A Role for the Nonsense-Mediated mRNA Decay Pathway in Maintaining Genome Stability in Caenorhabditis elegans

Abstract

Ionizing radiation (IR) is commonly used in cancer therapy and is a main source of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA damage. We have used Caenorhabditis elegans as an invertebrate model to identify novel factors required for repair of DNA damage inflicted by IR. We have performed an unbiased genetic screen, finding that smg-1 mutations confer strong hyper-sensitivity to IR. SMG-1 is a phosphoinositide-3 kinase (PI3K) involved in mediating nonsense-mediated mRNA decay (NMD) of transcripts containing premature stop codons and related to the ATM and ATR kinases which are at the apex of DNA damage signaling pathways. Hyper-sensitivity to IR also occurs when other genes mediating NMD are mutated. The hyper-sensitivity to bleomycin, a drug known to induce DSBs, further supports that NMD pathway mutants are defective in DSB repair. Hyper-sensitivity was not observed upon treatment with alkylating agents or UV irradiation. We show that SMG-1 mainly acts in mitotically dividing germ cells, and during late embryonic and larval development. Based on epistasis experiments, SMG-1 does not appear to act in any of the three major pathways known to mend DNA DSBs, namely homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ). We speculate that SMG-1 kinase activity could be activated following DNA damage to phosphorylate specific DNA repair proteins and/or that NMD inactivation may lead to aberrant mRNAs leading to synthesis of malfunctioning DNA repair proteins.

A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro -Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

“…HDR is an error-free repair pathway which is predominantly active during the late S phase and the entire G 2 phase of the cell cycle. In contrast, MMEJ is an error-prone pathway that is activated during G 1 and early S phases of the cell cycle ( 12 , 14 – 16 ). Another major difference between HDR and MMEJ is the size of the homology regions harbored within the linear DNA fragment.…”

Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation

“…To uncover new genes involved in the DDR, we performed an unbiased forward genetic screen. Upon ethyl methanesulfonate (EMS) mutagenesis of P0 wild-type ( N2 ) individuals, F2 animals were singled in 96-well plates ( González-Huici et al 2017 ). Progeny of singled L4 stages animals was split into aliquots ( González-Huici et al 2017 ).…”

“…Upon ethyl methanesulfonate (EMS) mutagenesis of P0 wild-type ( N2 ) individuals, F2 animals were singled in 96-well plates ( González-Huici et al 2017 ). Progeny of singled L4 stages animals was split into aliquots ( González-Huici et al 2017 ). One aliquot of L1 larvae was treated with 60 Gy of IR, a mutagenic agent that leads to a wide spectrum of DNA lesions including DNA double-strand breaks.…”

“…Mutagenesis and screening procedures were performed as described in González-Huici et al (2017) . Single nucleotide polymorphism (SNP) mapping was performed according to the protocol described in Davis et al (2005) .…”

“…DAPI staining of oocytes and RAD-51 immunostaining was performed as described in González-Huici et al (2017) . For DAPI staining of whole germlines, we used a procedure described in Craig et al (2012) .…”