Splicing of pre-mRNA is an essential process for dividing cells, and splicing defects have been linked to aging and various chronic diseases. Environmental stress has recently been shown to modify alternative splicing, and molecular mechanisms that influence stress-induced alternative splicing remain unclear. Using an in vivo RNA splicing reporter, we performed a genome-wide RNAi screen in Caenorhabditis elegans and found that protein translation suppression via silencing of the conserved eukaryotic initiation factor 4G (IFG-1/eIF4G) inhibits cadmium-induced alternative splicing. Transcriptome analysis of an ifg-1 deficient mutant revealed an overall decrease in intronic and intergenic reads and prevented cadmium-induced alternative splicing compared to the wild-type. We found that the ifg-1 mutant up-regulates>80 RNA splicing regulatory genes controlled by the TGF-β transcription factor SMA-2. The extended lifespan of the ifg-1 mutant is partially reduced upon sma-2 depletion and completely nullified when core spliceosome genes including snr-1, snr-2, and uaf-2 are knocked down. Depletion of snr-1 and snr-2 also diminished the enhanced cadmium resistance of the ifg-1 mutant. Together, these data describe a molecular mechanism through which translation suppression inhibits stress-induced alternative splicing, and demonstrate an essential role for RNA splicing in promoting longevity and stress resistance in a translation-compromised mutant.
CCR4‐NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4‐NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome‐wide RNAi screen, we identified a subunit of the CCR4‐NOT complex, ccf‐1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole‐transcriptome RNA sequencing, we show that the knockdown of ccf‐1 attenuates the activation of a broad range of stress‐protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and xenobiotic detoxification. Consistently, survival assays show that the knockdown of ccf‐1 decreases C. elegans stress resistance and normal lifespan. A yeast 2‐hybrid screen using a CCF‐1 bait identified the homeobox transcription factor PAL‐1 as a physical interactor. Knockdown of pal‐1 inhibits the activation of ccf‐1 dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of ccf‐1 and pal‐1 attenuates the activation of elt‐2 and elt‐3 under stress that encode master transcriptional co‐regulators of stress response in the C. elegans, and that overexpression of ELT‐2 can suppress ccf‐1's requirement for gene transcription in a stress‐dependent manner. Our findings reveal a new role for CCR4‐NOT in the environmental stress response and define its role in stress resistance and longevity in C. elegans.
CCR4-NOT is a versatile eukaryotic protein complex that controls multiple steps in gene expression regulation from synthesis to decay. In yeast, CCR4-NOT has been implicated in stress response regulation, though this function in other organisms remains unclear. In a genome-wide RNAi screen, we identified a subunit of the CCR4-NOT complex, ccf-1, as a requirement for the C. elegans transcriptional response to cadmium and acrylamide stress. Using whole-transcriptome RNA sequencing, we show that knockdown of ccf-1 attenuates the activation of a broad range of stress protective genes in response to cadmium and acrylamide, including those encoding heat shock proteins and glutathione s-transferases. Consistently, survival assays show that knockdown of ccf-1 decreases C. elegans stress resistance. A yeast-2-hybrid screen using a CCF-1 bait identified the homeobox transcription factor PAL-1 as a physical interactor. Knockdown of pal-1 inhibits the activation of ccf-1 dependent stress genes and reduces C. elegans stress resistance. Gene expression analysis reveals that knockdown of pal-1 down-regulates the mRNA levels of elt-2 and elt-3, which serves as the master transcriptional co-regulators of stress response in the C. elegans intestinal and epidermal tissues respectively. These results reveal a new role for CCR4-NOT in stress response regulation with PAL-1 through the transcriptional control of elt-2 and elt-3 in C. elegans.
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