Sakai Y et al. (2021) J Neurosci "BRCA1-BARD1 regulates axon regeneration in concert with the Gq-DAG signaling network."

Pastuhov SI, Matsumoto K, Hanafusa H, Shimizu T, Hisamoto N, Sakai Y

[J Neurosci, 2021]

The breast cancer susceptibility protein BRCA1 and its partner BARD1 form an E3 ubiquitin ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1-BARD1 in post-mitotic cells, such as neurons, remain poorly defined. Here we report that BRC-1 and BRD-1, the <i>Caenorhabditis elegans</i> orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gq-diacylglycerol (DAG) signaling pathway. This pathway is down-regulated by DAG kinase (DGK), which converts DAG to phosphatidic acid. We demonstrate that inactivation of DGK-3 suppresses the <i>brc-1 brd-1</i> defect in axon regeneration, suggesting that BRC-1-BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1-BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1-BRD-1 complex regulates axon regeneration in concert with the Gq-DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.<b>Significance Statement</b>BRCA1-BARD1 is an E3 ubiquitin ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1-BARD1 in post-mitotic cells, such as neurons, remain poorly defined. We show here that <i>C. elegans</i> BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase DGK-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1-BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1-BARD1 in fully-differentiated neurons.

Sakai Y et al. (2019) EMBO Rep "TDP2 negatively regulates axon regeneration by inducing SUMOylation of an Ets transcription factor."

Hisamoto N, Hanafusa H, Shimizu T, Sakai Y, Matsumoto K, Li C, Pastuhov SI

[EMBO Rep, 2019]

In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1. Here, we find that svh-14/mxl-1, a gene encoding a Max-like transcription factor, is required for activation of svh-2 expression in response to axonal injury. We show that MXL-1 binds to and inhibits the function of TDPT-1, a C.elegans homolog of mammalian tyrosyl-DNA phosphodiesterase 2 [TDP2; also called Ets1-associated protein II (EAPII)]. Deletion of tdpt-1 suppresses the mxl-1 defect, but not the ets-4 defect, in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, which inhibits ETS-4 transcriptional activity, and MXL-1 counteracts this effect. Thus, TDPT-1 interacts with two different transcription factors in axon regeneration.

Sakai Y et al. (2022) EMBO Rep "Histidine dephosphorylation of the Gβ protein GPB-1 promotes axon regeneration in C. elegans"

Sakai Y, Hisamoto N, Hanafusa H, Matsumoto K

[EMBO Rep, 2022]

Histidine phosphorylation is an emerging noncanonical protein phosphorylation in animals, yet its physiological role remains largely unexplored. The protein histidine phosphatase (PHPT1) was recently identified for the first time in mammals. Here, we report that PHIP-1, an ortholog of PHPT1 in Caenorhabditis elegans, promotes axon regeneration by dephosphorylating GPB-1 G&#946; at His-266 and inactivating GOA-1 Go&#945; signaling, a negative regulator of axon regeneration. Overexpression of the histidine kinase NDK-1 also inhibits axon regeneration via GPB-1 His-266 phosphorylation. Thus, His-phosphorylation plays an antiregenerative role in C. elegans. Furthermore, we identify a conserved UNC-51/ULK kinase that functions in autophagy as a PHIP-1-binding protein. We demonstrate that UNC-51 phosphorylates PHIP-1 at Ser-112 and activates its catalytic activity and that this phosphorylation is required for PHIP-1-mediated axon regeneration. This study reveals a molecular link from ULK to protein histidine phosphatase, which facilitates axon regeneration by inhibiting trimeric G protein signaling.

Sakai Y et al. (2023) PLoS Genet "Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans."

Tsunekawa M, Hisamoto N, Shimizu T, Matsumoto K, Sakai Y

[PLoS Genet, 2023]

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.

Sakai Y et al. (2021) J Neurosci "The integrin signaling network promotes axon regeneration via the Src-ephexin-RhoA GTPase signaling axis."

