Hammerquist, A.M. et al. (2019) International Worm Meeting "Roles for mafr-1in sperm quality and male fertility."

Hammerquist, A.M., Curran, S.P.

[International Worm Meeting, 2019]

RNA polymerase (pol) III negative regulator mafr-1has been shown to affect RNA pol III transcript abundance, lipid biosynthesis and storage, progeny output, and lifespan. However, all previous studies of mafr-1have been conducted using RNAi or a gain-of-function allele. We deleted mafr-1from the Caenorhabditis elegansgenome and found that animals lacking mafr-1replicated many phenotypes from previous research, but strikingly not the oocyte-related reproductive and lipid storage phenotypes. Interestingly, mafr-1null males have smaller spermatids that are less capable of activation. Furthermore, MAFR-1 may interact with sperm-enriched proteins to affect these phenotypes.

Hammerquist AM et al. (2021) Mol Biol Cell "Maf1 regulates intracellular lipid homeostasis in response to DDR activation."

Hammerquist AM, Curran SP, Escorcia W

[Mol Biol Cell, 2021]

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 <i>C. elegans</i> accumulate intracellular lipids in a dose dependent manner. The increase in intracellular lipids in response to exposure to UV light utilizes <i>mafr-1,</i> a negative regulator of RNA polymerase III and the apical kinases <i>atm-1</i> and <i>atl-1</i> of the DNA damage response (DDR) pathway. In the absence of exposure to UV light, the genetic ablation of <i>mafr-1</i> 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, <i>egl-1</i> and <i>ced-13</i>. Taken together, our study reveals a places <i>mafr-1</i> as a component the DDR pathway response to regulating lipid homeostasis following exposure to UV genotoxic stress.

Hammerquist AM et al. (2020) Sci Rep "Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans."

Hammerquist AM, Curran SP

[Sci Rep, 2020]

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.

Hammerquist AM et al. (2021) Bio Protoc "Analysis of Caenorhabditis elegans Sperm Number, Size, Activation, and Mitochondrial Content."

Curran SP, Hammerquist AM, Yen CA

[Bio Protoc, 2021]

Infertility is a widespread and often unexplained issue. Studying reproduction using <i>C. elegans</i> 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 <i>C. elegans</i> sperm quality, including number, size, rate of activation, and mitochondrial morphology. Studying sperm biology in a model system such as <i>C. elegans</i> 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. Graphic abstract: Flowchart depicting the preparation of <i>C. elegans</i> males and subsequent sperm quality assays.

Pradhan, A. et al. (2017) International Worm Meeting "The C-box region of MAF1 regulates transcriptional activity and protein stability."

Pradhan, A., Khanna, A., Curran, S.P., Hammerquist, A.M.

[International Worm Meeting, 2017]

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.

Wissmann AK et al. (1997) International C. elegans Meeting "Identification of Genes Involved in Small G-Protein Mediated Elongation of the C. Elegans Embryo"

Wissmann AK, Allen FL, Ingles J, Mains PE

[International C. elegans Meeting, 1997]

Elongation of C. elegans embryos appears to be driven by microfilament dependent cell shape changes in the hypodermis (1). Several mutations in let-502 have been identified that lead to arrest during early embryonic elongation (2,3). We have cloned let-502 and found that it encodes a serine/threonine kinase with significant similarity to a group of recently identified kinases activated by the binding of small G-proteins (Rho/Rac) and to human myotonic dystrophy kinase. let-502 mutations can be genetically suppressed by lf mutations in mel-11. We cloned mel-11 and identified its similarity to smooth muscle myosin phosphatase regulatory subunits. We propose a model in which binding of Rho activates LET-502, which in turn inhibits the activity of a smooth muscle-like myosin phosphatase complex via phosphorylation of MEL-11. This inactivation of the phosphatase complex leads to actin-myosin contraction resulting in the cell shape changes necessary for embryonic elongation (3). We have recently obtained evidence that MEL-11 interacts genetically with Rho or a similar G-protein. Double mutants of mel-11 and unc-73, a suspected Rho guanine nucleotide exchange factor, suggest that Rho may have a direct activating effect on MEL-11. We have also conducted a genetic screen for mel-11 suppressors resulting in a number of candidates that may define additional components in this small G-protein mediated pathway. 1 Priess, J.R. & Hirsh, D.L. Developmental Biology 117, 156-173 (1986) 2 Howell, A.M. & Rose, A.M. Genetics 126, 583-592 (1990) 3 Wissmann et al. Genes & Development 11, 409-422 (1997)

