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The Detection of Ribonuclease Cleavage Sites THE DETECTION OF RIBONUCLEASE CLEAVAGE SITES IN HOST AND VIRAL RNAS DURING VIRAL INFECTIONS by DAPHNE A. COOPER B.S., California Polytechnic State University, San Luis Obispo A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Microbiology Program 2015 ii This thesis for the Doctor of Philosophy degree by Daphne A. Cooper has been approved for the Microbiology Program by Thomas E. Morrison, Chair David J. Barton, Advisor Linda F. van Dyk Mario L. Santiago Hugo R. Rosen Jay R. Hesselberth Date 5/06/2015 iii Cooper, Daphne A (Ph.D., Microbiology) The Detection of Ribonuclease Cleavage Sites in Host and Viral RNAs during Viral Infections Thesis directed by Professor David J. Barton. ABSTRACT Ribonucleases are critical components for cellular responses to viruses. RNase L is an endoribonuclease activated during many types of viral infections, yet the RNAs targeted by RNase L to promote its antiviral activities are poorly characterized. To better understand the RNAs cleaved by RNase L during viral infections, I optimized and validated cDNA synthesis and deep sequencing methods that enrich for RNA fragments with terminal 2ˊ, 3ˊ-cyclic phosphates. RNase L, in addition to many other ribonucleases, produces RNA cleavage fragments with terminal 2ˊ, 3ˊ-cyclic phosphates, making 2ˊ, 3ˊ-cyclic phosphate cDNA synthesis and deep sequencing methods broadly applicable for the study of many types of ribonucleases. Using these methods, I characterized RNA cleavage from cells infected with poliovirus, influenza A virus, hepatitis C virus, and from liver tissue from a hepatitis C virus-infected patient. Deep sequencing of 2ˊ, 3ˊ-cyclic phosphate cDNA libraries revealed that RNase L activation during poliovirus and influenza A virus infections provoked cleavage of host and viral RNAs. Two sites in 18S rRNA, UU541 and UU743, were among the most frequently detected RNase L-dependent cleavage sites. Cleavage at these sites potentially inhibits protein synthesis and promotes apoptotic cell death of virally-infected cells. Although these studies focused on RNase L, I also detected RNA cleavage by other ribonucleases. Usb1, a nuclear 3ˊ→5ˊexoribonuclease, post-transcriptionally modifies the 3ˊ-end of U6 snRNA to generate a 2ˊ, 3ˊ-cyclic phosphate frequently detected in 2ˊ, 3ˊ-cyclic phosphate cDNA libraries. 2ˊ, 3ˊ-cyclic phosphates were also iv detected at the end of 5S rRNA, indicating that 5S rRNA might be post-transcriptionally modified in a manner similar to U6 snRNA. Finally, cleavage sites in tRNAs, rRNAs, and U3 snoRNA, consistent with the activity of angiogenin and other RNase A family endoribonucleases, were detected in hepatitis C virus-infected cells and tissues. To conclude, the data described in this thesis define some of the RNA substrates of RNase L and other ribonucleases during viral infections. 2ˊ, 3ˊ-cyclic phosphate cDNA synthesis and deep sequencing reveal the frequency, location, and specificity of ribonuclease cleavage sites in RNAs within cells, revealing, in part, how these enzymes contribute to health and disease. The form and content of this abstract are approved. I recommend its publication. Approved: David J. Barton v ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor, Dave Barton, for his guidance, support, and encouragement during my training period within his lab. I am very grateful to have had the opportunity to work with Dave, where under his guidance I have developed critical skills to help me during my professional career. This thesis research required a great deal of bioinformatic data processing, and almost everything I know in that regard is because of Jay Hesselberth. I am extremely grateful for all of his help and guidance during the course of my thesis work. Jay also authored several programs that I used to analyze data presented in this thesis. I am also very thankful for my thesis committee, who provided me with encouragement and invaluable ideas and suggestions over the years to keep my research progress on the right track. I would also like to recognize Shuvojit Banerjee from Robert Silverman’s Lab at Cleveland Clinic, Katelyn Leahy, formerly of Hugo Rosen’s Lab at University of Colorado Anschutz Medical Campus, Andrew Firth of University of Cambridge, and Ann Palmenberg of University of Wisconsin, Madison, for their contributions to the data presented in this thesis. I would also like to thank Brian Kempf of the Barton Lab. Without Brian, the lab would have been a very lonely place. I also want to thank Brian for his feedback and suggestions while I wrote this thesis. Finally, I want to thank my boyfriend, Jon Mann, and my family and friends for their support and encouragement over the past six years. vi TABLE OF CONTENTS CHAPTER I. INTRODUCTION…………………………………………...........................................1 2ˊ-5ˊ Oligoadenylate Synthetase (OAS)/RNase L system..................................... 