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Sequence and Structure Requirements of Y RNA-Derived Small RNA Biogenesis

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Sequence and Structure Requirements of Y RNA-Derived Small RNA Biogenesis Sequence and structure requirements of Y RNA-derived small RNA biogenesis Carly Turnbull A thesis submitted to the University of East Anglia for the degree of Doctor of Philosophy University of East Anglia Norwich School of Biological Sciences May 2014 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. ABSTRACT Numerous small non-coding RNAs have been identified in mammalian cells, includ- ing microRNAs and piwi-interacting RNAs. The patterns of gene expression within cells can be altered in response to cellular stress. To examine the effects of cellu- lar stress on all small RNA types, a number of human cell lines were treated with Poly(I:C), a mimic of viral infection, and the levels of small RNAs were examined by next generation sequencing. Surprisingly, we did not find many differentially ex- pressed microRNAs, but we discovered a new class of small RNAs that were 30-35 nucleotides and showed up-regulation following Poly(I:C) treatment. These slightly longer small RNAs were derived from many different types of non-coding RNA and only very few small RNAs were derived directly from messenger RNAs. Small RNAs derived from various types of RNA were validated by northern blot. Further sequencing libraries were prepared for Poly(I:C)-treated and untreated MCF7 cells, as well as Poly(I:C)-treated and untreated SW1353 cells. The human Y5 RNA gene was chosen as an example of a Poly(I:C)-induced small RNA-producing gene. This gene was cloned into an expression construct and systematically mutated to alter the sequence or secondary structure of the resulting Y RNA. These mutant plasmids were expressed in mouse cells and the effect on small RNA production determined. These individual mutants together helped to determine a region vital for cleavage, and that it is the structure rather than the sequence within this region that is impor- tant. A high-throughput method was also implemented, involving the generation of large pools of plasmids containing all possible sequences within a particular region of the RNA gene. These pools were expressed in mouse cells and the mutants that were expressed and processed into small RNAs were sequenced. These experiments showed that the formation of the large internal loop determines the internal cleavage site. 2 ACKNOWLEDGEMENTS I would like to thank my supervisor, Tamas Dalmay, for all his help and advice during this project, and for controlling the many ideas I had whilst undertaking it. I am grateful to Guy Wheeler for his help and advice and for forcing me to take regular breaks for coffee. I am very grateful to Ping Xu and Sara Lopez-Gomollon for their careers advice and their honest opinions on my work, as well as providing me with the enthusiasm and encouragement to continue in science. Many thanks to all those in the Munsteberg lab for providing me with samples and helping me to extract them, especially those that were sent from other labs. I am very grateful to my fiance Lee, for moving my entire house for me while I was doing my last few weeks of lab work. You have given me an amazing amount of support and I am not sure I would have finished without it! 3 ABBREVIATIONS 30mer 30-35 nucleotide small RNA AGO Argonaute ANG Angiogenin APS Ammonium persuphate ARG Analytical reagent grade ATP Adenosine triphosphate cDNA Complementary DNA CNBP Cellular nucleic acid binding protein CRM1 Chromosome region maintenance 1 (also exportin1 or Xpo1) ∆3’ Mutant with 3 nucleotide in 3 tail deleted ∆1S Mutant with one side of the stem deleted ∆2S Mutant with both sides of the stem deleted ∆ACC Mutant with ACC nucleotides deleted ∆Ro Mutant with Ro60 binding site deleted DGCR8 DiGeorge Syndrome Critical Region 8 DMEM Dulbecco's modified eagle medium dsRNA Double-stranded RNA dsDNA Double-stranded DNA DTT Dithiothreitol dNTPs Deoxyribonucleotides drY D. radiodurans homolog of Y RNA EBER Epstein-Barr virus-encoded small RNA EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDTA Ethylenediaminetetraacetic acid eIF4F Eukaryotic initiation factor 4F complex eIF4G/A Eukaryotic initation factor 4G/A complex Endo-siRNA Endogenous small interfering RNA FAM Fluorescein amidite FBS Fetal bovine serum gDNA Genomic DNA HDAC Histone deacetylase hnRNP Heterogeneous nuclear ribonucleoprotein particle hY Human Y RNA IFIT5 Interferon-induced protein with tetratricopeptide repeats 5 IRES Internal ribosome entry