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    DTPQDT02019019639.pdf

    i Translation of Partially-Decayed Messenger RNAs in Yeast __________ A Thesis Presented to the Faculty of Natural Sciences and Mathematics University of Denver __________ In Partial Fulfillment of the Requirements for the Degree Master of Science __________ By Ana L. Franklin June 2019 Advisor Erich G. Chapman     ProQuest Number     All rights reserved  INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.  In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.      ProQuest  Published by ProQuest LLC . Copyright of the Dissertation is held by the Author.   All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition ProQuest LLC.   ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 13897058 13897058 2019 ii Copyright by Ana L. Franklin 2019 All Rights Reserved ii Author Ana L. Franklin Title Translation of Partially-Decayed Messenger RNAs in Yeast Advisor Erich G. Chapman Degree Date June 2019 ABSTRACT Flaviviruses are positive-strand single-stranded RNA viruses that are known to form pseudo-knot RNA structures that halt the progression of 5’3’ exonuclease Xrn1. We show that these viral Xrn1-resistant structures xrRNAs can be used to protect specific homologously-expressed messenger RNAs from 5’3’ degradation. We investigated the effects of addition of xrRNAs, artificially-installed into the intergenic region of bicistronic mRNA reporters, in the observed levels of protein expression in yeast. The reporters also contain an internal ribosome entry site from the cricket paralysis virus CrPV IRES to allow for cap-independent translation of the decay-protected gene, LacZ, encoding the enzyme β-galactosidase. Through the use of primer extension, β- galactosidase assay, and western blots, the results indicate that the partially-decayed RNAs are successfully translated, and that addition of xrRNAs results in a 30-50 fold increase in measured enzymatic activity and an accumulation of decay-resistant transcripts in the cell. iii TABLE OF CONTENTS Chapter One Introduction. . 1 1.1 Eukaryotic mRNA 1 1.1.1 Transcription 2 1.1.2 Processing and Transport. .. 4 1.1.3 Translation .6 1.1.3.1 Eukaryotic Versus Prokaryotic mRNAs.. 9 1.1.3.2 Internal Ribosome Entry Sites IRES. .10 1.1.3.2.1 Mechanisms of Internal Ribosomal Entry 11 1.1.3.2.2 Translation Initiation without Initiator tRNA The CrPV IRES . 12 1.1.4 mRNA Half-Lives and Stability . 14 1.1.5 mRNA Turnover 16 1.2 Partially-Decayed RNAs . .18 1.2.1 Flaviviridae and Sub-Genomic RNAs . .. 18 1.3 S. cerevisiae as a Model Organism. 18 1.3.1 S. cerevisiae Genomics .. 20 1.3.2 Auxotrophy as a Selective Marker 21 1.3.3 IRES in Yeast . 22 1.4 Thesis Objective Investigation of the Effect of Artificially-Installed xrRNAs on Translation of Partially-Decayed mRNAs 22 1.4.1 Research Aim 1. Detection of Decay-Resistant Transcripts .. 23 1.4.2 Research Aim 2. Quantification of Enhancement in Protein Expression 24 Chapter Two Materials and Methods .26 2.1 Strains and Plasmids 26 2.2 Yeast Husbandry .. 28 2.3 Transformation of Yeast cells .. 29 2.4 Sequencing. 29 2.5 Colony PCR. 31 2.6 Quantification of Enhancement in Protein Expression . . 32 2.6.1 β-Galactosidase Assay . 32 iv 2.6.2 Western Blots. 33 2.7 Detection of Decay-Resistant Transcripts . 34 2.7.1 Fragment Analysis of Primer Extension Products . 34 Chapter Three Results .. 37 3.1 Vector Composition . 37 3.2 Methionine Initiator tRNA Genes are Knocked-Out in H2545 Strain. 39 3.3 Expression of Reporters Containing xrRNAs Yields Decay-Resistant Transcripts .. 41 3.4 Partially-Decayed Messenger RNAs Are Translated and Lead to Enhanced Protein Expression . 44 3.5 DXO1 Knockouts Display Increased Enzymatic Activity after Transformation with JPS 1480s Series Reporters .4 5 Chapter Four Discussion and Summary .51 v LIST OF FIGURES FIGURE 1. Assembly of the eukaryotic 80S ribosome capable of translation initiation. The eIF2GTPMet–tRNAiMet ternary complex is vital for proper formation of the cap- dependent translation initiation complex .7 FIGURE 2. Stereo view of ribosome-bound CrPV IRES structure PDB 2NOQ. Mutations that prevent formation of the small 3’end pseudoknot formation and inhibit internal ribosomal initiation are highlighted in yellow. . 