3 Angiogenin and other Ribonuclease A Family Enzymes…………...................…21 IRE1 and the Unfolded Protein Response...........................................................29 RNase T2, An Enzyme Involved in rRNA Processing………………....................32 Usb1, A 3ˊ→5ˊ exonuclease Important for Regulating the Length of U6 snRNA………………………………………….........................…33 Mechanisms of RNA Cleavage……………………………….............................…… 35 Scope of Thesis………………………...............................................................…38 II. METHODS TO DETECT RIBONUCLEASE L CLEAVAGE IN RNA….................40 Introduction………………………………………….……………………................... 40 Materials and Methods…………………………………….……………................… 43 Results……………………………………………..………………………….............. 49 Discussion…………………………………………………………………..............… 70 III. POLIOVIRUS INFECTION: RIBONUCLEASE CLEAVAGE SITES IN HOST AND VIRAL RNAS……………………..………………............… 76 Introduction………………………………............................................................... 76 Materials and Methods……………………............................................................ 79 Results…………………………………………..….................................................. 81 Discussion……………………………….…….........................................................99 IV. INFLUENZA A VIRUS INFECTION: RIBONUCLEASE CLEAVAGE SITES IN HOST AND VIRAL RNAs……...………………………………..............107 Introduction…..………………………………….……………………………............107 Materials and Methods………………………………………………………............110 Results………………………..……………….………………………………............114 vii Discussion………………………….......................................................................127 V. HEPATITIS C VIRUS INFECTION: RIBONUCLEASE CLEAVAGE SITES IN HOST AND VIRAL RNAS……………………………………….….....…135 Introduction……..……………….......................................................................…135 Materials and Methods………………..................................................................138 Results……………………...................................................................................140 Discussion……..………………....………….........................................................155 VI. SUMMARY AND FUTURE DIRECTIONS………………………..........................163 Characteristics of RNA Fragments Produced by RNase L Cleavage………......165 Impact of RNA Cleavage by RNase L……………………....................................166 Terminal 2ˊ, 3ˊ-Cyclic Phosphates on 5S rRNA…………...................................172 Angiogenin Stress Response………………………………..................................175 The Intersection of RNase L and Angiogenin…..………….................................177 REFERENCES………………………………………………………...............................180 viii LIST OF TABLES TABLE 1.1 The function and specificity of some of the RNases expressed by humans that produce RNA fragments with terminal 2ˊ, 3ˊ -cyclic phosphate.................. 6 1.2 Viral countermeasures to RNase L...................................................................18 2.1 TOPO-TA cloning of 2ˊ, 3ˊ-cyclic phosphate cDNA libraries from HCV RNA cleaved by RNase L…............................................................53 2.2. 2ˊ, 3ˊ-cyclic phosphate cDNA synthesis and Illumina sequencing: HCV RNA.......................................................................59 2.3 2ˊ, 3ˊ-cyclic phosphate cDNA synthesis and Illumina sequencing: PV RNA..........................................................................60 3.1 Frequency of 2ˊ, 3ˊ-cyclic phosphates in host and viral RNAs from M25 HeLa cells........................................................................................83 3.2 Frequency of 2ˊ, 3ˊ-cyclic phosphates in host and viral RNAs from W12 HeLa cells……................................................................................ 83 3.3 RNase L -dependent and -independent cleavage sites in 28S rRNA from W12 and M25 HeLa cells........................................................................ 88 3.4 RNase L -dependent and -independent cleavage sites in 18S rRNA from W12 and M25 HeLa cells........................................................................ 91 4.1 Ribonuclease cleavage of IAV RNAs.............................................................117 4.2 RNase L-dependent and -independent cleavage in 28S rRNA from A549 cells...............................................................................................124 4.3 RNase L -dependent and -independent cleavage in 18S rRNA from A549 cells………....................................................................................125
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