Biogenesis of Y RNA derived small RNAs

Biogenesis of Y RNA–derived small RNAs Adam Edward Hall A thesis submitted for the degree of Doctor of Philosophy University of East Anglia School ...
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Biogenesis of Y RNA–derived small RNAs

Adam Edward Hall

A thesis submitted for the degree of Doctor of Philosophy

University of East Anglia School of Biological Sciences

February 2013

© 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.

'Nothing has such power to broaden the mind as the ability to investigate systematically and truly all that comes under thy observation in life.' Marcus Aurelius, Roman Emperor, c. 161 A.D.

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Abstract

Small non-coding RNAs (sRNAs) constitute a significant portion of the transcriptome in eukaryotes. Many of these sRNAs regulate gene expression. Nextgeneration sequencing (NGS) has revealed a plethora of previously uncharacterised sRNAs with potential biological function, a number of which originate from longer RNAs. Here, the biogenesis of sRNAs derived from the non-coding Y RNAs (YsRNAs) was characterised as a model for understanding this emerging class of sRNA fragments. Y RNAs are highly conserved, 100 nt long molecules involved in DNA replication which bind to the autoimmune proteins Ro60 and La. YsRNAs are produced in cells undergoing apoptosis. Here, it was demonstrated that YsRNAs are generated from the 5’ and 3’ ends of all four Y RNAs in stressed and unstressed cells. Furthermore, production of these fragments was observed in both cancerous and non-cancerous cells. Although YsRNAs have been proposed to have gene silencing activity, experiments done here found that YsRNAs do not enter the microRNA pathway and are not generated by the gene silencing-related protein Dicer. Furthermore, experiments established that the enzyme which produces fragments from tRNAs, angiogenin, was also not responsible for YsRNA generation. Using mammalian cultured cells along with gene knockout and RNA interference (RNAi) technology, it was determined that RNase L contributed to YsRNA generation. Furthermore, the Y RNA binding protein Ro60 was shown to be essential for YsRNA production through a model of RNase protection. Analysis of deep sequencing data in Ro60 knockout cells revealed that many other sRNAs are also dependent on Ro60. Finally, a ‘high definition’ (HD) protocol to improve NGS detection of sRNAs was tested. The HD protocol was found to be better at detecting sRNAs than current methods. This will facilitate more efficient detection of novel sRNAs in the future.

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Table of Contents

ABSTRACT…………………..……………………………………………………3 LIST OF FIGURES…………………………..…..……………………..…….....10 PREFACE……………………………………..…………………..……..………14 ACKNOWLEDGEMENTS……………….….………………..……..…………15 PUBLICATIONS……………………………………...…….………………..….16 ABBREVIATIONS……………………………………...……………………….17

CHAPTER 1…………………...………………………………………..………..20 Introduction

1.1

Overview: A genome governed by RNA……………………..…………...21

1.2

Evolution, structure and biogenesis of Y RNAs…………………………..23

1.3

Y RNA binding proteins………………………………………..…………28 1.3.1 General anatomy of the Y RNA Ribonucleoprotein (Y RNP)…....29 1.3.2 Ro60…………………………………………………………….....34 1.3.3 The La protein…………………………………………………..…38 1.3.4 Ro52…………………………………………………………..…...41 1.3.5 hnRNP I & K…………………………………………………...…42 1.3.6 RoBPI……………………………………………………………..43 1.3.7 Nucleolin……………………………………………………….….43 1.3.8 L5……………………………………………………...…………..44 1.3.9 ZBP1………………………………………………………………45 4

1.4

The role of Y RNAs in chromosomal DNA replication…………..………45

1.5

Y RNAs in cancer…………………………………………………...…….51

1.6

Y RNAs in apoptosis…………………………………………………...…53

1.7

Small RNA biology…………………………………………………..…...55 1.7.1 The advent of ‘deep sequencing’ and the discovery of the ‘small RNA world’……………………………………………………….55 1.7.2 MicroRNAs (miRNAs): A small RNA case study………………..56

1.8

Small RNAs derived from longer RNAs……………………………….....58 1.8.1 Filtering of deep sequencing data reveals extensive RNA fragmentation……………………………………………...………58 1.8.2 tRNA fragmentation………………………………………………60 1.8.3 Y RNA-derived small RNAs………………………………...……63

1.9

Aims and objectives of the thesis…………………………………………65

CHAPTER 2…………………………………………………………….……..…66 Materials and Methods

2.1

Cell culture……………………………………………………...…………67 2.1.1 Cell lines and growth media…………………………………..…..67 2.1.2 PCR genotyping of RNase L-/- cell lines………………………….68 2.1.3 Passaging and cell growth conditions……………………………..70

2.2

siRNAs and transfection…………………………………………………..71

2.3

Poly(I:C) treatment………………………………………………………..73

2.4

RNA extraction……………………………………………………………73

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2.5

Assessment of RNA quality and concentration…………………………...75

2.6

Protein extraction and quantification……………………..……………….75

2.7

Small RNA Northern blotting……………………………………………..76 2.7.1 Polyacrylamide gels for small RNA Northern blotting …………..76 2.7.2 Northern blotting of RNA…………………………………………77 2.7.3 Carbodiimide-mediated cross-linking of RNA……………………78 2.7.4 Pre-hybridisation of Northern blot………………………..……….79 2.7.5 Northern blot probe synthesis…………………………...…...……79 2.7.6 Post-hybridisation

washing

of

Northern

blots

and

signal

detection………………………………………………………..….80 2.8

Quantitative Northern blotting and statistical analysis of band Intensities……………………………………………………………….…81

2.9

Oligonucleotide probe sequences for Northern blot hybridisations………82 2.9.1 Antisense DNA oligonucleotide probe sequences (Sigma)…….....82 2.9.2 Locked nucleic acid (LNA) modified oligonucleotide probes (Exiqon)…………………………………………...………………82

2.10

Western blotting……………………………………………...……………83 2.10.1 Polyacrylamide gel electrophoresis of proteins………...…………83 2.10.2 Blotting of proteins………………………………………………..83 2.10.3 Western blot antibody hybridisation………………………………84

2.11

Antibodies for Western blotting…………………………………………..85

2.12

Y5 expression construct…………………………………………...………86

2.13

Angiogenin expression construct………………………………………….86

2.14

Plasmid purification……………………………………………………….87

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2.15

Dideoxynucleotide chain termination (Sanger) sequencing………...…….88

2.16

Anion exchange chromatography of non-stressed and poly(I:C) treated cells…………………………………………………………………..……88

2.17

Ago2 pull-down assay……………………………………………...……..89

2.18

Angiogenin in vitro cleavage experiments……………………………..…90

2.19

Adjustment of pH and filter sterilisation of buffers……………………….91

2.20

Mouse organ dissection and subsequent RNA isolation…………………..91

2.21

Purification of sRNA (

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