Investigation of the Long Non Coding RNA Growth Arrest Specific 5 Control of Cell Fate Restricted; Files Only

Esposito, Regan (Summer 2018)

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Long non coding RNAs are a novel class of RNAs that are poorly understood and challenging to study. The long non coding RNA Growth Arrest Specific 5 (Gas5) is a known tumor suppressor. Gas5 has been shown to interact with two transcription factors: the Glucocorticoid Receptor (GR) and the Y-Box Binding protein 1 (YBX1) and potentially interacts with a wide variety of proteins. Gas5 structural interaction with the GR, its impact on transcription, and potential interactions with other proteins can explain how Gas5 controls apoptosis and proliferation as well as provide insight into the study of lncRNAs. This work examines the various activities of Gas5 and proposes a method to continue to study and characterize this lncRNA.

Table of Contents

Chapter 1: Investigating the Long Non Coding RNA Growth Arrest Specific 5 

Investigating the Long Non Coding RNA Growth Arrest Specific 5                            1

          LncRNA Growth Arrest Specific 5                                                                               1

LncRNA Growth Arrest Specific 5 is downregulated in multiple cancer types             1-2

The Glucocorticoid Receptor                                                                                      2-3

LncRNA Gas5 Interacts with Multiple Proteins                                                           3-4

References                                                                                                                  5-8

            Figures and Tables

                        1.1 Domain Map of GR                                                                                   2         

                        1.2 Mechanisms of Transcriptional Regulation by Gas                                   4

Chapter 2: Structural Characterization of GR DBD and Gas5 as DNA

            Introduction                                                                                                                 9

            Results                                                                                                                        10-11

            Discussion                                                                                                                   12

            Figures and Tables                                                                                                     13 - 25

Figure 2.1 Domain Map of GR                                                                        13

Figure 2.2. Gas5 as DNA has the Same Effect as Full Length Gas5

RNA                                                                                                    14

Figure 2.3. Gas5 DNA Constructs                                                                  15

Figure 2.4. FP with Gas5 as DNA and the GR DBD                                       16

Figure 2.5. FP with Gas5 as DNA with ancestral GR proteins                        17

Figure 2.6. Fluorescence polarization binding experiments with

the MR DBD                                                                                        18

Figure 2.7. Crystal Structure of the GR DBD and G549A DNA                      19

Figure 2.8. Crystal structure of the GR DBD and G549A mutation

as DNA                                                                                                20

Figure 2.9. Additional DNA Stem Constructs show Small

Crystals in Screening Trays                                                                21

Figure 2.10. GR DBD complexed with Gas5 stem 542                                   22

Figure 2.11. Competition of the GR DBD off of DNA with full

length Gas5 and G549A RNA                                                             23

Table 2.1. Gas5 Stem-Loop Constructs from the GR-DBD

Binding Region                                                                                    24

Table 2.2. The GR DBD and G549A as DNA Crystal

Structure Statistics                                                                              25

            Materials and Methods                                                                                                26-28

            References                                                                                                                  29

Chapter 3: Impact of Gas5 on the Glucocorticoid Receptor Transcriptional Activity           

            Introduction                                                                                                                 30-31

Results                                                                                                                        32

            Discussion                                                                                                                   33

            Figures and Tables                                                                                                     34-42  

                        Figure 3.1. Gas5 as DNA has the Same Effect as Full Length

Gas5 RNA                                                                                           34

Figure 3.2. Overexpression of Gas5 is inconsistent                                       35

Figure 3.3. Gas5 and G549A have no inhibitory effect of the

GR transcriptional activation in HeLa cells                                         36

Figure 3.4. Gas5 inhibits GR transcriptional activation in

HeLa cells, G549A does not                                                               37

Figure 3.5. Gas5 blocks transcriptional activation of the

GR in A549 cells                                                                                 38

Figure 3.6. Gas5 inhibits GR at the IL-6 promoter                                          39

Figure 3.7. Gas5 shows no significant effect on GR activity

in CV1 cells                                                                                         40

Figure 3.8. Schematic of luciferase constructs available in the lab.               41

Table 3.1. qRT-PCR Primers                                                                          42

Materials and Methods                                                                                                43

            References                                                                                                                  44-45

Chapter 4: Investigating other lncRNA Gas5 Interaction Partners                                 

            Introduction                                                                                                                 46       

Proposed Methodology                                                                                               47

            Discussion                                                                                                                   48

            Figures and Tables                                                                                                     49-52

Figure 4.1. Gene Ontology (GO) Analysis of Proteins

Observed in a Biotinylated Gas5 Pull Down Experiment                    49

Figure 4.2. RAP-MS Representative Schematic                                             50

Figure 4.3. Each probe set is amplified with a T7 polymerase

sequence added                                                                                 51

Figure 4.4. Probe sets A and B were in vitro transcribed                                52

            References                                                                                                                  53

Chapter 5: LncRNA Gas5 Could Control Cell Fate through the Glucocorticoid Receptor, but it isn’t the Only Answer                                                                                                        54-56

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