Takaharu Yamamoto et al. (2008) Development & Evolution Meeting "The san-1/MAD3 Synthetic Lethal Screen Identified a Kinetochore Receptor of MDF-1/MAD1 in C. elegans"

Risa Kitagawa, Sonoko Watanabe, Takaharu Yamamoto

[Development & Evolution Meeting, 2008]

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by delaying onset of anaphase until all kinetochores of chromosomes are properly attached to the spindles. A genomewide RNAi screen for synthetic genetic interactors with one of the SAC gene, san-1/MAD3 and a secondary screen for genes required to activate the SAC of C. elegans identified an uncharacterized gene, temporarily named tsl-1 (Mad three synthetic lethal-1). The tsl-1 encodes a coiled-coil protein (TSL-1) that localized to the kinetochore from prometaphase until anaphase. Kinetochore localization of TSL-1 depended on KNL-1, a highly conserved kinetochore protein and CZW-1/ZW10, a component of the RZZ (ROD-ZW10-ZWILCH) complex. TSL-1 was required for spindle defect--induced kinetochore localization of other SAC protein, MDF-1/MAD1. TSL-1 also coimmunoprecipitated with MDF-1/MAD1 in vivo, suggesting its physical association with MDF-1/MAD1 at unattached kinetochores. Together, these results indicate that TSL-1 functions in a kinetochore receptor of MDF-1/MAD1, thereby targeting MDF-1/MAD1 to the unattached kinetochore to exert SAC function in the RZZ complex pathway.

Ford, Jason R. et al. (2011) International Worm Meeting "Identifying the role of the Tousled-like kinase during the cell cycle."

Furuta, Tokiko, Ford, Jason R., Adams, Henry, Schumacher, Jill M.

[International Worm Meeting, 2011]

The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana via a mutation responsible for aberrant floral organ.. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1; the human kinases are nuclear proteins with maximal activity tied to ongoing DNA replication during S-phase. Very few substrates of TLK have been described with the most reported being the chromatin assembly factor ASF. Aside from its S-phase functions, we have shown that TLK-1 also contributes to chromosome segregation as a substrate and activator of the AIR-2/Aurora B kinase. AIR-2-dependent phosphorylation of TLK-1 is detectable at the centrosomes, kinetochore (KT), and KT-microtubules (MTs) from early prophase to metaphase. TLK-1 depletion phenotypes include embryonic lethality, a spindle assembly checkpoint (SAC)-dependent mitotic delay, chromosome segregation defects, and distended nuclei in late multicellular embryos. To further determine the consequences of TLK-1 depletion in developing embryos, we used live-cell analysis of early embryos subjected to tlk-1(RNAi). Our analysis revealed a spindle rotation defect in 50% of a TLK-1-depleted population during the first mitotic division. More specifically, the centrosome-pronuclear complex rotation in TLK-1-depleted embryos is significantly delayed with respect to nuclear envelope breakdown. This striking phenotype suggests a nascent role for a known chromatin regulator, TLK-1, in regulating microtubule behavior during mitosis, and the epistatic relationships between tlk-1 and other genes required for spindle rotation are being investigated. It has been reported that P0 embryos depleted of regulatory dynein subunits exhibit an inability to correctly rotate the centrosome-pronuclear complex in a manner strikingly similar to that of the TLK-1-depleted- P0 embryos, suggesting that TLK-1 functionality may influence dynein-dependent processes in the early embryo. Interestingly, co-depletion of TLK-1 with certain dynein subunits results in an inability to completely separate centrosomes and correctly rotate the mitotic spindle, an effect that forces MTs emanating from centrosomes to attach KTs in a monopolar-like fashion. With these data taken together, we hypothesize that TLK-1 is affecting vital cytoskeletal processes relating to centrosome dynamics and other dynein-related processes during mitosis, and relating the established roles of TLK-1 to its functions in cytoskeletal/spindle dynamics and cell cycle regulation will shed light on the consequences of TLK-1 action during development.

Paulini, Michael et al. (2013) International Worm Meeting "another can of worms - more genomes & sequences at WormBase."

Paulini, Michael, Howe, Kevin, Tuli, Mary Ann, Davis, Paul, Williams, Gareth

[International Worm Meeting, 2013]

Since 2011, the number of nematode species represented in WormBase increased by half from 12 to 19. These species can be divided into close relatives of Caenorhabditis elegans and parasitic nematodes. In addition the in-depth curation of a limited set of core species has been extended, with the support of the wider research community, to include Brugia malayi, a nematode of clade III and one of the causative agents of lymphatic filariasis in human. Support for multiple alternative reference genomes for a single species (like Ascaris suum somatic/germline assemblies) has been added. To support the extension of WormBase to non-Caenorhabditis species and provide a standardised nomenclature across nematodes, we have also extended our gene-naming service to include B.malayi and helped to provide a first pass gene nomenclature based on a combination of publications and predictions through orthologs. Another development focus was the mapping procedure for RNAi and expression probes, which now allows for more accurate mappings in a species-agnostic way. Also the use of RNASeq by WormBase increased and mappings of RNASeq libraries deposited in the Short Read Archive are not only provided as BAM files, but are also post processed and used in the curation process providing information on TSL sites, splicing, expression asymmetry, polycistronic transcripts and tissue and life-stage specific expression levels (available through SPELL). This is supplemented by the integration sequence features from modENCODE data, like transcription factor, RNA polymerase II and histone binding sites as well as genelets and transcripts.