site IPTG Isopropyl -D-1-thiogalactopyranoside LB Luria-Bertani LNA Locked nucleic acid 4 lncRNA Long intergenic non-coding RNA MBq Megabecquerel MCPIP1 Monocyte chemotactic protein-induced protein mESCs Mouse embryonic stem cells MIDAS Metal ion-dependent adhesion site miRNA MicroRNA MOV10 Moloney leukaemia virus 10, homolog (mouse) mRNA Messenger RNA mRo60 Mouse Ro60 mY Mouse Y RNA ncRNA Non-coding RNA NGS Next generation sequencing nt Nucleotides OptiMEM OptiMEM I reduced serum media PAGE Polyacrylamide gel electrophoresis PASR Promoter-associated small RNA PBS Phosphate buffered saline PCR Polymerase chain reaction PI Propidium iodide piRNA Piwi-interacting RNA Piwi P-element induced wimpy testes in Drosophila PKR Protein kinase RNA-activated PNK T4 polynucleotide kinase PNPase Polynucleotide phosphorylase Poly(I:C) Polyinosinic:polycytidylic acid Puf60 Poly(U)-binding splicing factor 60 kDa RISC RNA-induced silencing complex RNase Ribonuclease RNASET2 Human orthologue of Rny1p RNH1 Ribonuclease inhibitor of angiogenin RNP Ribonucleoprotein particle Rny1p Ribonuclease in yeast 1 RoBP1 Ro60-binding protein 1 RPA Replication protein A Rpm Revolutions per minute RRM RNA recognition motif rRNA Ribosomal RNA Rsr Ro-sixty related (D. radiodurans orthologue of Ro60) RT-PCR Reverse transcriptase PCR 5 QC Quality Control SAP Shrimp alkaline phosphatase sbRNA Stem bulge RNA SDS Sodium dodecyl sulfate siRNA Small interfering RNA sitRNA Stress-induced tRNA-derived RNA SLE Systemic lupus erythematosus sno-miRNA Small nucleolar RNA with microRNA-like activity snoRNA Small nucleolar RNA snRNA Small nuclear RNA sRNA Small RNA SS Sj¨ogrensSyndrome SSC Sodium chloride - sodium citrate buffer STS Staurosporine Sub1S Mutant with one side of the stem substituted Sub2S Mutant with both sides of the stem substituted svRNA Small vault RNA TBE Tris-borate-EDTA TEMED Tetramethylethylenediamine tiRNA Transcription initiation RNA TOP Terminal oligopyrimidine tract tRNA Transfer RNA Tsix Antisense RNA transcribed from Xist locus TTSaRNA Transcription start site-associated RNA UV Ultra violet vWFA Von Willebrand factor A-like motif X-gal 5-Bromo-4-chloro-3-indolyl -D-galactopyranoside XIAP X-linked inhibitor of apoptosis XiRNA X-inactivation RNA Xist X-chromosome inactivation-specific transcript xRo60 Xenopus sp. Ro60 xY Xenopus sp. Y RNA YB-1 Y box-binding protein 1 ydRNA Y RNA-derived RNA ZBP1 Zipcode-binding protein 1 6 Contents ABSTRACT . 2 ACKNOWLEDGEMENTS . 3 ABBREVIATIONS . 4 1 INTRODUCTION 17 1.1 Long non-coding RNAs . 18 1.2 Small non-coding RNAs . 18 1.2.1 MicroRNAs . 18 1.2.2 Small interfering RNAs . 19 1.2.3 Piwi-interacting RNAs . 19 1.2.4 Promoter-associated RNAs . 20 1.2.5 X-inactivation RNAs . 20 1.2.6 Small vault RNAs . 20 1.2.7 Transfer RNA-derived RNAs . 21 1.2.8 SnoRNA-derived RNAs . 21 1.3 Stress-induced small ncRNAs . 21 1.3.1 Transfer RNA 'halves' . 22 1.3.2 Ribosomal RNA-derived RNAs . 25 1.4 Origins of the project . 26 1.4.1 What are Y RNAs? . 28 1.4.1.1 Y RNA evolution . 29 1.4.2 Y RNA secondary structure . 30 1.4.3 Y RNA binding proteins . 32 1.4.3.1 Ro60 and misfolded ncRNA binding . 33 1.4.3.2 Ro60 and Y RNA binding . 34 1.4.3.3 La . 35 1.4.3.4 La and Y RNA binding . 36 1.4.3.5 Nucleolin . 37 1.4.3.6 Calreticulin . 37 1.4.3.7 Heterogeneous nuclear ribonucleoproteins . 38 1.4.3.8 Ro RNP binding protein I . 38 7 1.4.3.9 Interferon-induced protein with tetratricopeptide re- peats 5 . 38 1.4.3.10 Ribosomal protein L5 . 39 1.4.3.11 Zipcode binding protein I . 39 1.4.3.12 Polynucleotide phosphorylase . 39 1.4.3.13 Other Y RNA-binding proteins . 39 1.4.3.14 Y RNP localization . 40 1.4.4 Y RNA function . 42 1.4.4.1 Y RNAs in DNA replication . 42 1.4.4.2 Y RNAs in RNPs . 44 1.4.4.3 Y RNAs and Ro60 function . 44 1.4.4.4 Y RNAs in splicing . 45 1.4.4.5 Y RNAs and Apoptosis . 46 1.4.4.6 Y RNAs as microRNAs? . 46 2 MATERIALS AND METHODS 47 2.1 CELL CULTURE . 48 2.1.1 Cell lines and cell culture media . 48 2.1.2 Passaging of cells . 48 2.1.3 Seeding of cells . 48 2.1.4 Transfection of cells . 48 2.1.4.1 Transient transfection of cells with Lipofectamine2000 48 2.1.4.2 Transient transfection of cells with Fugene6 . 49 2.1.5 Stress treatments . 49 2.1.5.1 Hygromycin treatment . 49 2.1.5.2 PBS treatment . 49 2.1.5.3 Poly(I:C) treatment . 49 2.1.5.4 Staurosporine treatment . 50 2.1.5.5 Lipofectamine2000 control treatment . 50 2.1.5.6 Untreated control conditions . 50 2.1.6 Flow cytometry of cells treated with Poly(I:C) or Hygromycin 50 2.2 RNA EXTRACTION . 51 2.2.1 RNA extraction from cell lines for northern blot analysis .
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