Pastuhov SI, Matsumoto K, Hisamoto N, Tsunekawa M, Ohta K, Shimizu T, Sakai Y, Hanafusa H

[J Neurosci, 2021]

Axon regeneration is an evolutionarily conserved process essential for restoring the function of damaged neurons. In <i>Caenorhabditis elegans</i> hermaphrodites, initiation of axon regeneration is regulated by the RhoA GTPase-ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain phosphorylation signaling pathway. However, the upstream mechanism that activates the RhoA pathway remains unknown. Here, we show that axon injury activates TLN-1/talin via the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade and that TLN-1 induces multiple downstream events, one of which is integrin inside-out activation, leading to the activation of the RhoA-ROCK signaling pathway. We found that the non-receptor tyrosine kinase Src, a key mediator of integrin signaling, activates the Rho guanine nucleotide exchange factor (GEF) EPHX-1/ephexin by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the <i>C. elegans</i> integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.<b>Significance Statement</b>The ability of axons to regenerate after injury is governed by cell-intrinsic regeneration pathways. We have previously demonstrated that the <i>C. elegans</i> RhoA GTPase-ROCK pathway promotes axon regeneration by inducing MLC-4 phosphorylation. In this study, we found that axon injury activates TLN-1/talin through the cAMP-Epac-Rap GTPase cascade, leading to integrin inside-out activation, which promotes axonal regeneration by activating the RhoA signaling pathway. In this pathway, SRC-1/Src acts downstream of integrin activation and subsequently activates EPHX-1/ephexin RhoGEF by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the <i>C. elegans</i> integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.

Sakai, Y. et al. (2019) International Worm Meeting "C. elegans TDP2 homolog negatively regulates axon regeneration by inducing SUMOylation of an Ets transcription factor."

Hanafusa, H., Shimizu, T., Li, C., Matsumoto, K., Pastuhov, S., Sakai, Y., Hisamoto, N.

[International Worm Meeting, 2019]

In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively. In this study, we found that svh-14/mxl-1, a gene encoding a Max-like transcription factor, activates svh-2 expression in response to axon injury. Furthermore, we identified TDPT-1, a C. elegans homolog of mammalian Tyrosyl-DNA Phosphodiesterase 2 [TDP2; also called Ets1-Associated Protein II (EAPII)] as an MXL-1-binding protein. We further show that deletion of tdpt-1 suppresses the mxl-1 defect but not the ets-4 defect in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, resulting in the inhibition of its transcriptional activity. Thus, TDPT-1 interacts with two different transcription factors and modulates their function in axon regeneration.

Perreault J et al. (2007) Mol Biol Evol "Ro-associated Y RNAs in metazoans: evolution and diversification."

Perreault J, Perreault JP, Boire G

[Mol Biol Evol, 2007]

The Y genes encode small non-coding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.

Yamaguchi M et al. (2016) Biochim Biophys Acta "NF-Y in invertebrates."

Yoshida H, Ly LL, Yamaguchi M, Ali MS, Yoshioka Y

[Biochim Biophys Acta, 2016]

BothDrosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of egl-5 and tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized.

Burns RG (1996) Trends in Cell Biology "Reply: Defining the gamma-, delta- and epsilon-tubulins."

Burns RG

[Trends in Cell Biology, 1996]

Keeling and Logsdon propose that the y-like sequences from Caenorhabditis elegans and Saccharomyces cerevisiae are bona fide y-tubulins that have undergone rapid evolutionary divergence. Indeed, genetic and localization studies with the yeast epsilon-tubulin (encoded by the TUB4 gene) reveal striking similarities to the bona fide y-tubulins, whereas there is no apparent human analogue to the C. elegans delta-tubulin among the 60 available human y-tubulin expressed-sequence tags. (ESTs).

Gardiner TJ et al. (2009) RNA "A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication."

Langley AR, Gardiner TJ, Krude T, Christov CP

[RNA, 2009]

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.