Anthony S Rogers (2005) International Worm Meeting "Full Length Transcripts in WormBase"

Anthony S Rogers

[International Worm Meeting, 2005]

UnTranslated Regions (UTRs) of messenger RNAs have been shown to be important in the regulation of several C.elegans genes such as tra-2(1) and fem-3(2). To give an improved representation of UTR regions in WormBase predicted full length transcripts are now generated each release. These are built up automatically from the available transcript data and the current CDS structure. Non-genomic transcript sequence such as Trans-spliced leaders and polyA tails are masked before aligning the transcript to the genome using BLAT(3). Identifying TSL and polyA sequences prior to genome alignment helps to prevent misalignments and means individual features can be made to represent these sequences. UTR structures are built from the subset of transcripts which have been computationally shown to agree with the CDS prediction. This new method of representing full length transcripts allows us to build objects that more closely resemble their biological reality with trans-spliced leader sequence and polyadenylation information included where applicable. It is also possible to show where there is evidence of multiple UTRs for a single CDS.As of WS140 there are 25869 transcripts derived from 22410 CDSs representing the 19735 protein coding genes. 1) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C.elegans Goodwin EB, Okkema PG, Evans TC and Kimble J. Cell, 1993 75:329-339 2) Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region. Ahringer JA, Kimble JE Nature, 1991 349:346-348 3) BLAT The BLAST-Like Alignment Tool W. James Kent. Genome Research (2002) Vol. 12, Issue 4, 656-664

Marianne Land et al. (2001) International C. elegans Meeting "Depletion of PKL (KIN-4) in Muscle Results in L1 Paralysis"

Marianne Land, Charles S Rubin

[International C. elegans Meeting, 2001]

In C.elegans kin-4 encodes two isoforms of a PDZ containing S/T protein kinase (PKL). PKL1 and PKL2 are identical over 1333 amino acids but have different C termini that are generated by omission or inclusion of the penultimate exon of the gene. MAST205, a mammalian protein kinase, has catalytic and C terminal PDZ domains that are homologous with corresponding domains in PKL. However their N and C terminal sequences have limited but potentially significant homology with those of PKL. I have used C.elegans and mammalian systems to probe PKL functions. Immunostaining revealed that PKL1 is aligned along filaments of body-wall muscle (adjacent to actin) in C.elegans . PKL2 is enriched in pharyngeal muscle (adjacent to actin), the lateral surface of intestinal cells and the gonad. Both PKLs are associated with large, punctate structures in numerous cell types. MAST205 is expressed principally in testis, skeletal muscle and heart. In a skeletal muscle cell line MAST205 is enriched in focal adhesions and actin cytoskeleton. The kinase accumulates at "zipper like" cell-cell junctions in a spermatocyte-derived cell line. These junctions and MAST205 are dispersed in cells treated with cytochalasin B. Large punctate structures that contain MAST205 are also evident in these cells. 32 P-labeled PDZ domains from MAST205 and PKL avidly bind mouse skeletal muscle beta-tropomyosin (TM). Binding is dramatically reduced when C-terminal amino acids of beta-TM, (TSL, a potential PDZ ligand) are replaced with NNL, NSL or NSA. C.elegans TMs, CeTMI and CeTMIII bind both full length PKL1 and a 40kDa PKL1 fragment that includes the PDZ domain. CeTMI and CeTMIII are also phosphorylated by PKL. CeTMI and CeTMIII lack PDZ ligand sequences at their C-termini. Deletion analysis revealed that 16 C-terminal amino acids of CeTMI are not required for binding with the PDZ-region of PKL. However, deletion of 63 or 113 C-terminal amino acids from CeTMI decreased binding activity by 60 and 80 %, respectively. Immunofluorescence analysis showed that PKL1 and CeTMs are co-clustered in filaments of body wall muscle. Thus, PKLs may be targeted to actin filaments in body wall and pharyngeal muscle via interaction with tropomyosin. Anchored PKL/MAST205 may catalyze phosphorylation of tropomyosin and/or troponin subunits, thereby regulating contraction. Transgenic nematodes expressing anti-sense PKL RNA in muscle are paralyzed at L1 and move slowly at subsequent larval stages. This correlates with a >70% reduction in PKL1 protein. Wild-type PKL1 and "kinase-dead" PKL1 are being selectively expressed in body wall muscle of transgenic nematodes. Effects on C. elegans movement and muscle structure will be assessed in order to obtain additional clues regarding functions of PKL. PKL may be a novel regulator of muscle physiology.