In vivo Characterization of the Critical Interaction between the RNA Exosome and the Essential RNA Helicase Mtr4 Público
Farchi Behar, Daniela (Fall 2020)
Abstract
The RNA exosome is a conserved, exo/endoribonuclease complex that processes/degrades numerous coding and non-coding RNAs. The 10-subunit core RNA exosome is composed of three S1/KH cap subunits (EXOSC2/3/1), a lower ring of six PH-like subunits (EXOSC4/7/8/9/5/6), and a base 3’-5’ riboexo/endonuclease subunit, DIS3. RNA processing mutations are the second most common class of mutations linked to multiple myeloma, and mutations in the riboexo/endonuclease gene, DIS3, have been repeatedly identified in patients with this disease. Recently, a rare multiple myeloma patient missense mutation was identified in the cap subunit gene EXOSC2. This missense mutation results in a single amino acid substitution, M40T, in a highly conserved domain of EXOSC2. Structural studies suggest this M40 residue makes direct contact with the essential RNA helicase, hMtr4, and may stabilize an interaction in vivo. We hypothesize that the EXOSC2 substitution M40T destabilizes the interface between the complex and the hMtr4 helicase, thus affecting downstream targeting/processing of the RNA exosome. To test this hypothesis, we generated Saccharomyces cerevisiae that express the corresponding EXOSC2 M40T variant, Rrp4 M68T. Cells expressing Rrp4 M68T show no detectable growth defect, suggesting that the essential Mtr4-RNA exosome interaction is functional with the multiple myeloma modeled substitution. However, when we genetically delete other cofactors that are stabilizing partners for Mtr4, S. cerevisiae expressing Rrp4 M68T, cells show a severe growth defect. These data suggest that the introduction of the multiple myeloma mutation impairs the function of the essential RNA exosome, potentially by decreasing the stability of the binding interface between EXOSC2 and hMtr4. To further assess the functional consequences in S. cerevisiae Rrp4 M68T cells, we performed RT-qPCR to analyze known RNA exosome target transcripts. By investigating the M40T substitution in S. cerevisiae, we provide insight into the functional relevance the M40T substitution could have in disease progression and characterize a key cofactor interface with the RNA exosome in vivo.
Table of Contents
INTRODUCTION………………………………………………………………………………..1
MATERIALS AND METHODS………………………………………………………………..1
Saccharomyces cerevisiae strains, plasmids, and chemicals………………………....6
Site-directed mutagenesis…………………………………………………………….……..7
Saccharomyces cerevisiae transformations………………….…………………….……..8
Saccharomyces cerevisiae serial dilution and optical density growth assays….....8
Immunoblotting………………………………………………………………...……………..9
Immunoblot quantification…………………………………………………...…………....10
Total RNA isolation…………………………………………………...………………...…...10
Quantitative RT-PCR………………………………………………...………………...…....11
In silico predictions and analysis…………………………………...………………....….12
RESULTS……………………………………………..………………...………………...…....12
In silico analysis of EXOSC2-M40T and Rrp4-M68T mutations.…………….….....12
Protein levels and viability are not severely affected in Rrp4-M68T cells….…....14
Rrp4-M68T affects RNA exosome target TLC1 ncRNA steady state levels….…...15
Deletion of MPP6 and RRP47 cofactor genes causes rrp4-M68T growth defects..16
Overexpression of Mtr4 rescues growth defect of Dmpp6 rrp4-M68T cells….……17
DISCUSSION…………………………………………..………………...………………..…...17
REFERENCES…………………………………………..………………...………….......…...25
FIGURES…………………………………………..………………...…………….……....…...32
SUPPLEMENTAL MATERIAL…………………………………………..……………...…....41
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In vivo Characterization of the Critical Interaction between the RNA Exosome and the Essential RNA Helicase Mtr4 () | 2020-11-24 14:33:29 -0500 |
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