Burger, Julien et al. (2009) International Worm Meeting "Functional characterization of a novel cullin-based E3-ligase involved in cell cycle progression in C. elegans."

Richaudeau, Benedicte, Pintard, Lionel, Merlet, Jorge, Burger, Julien

[International Worm Meeting, 2009]

The COP9 signalosome (CSN) is an evolutionary conserved macromolecular complex that interacts with Cullin-RING ubiquitin-Ligases (CRLs) and regulates their activity by hydrolyzing cullin-Nedd8 conjugates. In particular, the CSN sequesters inactive CRL4Ddb2 ubiquitin-ligase, which rapidly dissociates from the CSN upon DNA damage. Whether other CRLs are similarly sequestered and regulated by the CSN is currently unknown. We have recently defined the protein interaction network of the mammalian CSN using a combination of affinity purification tagging and sensitive tandem mass spectrometry approaches (LC-MS/MS). Notably we identified a small subset of CRLs, which stably interact with the CSN, and thus might be specifically activated by dissociation from the CSN in response to specific cues, such as DNA damage. Consistent with this hypothesis, most of the CSN-interacting CRLs identified in this study appear to be involved in cell cycle progression and DNA metabolism; however, the function of others remains elusive. We have identified worm orthologues of most of these CRLs and we are now using C. elegans as a model system to dissect their function during cell cycle progression. In particular, we are currently focusing on a CUL-2-based ubiquitin-ligase that uses F33G12.4/LRR-1 as a substrate-recognition subunit. LRR-1 contains a typical BC/CUL-2 box and binds CRL2 components in vitro and in vivo. RNAi-mediated depletion of lrr-1 results in an embryonic lethal phenotype, with severe delays in S-phase, as revealed by time-lapse video-microscopy. Importantly, this delay is entirely suppressed by inactivation of the DNA replication checkpoint, suggesting that the CRL2LRR-1 complex might control DNA replication in the early embryo. Consistent with this hypothesis, lrr-1(RNAi) treated embryos are hypersensitive to sublethal doses of the replication inhibitor hydroxyurea (HU) and the DNA-damaging agent methyl methanesulfonate (MMS). Experiments are under way to elucidate the function of this ubiquitin-ligase and to identify its critical substrate(s).

Merlet, Jorge et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The CRL2LRR-1 ubiquitin-ligase regulates cell cycle progression during C. elegans development"

Merlet, Jorge, Gomes, Jose-Eduardo, Tavernier, Nicolas, Pintard, Lionel, Burger, Julien, Richaudeau, Benedicte

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In early C. elegans embryos, the ATR (ataxia telangectasia mutated and rad3 related) checkpoint pathway controls the timing of cell division in the P lineage, or future germ line of the animal. However, the mechanisms regulating the ATR checkpoint pathway are still poorly understood. Here, we establish that the Leucine Rich Repeats protein, LRR-1, promotes cell cycle progression during development in C. elegans , both in the adult germ line and in the early embryo. Our results indicate that LRR-1 is a substrate-recruitment factor of a cullin 2-RING E3-ligase (CRL2), which down regulates the ATR signaling pathway in C. elegans . LRR-1 contains a typical BC/Cul2-box through which it binds CRL2 components in vitro and in vivo . Lo ss of lrr-1 function causes a cell-cycle arrest in the mitotic region of the germ line resulting in sterility owing to depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATR/ATL-1 and Chk1/CHK-1 suppresses this cell cycle arrest and fully restores lrr-1 mutant fertility. Likewise, in early embryos, lrr-1 inactivation leads to hyper-activation of the ATL-1/Chk1 pathway, which delays mitotic entry and causes embryonic lethality. Our findings suggest a model in which the CRL2 LRR-1 E3 ligase negatively regulates the DNA replication checkpoint and thereby coordinates cell cycle progression and development.

Gomes, Jose-Eduardo et al. (2009) International Worm Meeting "The protein-phosphatase 4 subunit PPFR-1 is a potential activator of MEI-1/Katanin."

Gomes, Jose-Eduardo, Formstecher, Etienne, Mains, Paul E., Han, Xue, Pintard, Lionel, Richaudeau, Benedicte

[International Worm Meeting, 2009]

Upon fertilization the C. elegans oocyte resumes and completes meiosis, and twenty minutes latter the first embryonic mitosis takes place. Despite sharing the same cytoplasm, the meiotic and mitotic spindles are radically different: while the small asterless meiotic spindle assembles close to the cell cortex, the mitotic spindle is large, with robust asters, and is positioned in the center of the embryo. MEI-1/Katanin is a microtubule-severing protein essential for meiotic spindle formation; however its activity is incompatible with the assembly of a fully functional mitotic spindle. In wild-type embryos, MEI-1 is inactivated at the meiosis-to-mitosis transition allowing for mitotic spindle assembly. We have previously established that, after meiosis, a Cullin-3-based E3-ligase targets MEI-1 for ubiquitin-mediated proteasomal degradation. MEI-1 is recruited by the CUL-3-based complex through the adaptor protein MEL-26. In order to identify new components of the pathway, we conducted a Yeast Two-Hybrid screen using MEL-26 as bait. In this screen we identified PPFR-1, a regulatory subunit of the serine/threonine phosphatase PP4 complex. Loss-of-function of ppfr-1, through both RNA-interference or a putative null allele, suppresses the lethality of ectopic MEI-1 expression during mitosis, either due to mel-26 loss-of-function or a to mei-1 gain-of-function allele encoding a protein refractory to MEL-26 mediated degradation. This suppression suggests the PP4 phosphatase is an activator of MEI-1. In addition, the ppfr-1 putative null allele displays a partly penetrant meiosis failure phenotype, again consistent with PP4 being an activator of MEI-1. To test whether PP4 modifies MEI-1 protein in vivo, we performed 2D gels on embryonic extracts; we were able to show that MEI-1 is phosophorylated in vivo and that these modifications are dependent on PPFR-1. Our results also show that PPFR-1 regulates MEI-1 independently of protein degradation. We are currently testing whether PPFR-1 is targeted for degradation by the CUL-3MEL-26 ligase at the meiosis-to-mitosis transition.