TEMPORAL PATTERNS OF SHORT NON-CODING RNA MODIFICATIONS AND EXPRESSION by AMMAR S. NAQVI 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 ________________________ Andrey Grigoriev ________________________ Jongmin Nam ________________________ Nancy Bonini Camden, New Jersey May 2015 ABSTRACT OF THE DISSERTATION Temporal Patterns of non-coding RNA Modifications and Expression by AMMAR S. NAQVI Dissertation Director: Dr. Andrey Grigoriev We investigated the function and properties of small RNAs, particularly microRNAs and tRNA-derived fragments (tRFs) with age. We report the characterization of a novel 3'-to- 5' exonuclease, Nibbler (Nbr), that generates differing isoforms of miRNAs in Drosophila. We developed a robust approach to help identify and characterize 3' heterogeneity in microRNAs controlled by Nbr, which assisted in identifying age- associated traits, including neurodegeneration and lifespan. Subsequently, given the fact Nbr interacts with Ago1 and not Ago2, we observed an accumulation of certain isoforms, which lead us to ask if there were particular patterns and trends that were Ago-specific. Interestingly, we report a novel age-associated change of select isoforms with age that is Ago2 specific. RNA deep-sequencing analysis coupled with experimental evidence reflected an increased loading of miRNA isoforms into Ago2 with age. Essentially, the loss of methylated miRNAs led to accelerated brain degeneration and shortened lifespan. Intriguingly, we also observed and identified Ago-loaded tRFs, which appear to have properties similar to those of miRNAs. We found this class of small RNAs to also display age-associated changes. For the first time, we found that differentially loaded Drosophila tRFs mapping to both nuclear and mitochondrial tRNA genes associating with all 20 amino acids. These tRFs show a number of similarities with miRNAs, including seed ii sequences, suggesting a similar role and function. Moreover, we further characterized and predicted targets for these tRFs and show a significant enrichment in development and neuronal function, suggesting a role in brain-related processes with age. In sum, we discovered a novel component of the canonical microRNA biogenesis pathway, responsible for the generation of multiple isoforms. We also connected specific age- associated patterns and trends of select microRNA isoforms, which were found to impact proper brain development and lifespan. Moreover, we identified differentially loaded tRFs and elucidated their structures, loading, and expression patterns, which corresponded closely with microRNAs. Finally, we were able to identify tRF seed regions that potentially play a role in brain activity or brain changes with age. iii ACKNOWLEDGEMENTS I am extremely thankful for my advisor, Dr. Andrey Grigoriev. His openness and ideas allowed me to pursue the study of small non-coding RNAs in a very meaningful way. Through his mentorship, I was able to contribute to the field. Under his supervision, I have learned a great amount that has given me the confidence and skill set to continue as a scientist. I also would like to thank Dr. Nancy Bonini for her support and leadership that helped me succeed as a graduate student. Her lab's experimental expertise and contributions were invaluable. I would also like to thank Dr. Jongmin Nam for his ideas and feedback throughout my tenure as a graduate student. The combined support of my committee members has really helped me grow as a scientist and I am forever indebted for their help. I would also like to thank the Grigoriev Lab members for their added insight and assistance and members of the Bonini Lab, Masashi Abe and Virzhiniya Feltzin, particularly for their experimental help. Finally, I want to thank my family and friends for their unconditional support that made my research possible. iv Table of Contents Abstract.................................................................................................................. vi Acknowledgements…………………………………..…………………………...iv List of Tables……………………………………………………………………...ix List of Figures..........................................................................................................x Chapter 1 Introduction…………………………………………………………....01 microRNA biogenesis...................................................................................01 microRNA modifications and diversity……………………………………03 tRNA-derived small RNAs...........................................................................07 Aging, neurodegeneration and small RNAs .................................................11 Chapter 2 Visualization of nucleotide substitutions in the micro(transcriptome)..18 Abstract.........................................................................................................19 Background...................................................................................................20 Results and Discussion .................................................................................25 Chapter 3 The exoribonuclease Nibbler controls 3' end processing of microRNAs in Drosophila…………………………………………………….……38 Summary.......................................................................................................38 Background...................................................................................................39 Results and Discussion .................................................................................40 Chapter 4 Impact of age-associated increase in 2'-O-methylation of miRNAs v on aging and neurodegeneration in Drosophila……………..…………67 Abstract.........................................................................................................68 Background...................................................................................................69 Results...........................................................................................................72 Discussion.....................................................................................................82 Chapter 5 Age-driven modulation of tRNA-derived fragments in Drosophila and their potential targets………………………………………….....98 Abstract.........................................................................................................99 Background.................................................................................................100 Results.........................................................................................................103 Discussion...................................................................................................113 Chapter 6 Materials and Methods........................................................................125 vi Chapter 2 Materials and methods…………………………………………125 Chapter 3 Materials and methods…………………………………………126 Chapter 4 Materials and methods………………………………………....135 Chapter 5 Materials and methods………………………………………....143 Chapter 7 Discussion and Future Directions..………………………………….146 Select isoforms impacted by Nbr and other components involved.………147 3’ processing in other species……………………………………….….....148 Other small RNAs impacting the brain…………………………………...148 tRF and its miR-like function……………………………………………..150 Further characterization of Ago2-loaded tRF targets……………………..151 Tissue-specificity of tRFs…………………………………………………151 Development-associated Profiling oftRFs………………………………..152 tRF silencing with identified seeds……………………………………….153 tRF evolution and modifications………………………………………….155 Conclusion………………………………………………………………...156 Bibliography ........................................................................................................157 vii List of Tables Table 2.1: Small RNA Libraries ...........................................................................34 Table 2.2: microRNA Candidates for Editing .......................................................35 Table 3.1: miRNA Reads from Deep Sequencing Data………………………….47 Table 3.2: Sequence Counts of miRNAs from Ends of the Ratio Plot in Control (WT) and Nbr Mutants……………………………………………………47 Table 3.3: Summary of Results on miRNAs from the Two Ends of the Length Ratio Plot……….…………………………………………………………52 Table 3.4: Genes Identified by Microarray that Changed in Cells upon Nbr Knockdown………………………………………………………….53 viii List of Figures Figure 2.1: General Display Features ....................................................................34 Figure 2.2: The bantam stem-loop edited regions and NGS reads ........................35 Figure 3.1: miRNA Reads from Deep Sequencing Data .......................................57 Figure 3.2: nbr Is Required to Generate the Isoforms of miR-34..........................58 Figure 3.3: Nbr Interacts with Ago1-RNA-Induced Silencing Complex ..............59 Figure 3.4: Nbr Is Required In Vivo to Process Select miRNAs and Silence Target Messenger RNAs ..............................................................................61 Figure 3.5: Reduction of nbr Affects Biogenesis of miR-34 Shorter Isoforms.....63 Figure 3.6: The Interaction between Nbr and Ago1 Is Not Dependent on RNA...64 Figure 3.7: New nbr-Dependent Candidate miRNAs............................................65 Figure 4.1: Nbr-dependent miRNAs show distinct isoform patterns with age......87 Figure