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. 2014 Feb 19;4(2):231-41.
doi: 10.1534/g3.113.009126.

Identification of suppressors of mbk-2/DYRK by whole-genome sequencing

Yuemeng Wang  1 , Jennifer T Wang, Dominique Rasoloson, Michael L Stitzel, Kevin F O' Connell, Harold E Smith, Geraldine Seydoux
Affiliations

Identification of suppressors of mbk-2/DYRK by whole-genome sequencing

Yuemeng Wang et al. G3 (Bethesda). .

Abstract

Screening for suppressor mutations is a powerful method to isolate genes that function in a common pathway or process. Because suppressor mutations often do not have phenotypes on their own, cloning of suppressor loci can be challenging. A method combining whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) mapping (WGS/SNP mapping) was developed to identify mutations with visible phenotypes in C. elegans. We show here that WGS/SNP mapping is an efficient method to map suppressor mutations without the need for previous phenotypic characterization. Using RNA-mediated interference to test candidate loci identified by WGS/SNP mapping, we identified 10 extragenic and six intragenic suppressors of mbk-2, a DYRK family kinase required for the transition from oocyte to zygote. Remarkably, seven suppressors are mutations in cell-cycle regulators that extend the timing of the oocyte-to-zygote transition.

Keywords: C. elegans; DYRK kinase; MBK-2; single nucleotide polymorphism mapping; suppressors; whole-genome sequencing.

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Figures

Figure 1
Figure 1
Isolation of suppressors of mbk-2(dd5). (A) Schematic of the oocyte-to-embryo transition comparing the distribution of MEI-1 (red, high level; pink, intermediate level; white, absent) and P granules (purple) in wild-type and mbk-2(dd5) zygotes. Pronuclei (○) and spindle are also shown. (B) Scheme for the isolation of mutants that suppress the embryonic lethality of mbk-2(dd5). (C) Scheme for the isolation of F2 Hawaiian/N2 recombinants carrying the suppressor mutations. Blue represents chromosomes region with N2 SNPs, and green represents chromosomes with Hawaiian SNPs. Note that for semi-dominant suppressors, a proportion of mbk-2(dd5); sup/Hw recombinants will also be present in the sequenced pool. Graphs show the percentage of Hawaiian SNPs (Y axis) along linkage group (LG) II (X axis) in a population of F2 recombinants [derived as in (C)] carrying no suppressor (D) or the recessive suppressor ax2014 (E). Each dot represents a unique Hawaiian SNP. Only SNPs with frequencies >20% are displayed.
Figure 2
Figure 2
Identification of the suppressor loci for the dominant suppressors ax2009 and ax2001. (A–C) Percent of hatched embryos among the progeny of hermaphrodites of the genotypes shown and fed dsRNA against the genes shown. (A) tat-4(RNAi) reverses the suppression of ax2009. cdc-37(RNAi) reverses the suppression of ax2001 (B) and enhances the embryonic lethality of mbk-2(dd5) at the semi-permissive temperature of 20° (C).
Figure 3
Figure 3
Rescue of the MEI-1 and P granule defects by the suppressors. (A) The genotypes indicated were scored for GFP::MEI-1 on the mitotic spindle. Black indicates no GFP::MEI-1 on spindle (as in wild-type) and white indicates GFP::MEI-1 on spindle [as in mbk-2(dd5)]. Ten zygotes scored for each genotype (see Figure S3 for representative images). The intragenic suppressor ax2004 shows the most efficient rescue. (B) Zygotes at pronuclear meeting (P) or metaphase of the first mitosis (M) were scored for P granule asymmetry. Black indicates full segregation (all P granules in posterior half of the zygote as in wild-type), gray indicates partial segregation (some P granules remaining in anterior half of the zygote), and white indicates no segregation (P granules uniformly distributed throughout the cytoplasm. The numbers of zygotes scored are indicated below each bar (see Figure S3 for representative images). The intragenic suppressor ax2004 was also the most efficient suppressor by this assay.
Figure 4
Figure 4
Cell-cycle suppressors extend the timing of the oocyte-to-zygote transition. Time spent from ovulation to pronuclear formation (A) and from the onset of pronuclear migration to pronuclear breakdown (B) for zygotes of the indicated genotypes (see Materials and Methods). Each dot represents a single zygote. Blue lines show the average time and red lines show the 95% confidence interval for wild-type zygotes.
Figure 5
Figure 5
Rescue of kinase activity by intragenic suppressors ax2004 and ax2006. (A) Representative example of a kinase assay using recombinant MBP-tagged MBK-2 expressed in E. coli. Upper panel shows ATP incorporation into the substrate MBP::MEI-1. Lower panel shows Coomassie blue–stained MBP::MBK-2 used in the assay to control for loading. KD is MBK-2(K196R) with a mutation in the ATP binding site. (B) Graph showing the relative activity (arbitrary units) of MBP::MBK-2 fusions obtained from two independent kinase assays [as in (A)]. Double-tail Student t test was applied to compare each suppressor to MBK-2(dd5).
Figure 6
Figure 6
Position of the intragenic suppressors in MBK-2. C. elegans MBK-2 sequence was superimposed onto the structure of human DYRK2 (MMDB ID: 77389; PDB ID: 3K2L). The catalytic loop, ATP binding region, and activation loop are shown in different colors. The phosphorylated tyrosine in the activation loop is shown in red. Amino acids mutated in dd5 and the four intragenic suppressors are highlighted in yellow. ax2005 maps near the activation loop on the opposite side of the molecule and is not visible in this view.
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