MAF1 is a conserved negative regulator of RNA polymerase (pol) III and intracellular lipid homeostasis across species. Here, we show that the MAF1 C-box region negatively regulates its activity. Mutations in Caenorhabditis elegans mafr-1 that truncate the C-box retain the ability to inhibit the transcription of RNA pol III targets, reduce lipid biogenesis, and lower reproductive output. In human cells, C-box deletion of MAF1 leads to increased MAF1 nuclear localization and enhanced repression of ACC1 and FASN, but with impaired repression of RNA pol III targets. Surprisingly, C-box mutations render MAF1 insensitive to rapamycin, further defining a regulatory role for this region. Two MAF1 species, MAF1L and MAF1S, are regulated by the C-box YSY motif, which, when mutated, alters species stoichiometry and proteasome-dependent turnover of nuclear MAF1. Our results reveal a role for the C-box region as a critical determinant of MAF1 stability, activity, and response to cellular stress.

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Surveillance of DNA damage and maintenance of lipid metabolism are critical factors for general cellular homeostasis. We discovered that in response to DNA damage-inducing UV light exposure, intact C. elegans accumulate intracellular lipids in a dose dependent manner. The increase in intracellular lipids in response to exposure to UV light utilizes mafr-1, a negative regulator of RNA polymerase III and the apical kinases atm-1 and atl-1 of the DNA damage response (DDR) pathway. In the absence of exposure to UV light, the genetic ablation of mafr-1 results in the activation of the DDR including increased intracellular lipid accumulation, phosphorylation of ATM/ATR targets proteins, and expression of the Bcl-2 homology region genes, egl-1 and ced-13. Taken together, our study reveals a places mafr-1 as a component the DDR pathway response to regulating lipid homeostasis following exposure to UV genotoxic stress.

The negative regulator of RNA polymerase (pol) III mafr-1 has been shown to affect RNA pol III transcript abundance, lipid biosynthesis and storage, progeny output, and lifespan. We deleted mafr-1 from the Caenorhabditis elegans genome and found that animals lacking mafr-1 replicated many phenotypes from previous RNAi-based studies and discovered a new sperm-specific role. Utilizing a yeast two-hybrid assay, we discovered several novel interactors of MAFR-1 that are expressed in a sperm- and germline-enriched manner. In support of a role for MAFR-1 in the male germline, we found mafr-1 null males have smaller spermatids that are less capable in competition for fertilization; a phenotype that was dependent on RNA pol III activity. Restoration of MAFR-1 expression specifically in the germline rescued the spermatid-related phenotypes, suggesting a cell autonomous role for MAFR-1 in nematode male fertility. Based on the high degree of conservation of Maf1 activity across species, our study may inform similar roles for Maf1 and RNA pol III in mammalian male fertility.

Infertility is a widespread and often unexplained issue. Studying reproduction using C. elegans males offers insight into the influence of individual factors on male fertility in humans. We have created a collection of protocols to assess several aspects of C. elegans sperm quality, including number, size, rate of activation, and mitochondrial morphology. Studying sperm biology in a model system such as C. elegans allows access to the wealth of resources and techniques that have been optimized for that organism while providing valuable biological information that may be applicable to other systems.