation. We have applied our new method to the study of ribose methylation in both Trypanosoma brucei and Mus musculus liver, and successfully obtained rRNA 2’-OMe profiles for both organisms. Our sequencing data strongly support experimentally validated known sites, while providing several candidates of novel sites, as well as evidence for differential methylation. In conclusion, our method is able to reliably identify 2’- OMe sites in a high-throughput manner. We are also interested in using the method to investigate potential mRNA internal sites, the targets of “orphan” snoRNAs, which have no currently known targets. Development and Application of Ribose Oxidation Sequencing (RibOxi-seq) Yinzhou Zhu B.S., University of Wisconsin-Madison, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2018 Copyright by Yinzhou Zhu 2018 ii APPROVAL PAGE Doctor of Philosophy Dissertation Development and Application of Ribose Oxidation Sequencing (RibOxi-seq) Presented by Yinzhou Zhu B.S. Major Advisor ___________________________________________________________________ Gordon G. Carmichael Associate Advisor ___________________________________________________________________ Arthur Gûnzl Associate Advisor ___________________________________________________________________ James Li Associate Advisor ___________________________________________________________________ Sandra K. Weller Associate Advisor ___________________________________________________________________ Stormy Chamberlain University of Connecticut 2018 iii TABLE OF CONTENTS APPROVAL PAGE…………………………………………………………………………........iii TABLE OF CONTETNTS……………………………………………………………………….iv LIST OF FIGURES……………………………………………………………………………...vii LIST OF TABLES………………………………………………………………………….…….ix I. INTRODUCTION………………………………………………………………………………1 A. Emergence of epitranscriptomics……………………………………………………...2 B. Ribose methylation/2’-O-methylation……………………………………………….18 C. Insights into 2’-OMe in humans……………………………………………………..36 D. Insights into 2’-OMe in other organisms…………………………………………….42 E. Methods to study 2’-OMe……………………………………………………………43 F. Thesis Objectives…………………………………………………………………….51 II. DEVELOPMENT OF RIBOSE OXIDATION SEQUENCING (RIBOXI-SEQ) FOR PROFILING 2’-OME SITES A. Abstract………………………………………………………………………………52 B. Background…………………………………………………………………………..52 C. Method development, Methods and Materials……………………………………….55 1. Methodology development………………………………………………….55 2. Cell culture and RNA extraction……………………………………………72 3. PCR validation of cDNA……………………………………………………72 4. Anti-sense oligo (ASO) knock down of U63 snoRNA……………………...73 iv 5. Radioactive primer extension……………………………………………….73 D. Results………………………………………………………………………………..76 1. RibOxi-seq accurately identifies annotated 2’-O-methylation sites within 18S and 28S rRNAs………………………………………………………………76 2. RibOxi-seq results confirm methylation heterogeneity within the same cell line……………………………………………………………………………83 3. RibOxi-seq requires modest input material but not high sequencing depth...88 4. RibOxi-seq is sensitive to methylation changes that are induced by complete depletion of specific snoRNA guides………………………………………..88 E. Discussion……………………………………………………………………………93 F. Complete protocol……………………………………………………………………94 III. APPLICATION AND EXTENSION OF RIBOXI-SEQ A. Abstract……………………………………………………………………………..107 B. Background…………………………………………………………………………107 C. Methods and Materials……………………………………………………………...110 1. RNA extraction and PolyA RNA isolation………………………………...110 2. RibOxi-seq…………………………………………………………………110 D. Results………………………………………………………………………………111 1. Profiling of T. brucei 2’-OMe rRNA landscape supports life stage-specific and site-specific differential methylation…………………………………...111 2. RibOxi-seq can detect mRNA 2’-OMe sites for genes abundantly expressed in corresponding cell lines or tissues……………………………………….117 E. Discussion…………………………………………………………………………..128 v IV. CONCLUSIONS AND FUTURE DIRECTIONS…………………………………………129 A. rRNA 2’-OMe landscape profiling of primary cells involved in diseases such as cancers………………………………………………………………………………129 B. Strategies to properly adapt RibOxiseq to transcriptome-wide applications……….130 V. BIBLIOGRAPHY…………………………………………………………………………...147 vi LIST OF FIGURES 1. Functional Roles of m6A……………………………………………………………...……...11 2. Box C/D snoRNA guided 2’-O-methylation and Box C/D snoRNP biogenesis…………….18 3. Box C/D snoRNP trafficking and its roles in RNA processing……………………………...21 4. Typical box C/D snoRNA guide and target………………………………………………….24 5. Models for potential roles of 2’-O-methylation……………………………………………...32 6. Diagram for the genomic locus of the Prader-Willie Syndrome critical region……………..39 7. Illustrations of conventional methods used for 2’-OMe detections and validation………….44 8. Available high-throughput methods for 2’-OMe detection………………………………….49 9. Chemical principle of the RibOxi-seq……………………………………………………….59 10. Continuation of RibOxi-seq………………………………………………………………….63 11. Protocol optimization………………………………………………………………………...67 12. cDNA Library validation using PCR………………………………………………………...70 13. QPCR verification of U63 KD efficiency……………………………………………………74 14. Volcano plot of the –log10 p value vs log2 fold change…………………………………….84 15. Radioactive dNTP concentration dependent primer extension experiment………………….86 16. RibOxi-seq on snoRNA KO 293 total RNA samples………………………………………..90 17. RibOxi-seq on T. brucei BF and PF total RNA samples…………………………………...113 18. New Benzonase digestion optimization for RibOxi-seq……………………………………119 19. RibOxi-seq on poly(A)+ enriched mouse liver RNAs and human H9 cell line RNAs…….122 20. Common artefact occurred during RibOxiseq for transcriptomic 2’-OMe mapping………124 21. Examples of genes with likely mRNA 2’-OMe sites……………………………………….126 vii 22. Alternative strategy for validating low abundance RNA 2’-OMe………………………….132 23. Strategy to adapt RibOxi-seq to Oxford Nanopore direct RNA sequencing……………….135 viii LIST OF TABLES 1. List of major RNA modifications for Archaea, Bacteria and Eukarya………………………..6 2. Summary of snoRNAs from curated database……………………………………………….27 3. List of 2’-O methylation sites found in 18S and 28S rRNA from PA1 cells………………...78 4. List of potential novel sites found in PA1 cells using RibOxi-seq………………………......82 5. List of promising differentially methylated sites to follow up……………………………..116 6. List of reagents and equipment used in RibOxi-seq………………………………………..137 7. List of primers and other oligos used……………………………………………………….139 8. Table 8. List of 2'-OMe sites detected in T. brucei using RibOxi-seq….………………….141 9. List of potential H9 mRNA sites…..……………………………………………………….146 ix Chapter I Introduction Since establishment of the foundations of molecular biology in the mid-1900s, additional modes of gene regulation, which operate beyond the routes of information flow described in the original central dogma, have been discovered. Layers of complexity to the seemingly simple sequences of bidirectional information transfer from DNA to RNA messages, as well as unidirectional translation from RNA molecules to proteins has been added. While Francis Crick’s central dogma of molecular biology remains technically valid, the interconnections between these three biomolecules have transcended generalization that the conventional pathways of information flow. With progress made in the past few decades, we have come to establish a more robust representation of cellular networks, which newly integrates transcriptional and post-transcriptional regulation networks. These two aspects specifically, have grown exponentially partially due to breakthroughs in high-throughput sequencing. Numbers of novel non-coding RNAs such as long non-coding RNAs (lncRNAs)