Zappaterra MD et al. (2000) West Coast Worm Meeting "Loss of a dynamin related protein MGM-1 causes excessive mitochondrial fragmentation"

van der Bliek A, Zappaterra MD

[West Coast Worm Meeting, 2000]

Our lab studies the functions of dynamin and dynamin related proteins. These proteins form a small family of large GTP-binding proteins. We have recently shown that the C. elegans dynamin related protein DRP-1, is involved in the scission of the mitochondrial outer membrane (1). Since little is known about mitochondrial division and mitochondrial morphology in animal cells, we are investigating the mechanisms that regulate these processes. MGM-1 is another member of the dynamin family present in animal cells. As of now we know that this protein is present in yeast, C. elegans, and humans. Unlike other members of the dynamin family, MGM-1 has an N-terminal mitochondrial leader sequence. MGM-1 is also more closely related to bacterial dynamin-like proteins than it is to dynamin or DRP-1, suggesting that this protein has followed a different evolutionary path. We speculate that MGM-1 was introduced into eukaryotic cells by the progenitors of mitochondria. Expression studies using -galactosidase under the control of the mgm-1 promoter show high levels of expression in intestines, body wall muscles, and neurons. These high levels might reflect high metabolic rates in those tissues, because the MGM-1 expression pattern is similar to that of another dynamin-related protein, DRP-1, which is also important for mitochondrial maintenance. Mgm-1 RNAi drastically slows the growth rate and approximately doubles the life span of C. elegans. We show that excessive fragmentation of mitochondria is induced by RNAi and mgm-1 antisense cDNA. We present evidence that MGM-1 is localized to the mitochondrial matrix where it might regulate the morphology of the mitochondrial inner membrane. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular Cell 4: 815-826. 1999.

Broeks A et al. (1993) Worm Breeder's Gazette "Transposon Induced Deletion Mutagenesis, a General Method"

Zwaal RR, Plasterk RHA, Broeks A

[Worm Breeder's Gazette, 1993]

To investigate the function of a gene one can analyze the phenotype of a null allele of that gene. Methods have been described to obtain Tc1 insertion alleles of genes (PCR and sib selection, A.M. Rushforth et al. WBG 11(5):65; mutant bank, R.H.A. Plasterk et al. WBG 12(4):11). Tc1 insertions are most frequently found in intron sequences. In some cases all or part of Tc1 can also be spliced out from exons, with the possibility that the function is not completely lost (A.M. Rushforth et al. in press). Therefore neither Tc1 intron nor exon insertions can be considered guaranteed null alleles. Some revertants of unc-22 ::Tc1alleles were found to have lost Tc1 plus 1-2 kb flanking sequences (J.E. Kiff et al. Nature 331:361-363 '8o. These deletions probably occur during repair of DNA breaks caused by Tc1 excision. Based on this observation we used Tc1 insertions as a starting point to isolate deletion mutants. 100 Cultures are started with 10 animals each; the DNA is analyzed using PCR. The primers for PCR are chosen a) to select for unidirectional deletions, or b) to select for bidirectional deletions. The distance between the primers is chosen 4.6 kb (3 kb genomic sequences + 1.6 kb Tc1 sequences), thus no product is found without a flanking deletion. A 3 kb somatic excision product can be found, but it is outcompeted in the PCR if a deletion allele is present. [See Figures] Deletions occurred with a frequency of 10 2 to 10-3 per allele per generation. Both unidirectional and bidirectional deletions have been found, the size varying from 900 bp to 2 kb. Thus far we have obtained 3 clonal deletion mutants (1 for pgp-1 and 2 for gpa-1 ) and we are in the course of cloning several other deletion derivates that have been detected (pgp-1 ,pgp-3 ,ceh-13 and gpa-2 ). More Tc1 insertions will be studied to investigate whether the unexpected high frequency of deletion alleles generated from Tc1 alleles is a general phenomenon. Since we have observed deletion derivates in all genes we looked at, it seems to be a general method. This method allows the use of all exon and intron insertions isolated from the mutant bank for the isolation of null alleles.

