Establishing the optimal conditions for in vivo RNA structural probing of guanine and uracil base-pairing in yeast Open Access

Abstract

RNAs can have catalytic or regulatory functions in the cell and play critical roles in many steps of gene regulation. RNA structure can play a key role in its function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structural probing is an indirect method to probe the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. The chemical modifications can target either the RNA backbone or the Watson-Crick face of nucleotides. The selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) can probe the ribose sugar in RNA nucleotides. Dimethyl sulfate (DMS) alkylates adenine and cytosine, but it is not reactive to guanine or uracil. Recently, new compounds were used to modify Gs and Us in the plant model system rice Oryza sativa. To complement the scope of RNA structural probing by chemical modifications in yeast, as part of my honors thesis dissertation work, I analyzed the effectiveness of guanine modification by a family of aldehyde derivatives, the glyoxal family, in Saccharomyces cerevisiae and Candida albicans in vivo. We also explored the effectiveness of uracil modification by carbodiimide derivatives in these species in vivo. We show that among the glyoxal family, phenylglyoxal (PGO) is the best guanine probe for in vivo structure probing as the guanine modification effectiveness demonstrates a concentration-dependence in S. cerevisiae, and C. albicans. We also demonstrate a concentration-dependent relationship for uracil modification by carbodiimide N-cyclohexyl-N- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) in S. cerevisiae and C. albicans in vivo. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. Our results provide the conditions for probing the reactivity of guanine and uracil in RNA structures in yeast and offer a useful tool for studying RNA structure and function in two widely used yeast model systems.

Table of Contents

Table of Contents

Background and Introduction………………………………………………………......1

1.1 Introduction of RNAs and their three-dimensional structures………...…….….1

1.2 The importance of understanding RNA three-dimensional structures….…….2

1.3 An overview of RNA structural probing techniques…………………………..3

1.4 Established in vivo RNA structural probing reagents for modification of guanines and uracils………………………………..……………………........3

1.5 Research plan…………….……………………………………………………4

Materials and Methods…………………………………………………………….…….4

2.1 Yeast Cell Culture……………………………………………………………..4

2.2 Incubation with Chemical Probes……………………………………………..5

2.3 Total RNA Extraction and Purification………...………………..……………5

2.4 Reverse Transcription…………………………………………………………6

2.5 Northern blot for snoRNA and tRNA……………………………………….…7

2.6 Serial Dilution Spot Test for Chemical Toxicity Assessment……………...…..7

Results……………………………………………………………...………………….….7

3.1 In vivo modification of yeast S. cerevisiae 5.8S rRNA by glyoxal, methylglyoxal, and phenylglyoxal characterized by denaturing UREA-PAGE of cDNAs………………………...………………………………………………...…8

3.2 In vivo modification of yeast S. cerevisiae 18S rRNA by glyoxal and derivatives characterized by denaturing UREA-PAGE of cDNAs………………..9

3.3 In vivo modification of yeast C. albicans 5.8S rRNA by phenylglyoxal by denaturing UREA-PAGE of cDNAs……………………………………….…..…10

3.4 Northern blot analysis for U3-3’, U3-5’ snoRNAs and tRNAs………...…….11

3.5 Serial dilution spot test for toxicity analysis in PGO-treated S. cerevisiae….12

3.6 In vivo modification of yeast S. cerevisiae 5.8S rRNA characterized by CMCT by denaturing UREA-PAGE of cDNAs………………………………...………………………………………….13

Discussion………………….………………………………………………………..…..14

Acknowledgement……………………………………………..…...……………….…..16

Contributions……………………………………………………………..………….…17

Reference………………………………...……………..….....................................17 

Table of Figures

Figure 1. In vivo modification of 5.8S rRNA by GO derivatives in S. cerevisiae.....................…8

Figure 2. In vivo modification of 18S rRNA by GO derivatives in S. cerevisiae …………….....9

Figure 3. In vivo modification of 5.8S rRNA by PGO in C. albicans …….……..……………..10

Figure 4. Northern blot analysis of the processing of U3 snoRNA and proline tRNA............…11

Figure 5. Serial dilution growth assay for toxicity analysis of PGO-treated S. cerevisiae……...12

Figure 6. In vivo modification of 5.8S rRNA by CMCT in S. cerevisiae …………………...…13

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Chemistry, Biochemistry Biology, General
Keyword	Medicine
Committee Chair / Thesis Advisor	Ghalei, Homa, Emory University Khoshnevis, Sohail, Emory University
Committee Members	Corbett, Anita, Emory University

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