Alternative splicing of coq-2 controls the levels of rhodoquinone in animals
- The Donnelly Centre, University of Toronto, Canada
- Laboratorio de Biología de Gusanos. Unidad Mixta, Departamento de Biociencias, Facultad de Química, Universidad de la República - Institut Pasteur de Montevideo, Uruguay
- Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, United States
- Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, United States
- Department of Chemistry and Biochemistry, Gonzaga University, United States
Figures
Figure 1
Rhodoquinone and ubiquinone biosynthesis and function in electron transport chains.
(A) In aerobic metabolism, ubiquinone (UQ) shuttles electrons in the ETC from Complex I (CI; yellow box) and quinone-coupled dehydrogenases (QDHs), such as Complex II. These electrons are ultimately …
Figure 2
C. elegans coq-2 gene model.
(A) The coq-2 gene contains two mutually exclusive exons, 6e (blue box) and 6a (red box), that are alternatively spliced (blue and red lines, respectively) generating two COQ-2 isoforms. Light gray …
Figure 3
Gene models for coq-2 orthologs in various species.
Parasitic helminths, as well as annelids and mollusks, have two internal exons that are spliced in a mutually exclusive manner. By contrast, humans and other hosts only have one exon that is …
Figure 4
Conserved changes between a- and e-form exons across RQ-producing species.
Amino acid sequences of COQ-2 orthologs were aligned using Clustal Omega (Madeira et al., 2019). The sequences of exons homologous to exon 6a/e in C. elegans, as well as the flanking five amino acid …
Figure 5 with 1 supplement
The C. elegans coq-2 edited strains and effects of exon 6a and 6e deletions on quinone biosynthesis.
(A) Mutant strains were generated in C. elegans by deletion of exon 6a (coq-2∆6a) or exon 6e (coq-2∆6e). (B) Deletion of exon 6a from the coq-2 gene significantly increased the level of RQ9 …
Figure 5—figure supplement 1
Deletion of coq-2 exon 6e affects the ability of worms to survive extended KCN treatment at various KCN concentrations.
Wild-type (N2), coq-2 and kynu-1(e1003) mutant L1 worms were exposed to 150–500 µM KCN for 15 hr. KCN was then diluted 6-fold and worm movement was measured over 3 hr to track recovery from KCN …
Figure 6
Correlation of COQ-2 splicing with change from aerobic to anaerobic life stages.
(A) Schematic of the life cycle of A. suum. ‘OUT’ denotes aerobically respiring free-living stages; ‘IN’ indicates stages living inside the host intestine. (B) Graph indicates the percentage of all …
Figure 7
Discrimination between RQ and UQ biosynthesis in C.
elegans. There are two variants of exon 6 in the C. elegans coq-2 gene (6a and 6e) which undergo mutually exclusive alternative splicing leading to COQ-2a and COQ-2e isoforms, respectively. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Caenorhabditis elegans) | coq-2 | WormBase | WBGene00000762 | |
| Strain, strain background (Caenorhabditis elegans) | N2 | Caenorhabditis Genetics Center (CGC) | N2 | Wild-type |
| Strain, strain background (Caenorhabditis elegans) | coq-2 (syb1715) | This paper | PHX1715 | coq-2∆6a Supplementary file 1 |
| Strain, strain background (Caenorhabditis elegans) | coq-2 (syb1721) | This paper | PHX1721 | coq-2∆6e Supplementary file 1 |
| Strain, strain background (Escherichia coli) | OP50 | Caenorhabditis Genetics Center (CGC) | OP50 | |
| Sequence-based reagent | sgRNAs used in this study | This paper | Supplementary file 1 | |
| Sequence-based reagent | PCR primers used in this study | This paper | Supplementary file 1 | |
| Chemical compound, drug | Potassium cyanide (KCN) | Sigma-Aldrich | 60178–25G | Stock solution: 50 mM in PBS buffer |
| Other | Hexane, HPLC grade | Sigma-Aldrich | 650552–1L | |
| Other | Acetonitrile, LC-MS grade | Fisher Scientific | A955-4 | |
| Other | Ubiquinone-3 standard | Campbell et al., 2019 | ||
| Other | Ubiquinone-9 standard | Sigma-Aldrich | 27597–1 MG | |
| Other | Rhodoquinone-9 standard | Roberts Buceta et al., 2019 | Isolated from Ascaris suum | |
| Software, algorithm | Whippet | Sterne-Weiler et al., 2018 | RRID:SCR_018349 | https://github.com/timbitz/Whippet.jl |
| Software, algorithm | HISAT2 | Kim et al., 2019 | RRID:SCR_015530 | https://daehwankimlab.github.io/hisat2/ |
| Software, algorithm | UCSF Chimera | Resource for Biocomputing Visualization and Informatics Pettersen et al., 2004 | RRID:SCR_004097 | http://plato.cgl.ucsf.edu/chimera/ |
| Software, algorithm | MODELLER | University of California at San Francisco Webb and Sali, 2016 | RRID:SCR_008395 | https://salilab.org/modeller/ |
| Software, algorithm | Image analysis pipeline | Spensley et al., 2018 | https://github.com/fraser-lab-UofT/acute_assay | |
| Software, algorithm | Python | Python | RRID:SCR_008394 | https://www.python.org/ |
| Software, algorithm | NIS-Elements | Nikon | RRID:SCR_014329 | https://www.microscope.healthcare.nikon.com/products/software/nis-elements |
| Software, algorithm | ImageMagick | ImageMagick | RRID:SCR_014491 | https://imagemagick.org/ |
| Software, algorithm | BioFormats | OME - Open Microscopy Environment | RRID:SCR_000450 | https://docs.openmicroscopy.org/bio-formats/5.7.1/users/comlinetools/index.html |
Additional files
-
Supplementary file 1
C. elegans strains.
- https://cdn.elifesciences.org/articles/56376/elife-56376-supp1-v2.elegansstrains.docx
-
Supplementary file 2
Statistical analysis of RQ9 and UQ9 levels in coq-2 mutant strains.
- https://cdn.elifesciences.org/articles/56376/elife-56376-supp2-v2.docx
-
Supplementary file 3
Genomic coordinates of known and predicted mutually exclusive coq-2 a/e exons.
- https://cdn.elifesciences.org/articles/56376/elife-56376-supp3-v2.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/56376/elife-56376-transrepform-v2.docx