13 FIGURE 3. The concentration of mRNAs in the cell is highly dependent on the rates of two opposing processes transcription and mRNA decay. Similarily, the major factors affecting protein concentrations are mRNA abundance, and rates of translation and protein degradation. .15 FIGURE 4. Overview of 5’3’ mRNA decay pathway. The mRNA is committed to decay after progressive deadenylation of the polyA tail results in a short oligoA tail. Degradation continues by removal of the m7G cap by a decapping enzyme, which exposes a 5’ monophosphate that is the substrate for the exoribonuclease, XRN1. XRN1 is the major 5’3’ exoribonuclease and completely degrades the remainder of the mRNA sequence by cleaving off one nucleotide at a time1 7 FIGURE 5. ZIKV xrRNA front and side views. 5’ end blue threaded through the center of a 3-helix junction 3’ labeled red. The pseudoknot forms a mechanical barrier which prevents progression of the 5’3’ exonuclease XRN1. .19 FIGURE 6. Translation of partially-decayed messenger RNAs. Addition of the decay- resistant structures xrRNAs to the reporters should result in accumulation of partially- decayed transcripts. Since these transcripts contain the CrPV IRES structure to allow for cap-independent translation, translation of partially-decayed mRNAs should result in increased protein synthesis2 3 FIGURE 7. JPS 1480 series reporters. All plasmids were kindly provided by the Jonathan P. Staley lab . 27 vi FIGURE 8. Map of reporter vectors JPS 1481 top and 1481X bottom, containing all key features. All reporters contain the following elements, listed in the 5’3’ direction the GAP promoter white arrow, ACT1 black, CrPV IRES blue, LacZ magenta, CUP1-1 brown, and the PGK1 terminator yellow. In addition, all reporters designated as “X” or “M” contain the xrRNAs red, located between ACT1 and the CrPV IRES.38 FIGURE 9. Sequence alignments of the different JPS 1480 series reporters confirming the presence of point mutations in the mutant structures. The mutant xrRNAs contain AGTTCA mutations in bases 30-32 and 103-105 shown in red, 1481M, and the mutant CrPV IRES contains a CCGG mutation in bases 190-191 shown in blue, 1482 and 1482X. 39 FIGURE 10. Colony PCR results confirming that imt3 and imt4 genes are knocked-out in H2545 yeast, priming that strain for successful cap-independent translation of the bicistronic reporter. Large individual colonies of S288C and H2545 were resuspended in lysis buffer and lysed by heating to 95 C. Lysates were used as templates for PCR amplification using imt3- and imt4-specific primers and subjected to equal reaction conditions. PCR products were analyzed through gel electrophoresis and examined for presence of bands at different lengths between the two strains . 40 FIGURE 11. A Fragment analysis data showing the presence of transcripts protected from degradation by one and two copies of the xrRNAs shown by green box. MW size standard shown as orange peaks, and corresponding lengths of the standard peaks are indicated by black arrows B Expected fragment length of transcripts containing the xrRNAs. Total yeast RNA content was isolated and reverse-transcribed using a fluorescently-labeled primer. The resulting fluorescently-labeled cDNA fragments were analyzed by capillary electrophoresis. The green arrow illustrates the approximate binding site of the primer. Results confirm the accumulation of xrRNA-protected fragments 42 FIGURE 12. Western Blot of H2545 cells expressing three different reporters from the JPS 1480 series. Whole cell protein extracts were incubated with either anti-β- Galactosidase or anti-GAPDH primary antibodies. A prominent band corresponding to the molecular weight of β-Galactosidase was observed in the cells expressing the reporter containing the fully-functioning xrRNAs 1481X, but no significant bands were observed if the reporter was lacking the xrRNAs 1481 or contained the mutant copy 1481M. This confirms that addition of the xrRNAs leads to increased protein expression of the target protein 43 FIGURE 13. Enzymatic activity of β-galactosidase in cell cultures expressing different reporters from the JPS 1480 series. Total yeast protein lysates were incubated with vii ONPG for 3 hours. Enzymatic activity was quantified as absorbance at 420 nm and normalized for reaction time and initial cell concentration, measured as absorbance at 600 nm. The reporters include either the active form of each element , the mutant or inactive form -, or are missing the element listed left blank. Increased enzymatic activity only observed upon transformation with reporters with fully-functioning copies of both xrRNAs and IRES. 44 FIGURE 14. β-galactosidase activity of WT CRY1 cells and three different knockout strains expressing three of the reporters from the JPS 1480 series. Total yeast protein lysates were incubated with ONPG for 3 hours. Enzymatic activity was quantified as absorbance at 420 nm and normalized for reaction time and initial cell concentration, measured as absorbance at 600 nm. β-galactosidase activity of WT CRY1 cells and three different knockout strains expressing three of the reporters from the JPS 1480 series. The increased enzymatic activity observed in the DXO1 knockout strain suggests that cap- independent translation of the CrPV IRES element is active in this strain . 