Alan M Zahler et al. (2001) International C. elegans Meeting "Molecular, genomic and genetic analysis of alternative splicing in C. elegans"

W James Kent, Alan M Zahler, Tracy Farrer, A Brock Roller, Jordan Benjamin

[International C. elegans Meeting, 2001]

Our lab is interested in regulation of alternative splicing and we have begun several projects to study alternative splice site choice using C. elegans as a model system. To this end, we have developed the Intronerator as a computational system for using EST information in C. elegans to identify alternatively spliced genes. We have identified over 800 alternatively spliced genes using this system (1). By comparing genomic sequence of C. briggsae and C. elegans we have identified regions of conservation near some of these spliced genes that are candidates for putative cis -regulatory elements for alternative splicing (2). We are developing splicing reporter assays to test the activity of these conserved elements in regulation of alternative splicing. One alternatively spliced gene we are studying in depth is the let-2 gene. Alternative splicing involves the use of two mutually exclusive exons, exons 9 and 10. In embryos, exon 9 is primarily used and in adults exon 10 is primarily used, with a gradual switch in the usage occurring during the larval stages (3). We have created a splicing reporter construct in which the alternatively spliced region of let-2 is fused to green fluorescent protein. The almost exclusive usage of exon 9 in embryos is maintained in this construct. A series of cis -mutations that we have devised in this reporter indicate that the unusual beginning of the intron between exon 10 and exon 11, the intron starts with GC instead of the canonical GT, is important for promoting the splicing of exon 9. Cis mutagenesis of the alternatively spliced regions of this gene to identify splicing regulatory elements is continuing. In addition, we have made a splicing reporter for this gene in which only the adult form of the message is in frame with GFP. Animals containing this reporter do not express detectable GFP until the L2 stage. We are using this reporter in a visual screen to identify genes involved in the regulation of let-2 alternative splicing. Our progress in this screen will be presented. 1. Kent, W.J. and Zahler, A.M. 2000. Nucleic Acids Research 28: 91-93. 2. Kent, W.J. and Zahler, A.M. 2000. Genome Research 10: 1115-1125. 3. Sibley M.H. et al. 1993. Journal of Cell Biology 123: 255-264

Ayako Hasegawa et al. (2003) International Worm Meeting "C.elegans homologue of yeast Mdm38 and mammalian Letm1 is essential for mitochondrial morphology"

Ayako Hasegawa, Alexander M van der Bliek

[International Worm Meeting, 2003]

Mitochondria are dynamic structures that divide and fuse throughout the life of a cell. Several proteins have been identified to affect mitochondrial outer membrane division and fusion. Our lab discovered that dynamin related protein called DRP-1 plays a key role in mitochondrial outer membrane division (1). The detailed analysis of the dominant-negative phenotype of DRP-1, which causes a block in outer membrane division, indicates that inner membrane division occurs independent of outer membrane division. 3D reconstruction of mitochondrial inner membrane and cristae by electron tomography also indicates that fusion occurs within mitochondria. Cristae often fuse to each other or to inner membrane (2). These data suggest that there are separate mechanisms of division and fusion for inner membrane aside from those for outer membrane, and a mechanism that coordinates different membrane remodelling. Our lab has been looking for novel proteins that are involved in regulation of mitochondrial morphology. C.elegans body wall muscle cells are used because they are large and flat, and their mitochondria are fairly well organized. Mitochondrially targeted fluorescence protein marker under muscle-specific promoter was used to visualize the mitochondria with minimum background. We are analyzing the function of a C.elegans homologue of the yeast Mdm38 protein, which has previously been implicated in mitochondrial morphology (3). This protein is also homologous to the mammalian protein Letm1, which is deleted in some patients with Wolf Hirschhorn Syndrome, a disease of as yet unknown etiology. We find that this protein is localized to mitochondria and affect mitochondrial morphology. Strong MDM38 loss-of-function causes abnormally enlarged and distended mitochondria, while mild loss-of-function causes enlarged and indented mitochondria, resembling oxidation-phophorylation complex loss-of-function phenotypes. Over expression of MDM38 appears to cause loss of mitochondrial potential and shriveling of matrix and/or swelling of outer membrane. We suspect that MDM38 homologous protein affects cristae remodeling, however the functionality of this protein is yet to be discovered. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular, Cell 2: 815-826. 1999. 2. Frey, T.G., Mannella, C.A. Trends Biochem Sci 25(7): 319-324. 2000. 3. Dimmer, K.S., Fritz, S., Fuchs, F., Messerschmitt, M., Weinbach, N., Neupert, W., Westermann, B. Mol Biol Cell 13(3): 847-853. 2002.