46 FIGURE 15. Colony PCR confirmation of ∆XRN1 genotype xrn1∆HygMX. Large individual colonies of wild-type CRY1 cells, as well as knockouts for XRN1, DXO1 and XRN1/DXO1, were resuspended in lysis buffer and lysed by heating to 95 C. Lysates were used as templates for PCR amplification using XRN1-specific primers and subjected to equal reaction conditions. PCR products were analyzed through gel electrophoresis. The PCR product obtained at around 2202 bp left in the ∆XRN1 and ∆XRN1/∆DXO1 strains confirms that, in these cells, the XRN1 ORF has been replaced with the HygMX cassette, yielding the knockout genotype. The PCR product observed at around 460 bp in the CRY1 and ∆DXO1 cells confirms that the wild-type cells contain the XRN1 ORF 47 FIGURE 16. Colony PCR confirmation of the ∆DXO1 genotype dxo1∆KanMX. Large individual colonies of wild-type CRY1 cells, as well as knockouts for XRN1, DXO1 and XRN1/DXO1, were resuspended in lysis buffer and lysed by heating to 95 C. Lysates were used as templates for PCR amplification using DXO1-specific primers, subjected to equal reaction conditions, and analyzed through gel electrophoresis. The results confirm the WT and mutant genotype, as observed in the difference in length between the PCR products observed at around 1980 bp in the CRY1 and ∆XRN1 cells and the products observed at around 2008 bp in the two ∆DXO1 strains. 48 FIGURE S1. Comprehensive map of reporter vectors JPS 1481 top and 1481X bottom, containing all key features, restriction enzyme cleavage sites, and primers used for sequencing shown by lime green arrows indicating direction of primer. New LacZ PE primer was also used for reverse transcription. 62 FIGURE S2. Qualitative results of β-galactosidase assay of H2545 cells expressing different. The tubes, listed from left-to-right, belong to cells expressing the following JPS viii 1480 series reporters 1481, 1481X, 1481M, 1482, 1482X, and empty cells. As results indicate, cells containing the IRES-dead elements have close to the same enzymatic activity as the empty cells 1482, 1482X. The cells containing the functioning IRES but inactive xrRNAs have some increased activity, as evidenced by the light yellow color 1481, 1481M. The cells with the highest activity are those in which both elements are active 1481X, second tube from the left. 63 FIGURE S3. β-galactosidase activity of WT CRY1 cells and three different knockout strains expressing three of the reporters from the JPS 1480 series, and the double- knockout strains also expressing two different plasmids containing the XRN1 ORF. Only the DXO1 knockout appears to have increased enzymatic activity when compared to the empty cells, suggesting that adding the XRN1 ORF to the double-knockout strain does not genetically rescue IRES activity. The activity of empty cells was also tested to establish a reference point of enzymatic activity. 64 FIGURE S4. Sequencing map of BJH 872 plasmid, encoding pXRN1. Map shows entire length of yeast wild-type XRN1gene black line. Colored arrows indicate fragment that was sequenced using each corresponding primer, as well as direction of the primer .64 ix LIST OF ABBREVIATIONS Abbreviation Meaning ATP Adenosine triphosphate ATPase Adenosine triphosphatase BP Base pair CBC Cap-binding complex cDNA Complementary DNA CPSF Cleavage and polyadenylation specificity factor CrPV Cricket paralysis virus CStF Cleavage stimulation factor CTD Carbon-terminal domain DNA Deoxyribonucleic acid DSE Downstream sequence element DXO1 Decapping exonuclease 1 EDTA Ethylenediaminetetraacetic acid eEF-1α Eukaryotic elongation factor 1α eEF-2 Eukaryotic elongation factor 2 eIF1 Eukaryotic initiator factor 1 eIF1A Eukaryotic initiator factor 1A eIF2 Eukaryotic initiator factor 2 eIF3 Eukaryotic initiator factor 3 eIF4A Eukaryotic initiator factor 4A eIF4F Eukaryotic initiator factor 4F eIF4F Eukaryotic initiator factor 4F eIF4G Eukaryotic initiator factor 4G eIF5 Eukaryotic initiator factor 5 eRF1 Eukaryotic release factor 1 eRF3 Eukaryotic release factor 3 GA

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