Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote.

Plk1 (polo-like kinase 1) is an evolutionarily conserved serine/threonine kinase instrumental for mitotic entry and progression. Beyond these canonical functions, Plk1 also regulates cell polarization and cell fate during asymmetric cell divisions in C. elegans and D. melanogaster. Plk1 contains a specialized phosphoserine-threonine binding domain, the polo-box domain (PBD), which localizes and concentrates the kinase at its various sites of action within the cell in space and time. Here we present protocols to express and purify the C. elegans Plk1 kinase along with biochemical and phosphoproteomic approaches to interrogate the PBD interactome and to dissect Plk1 substrate interactions. These protocols are most suitable for the identification of Plk1 targets in C. elegans embryos but can be easily adapted to identify and study Plk1 substrates from any source."

Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) in C. elegans embryos.

The nuclear envelope, which protects and organizes the interphase genome, is dismantled during mitosis. In the C. elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the parental genomes. During NEBD, Nuclear Pore Complex (NPC) disassembly is critical for rupturing the nuclear permeability barrier and removing the NPCs from the membranes near the centrosomes and between the juxtaposed pronuclei. By combining live imaging, biochemistry, and phosphoproteomics, we characterized NPC disassembly and unveiled the exact role of the mitotic kinase PLK-1 in this process. We show that PLK-1 disassembles the NPC by targeting multiple NPC sub-complexes, including the cytoplasmic filaments, the central channel, and the inner ring. Notably, PLK-1 is recruited to and phosphorylates intrinsically disordered regions of several multivalent linker nucleoporins, a mechanism that appears to be an evolutionarily conserved driver of NPC disassembly during mitosis. (149/150 words). One-Sentence Summary: PLK-1 targets intrinsically disordered regions of multiple multivalent nucleoporins to dismantle the nuclear pore complexes in the C. elegans zygote.

Cortical microtubule pulling forces contribute to the union of the parental genomes in the Caenorhabditis elegans zygote.

Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single Caenorhabditis elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required to form a pronuclear envelope scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera et al., 2020). Here, we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation, and conversely, nuclear envelopes remodeling facilitates spindle elongation. We also demonstrate that weakening the pronuclear envelopes via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly.

Life of sexually reproducing organisms starts with the fusion of the haploid egg and sperm gametes to form the genome of a new diploid organism. Using the newly fertilized Caenorhabditis elegans zygote, we show that the mitotic Polo-like kinase PLK-1 phosphorylates the lamin LMN-1 to promote timely lamina disassembly and subsequent merging of the parental genomes into a single nucleus after mitosis. Expression of non-phosphorylatable versions of LMN-1, which affect lamina depolymerization during mitosis, is sufficient to prevent the mixing of the parental chromosomes into a single nucleus in daughter cells. Finally, we recapitulate lamina depolymerization by PLK-1 in vitro demonstrating that LMN-1 is a direct PLK-1 target. Our findings indicate that the timely removal of lamin is essential for the merging of parental chromosomes at the beginning of life in C. elegans and possibly also in humans, where a defect in this process might be fatal for embryo development.

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans.

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.

Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy.

Autophagy, the process by which proteins or organelles are engulfed by autophagosomes and delivered for vacuolar/lysosomal degradation, is induced to ensure survival under starvation and other stresses. A selective autophagic pathway for 60S ribosomal subunits elicited by nitrogen starvation in yeast-ribophagy-was recently described and requires the Ubp3-Bre5 deubiquitylating enzyme. This discovery implied that an E3 ligases act upstream, whether inhibiting the process or providing an initial required signal. In this paper, we show that Ltn1/Rkr1, a 60S ribosome-associated E3 implicated in translational surveillance, acts as an inhibitor of 60S ribosomal subunit ribophagy and is antagonized by Ubp3. The ribosomal protein Rpl25 is a relevant target. Its ubiquitylation is Ltn1 dependent and Ubp3 reversed, and mutation of its ubiquitylation site rendered ribophagy less dependent on Ubp3. Consistently, the expression of Ltn1-but not Ubp3-rapidly decreased after starvation, presumably to allow ribophagy to proceed. Thus, Ltn1 and Ubp3-Bre5 likely contribute to adapt ribophagy activity to both nutrient supply and protein translation.

Cdc48/p97, a key actor in the interplay between autophagy and ubiquitin/proteasome catabolic pathways.

The AAA-ATPase Cdc48/p97 controls a large array of cellular functions including protein degradation, cell division, membrane fusion through its ability to interact with and control the fate of ubiquitylated proteins. More recently, Cdc48/p97 also appeared to be involved in autophagy, a catabolic cell response that has long been viewed as completely distinct from the Ubiquitine/Proteasome System. In particular, conjugation by ubiquitin or ubiquitin-like proteins as well as ubiquitin binding proteins such as Cdc48/p97 and its cofactors can target degradation by both catabolic pathways. This review will focus on the recently described functions of Cdc48/p97 in autophagosome biogenesis as well as selective autophagy.

The Rsp5 ubiquitin ligase and the AAA-ATPase Cdc48 control the ubiquitin-mediated degradation of the COPII component Sec23.

Ubp3/Bre5 complex is a substrate-specific deubiquitylating enzyme which mediates deubiquitylation of Sec23, a component of the COPII complex involved in the transport between endoplasmic reticulum and Golgi apparatus. Here we show that ubiquitylation of Sec23 is controlled by the Rsp5 ubiquitin ligase both in vivo and in vitro. We have recently identified Cdc48, a chaperone-like that plays a key role in the proteasomal escort pathway, as a partner of the Ubp3/Bre5 complex. We now found that cdc48 thermosensitive mutant cells not only accumulate ubiquitylated form of Sec23 but also display a stabilization of this protein at the restrictive temperature. This indicates that Cdc48 controls the proteasome-mediated degradation of Sec23. Our data favor the idea that Cdc48 plays a key role in deciphering fates of ubiquitylated Sec23 to degradation or deubiquitylation/stabilization via its cofactors.

Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy.

Ubiquitin-dependent processes can be antagonized by substrate-specific deubiquitination enzymes involved in many cellular functions. In this study, we show that the yeast Ubp3-Bre5 deubiquitination complex interacts with both the chaperone-like Cdc48, a major actor of the ubiquitin and proteasome system, and Ufd3, a ubiquitin-binding cofactor of Cdc48. We observed that these partners are required for the Ubp3-Bre5-dependent and starvation-induced selective degradation of yeast mature ribosomes, also called ribophagy. By contrast, proteasome-dependent degradation does not participate in this process. Our data favour the idea that these factors cooperate to recognize and deubiquitinate specific substrates of ribophagy before their vacuolar degradation.

Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme.

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3-Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped beta-strand-turn-alpha-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel beta-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3-Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.

Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription.

The mRNA nuclear export receptor Mex67/Mtr2 is recruited to mRNAs through RNA-binding adaptors, including components of the THO/TREX complex that couple transcription to mRNA export. Here we show that the ubiquitin-associated (UBA) domain of Mex67 is not only required for proper nuclear export of mRNA but also contributes to recruitment of Mex67 to transcribing genes. Our results reveal that the UBA domain of Mex67 directly interacts with polyubiquitin chains and with Hpr1, a component of the THO/TREX complex, which is regulated by ubiquitylation in a transcription-dependent manner. This interaction transiently protects Hpr1 from ubiquitin/proteasome-mediated degradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early messenger ribonucleoproteins assembly.

Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor.

The Bre5 protein is a cofactor for the deubiquitinating enzyme Ubp3, and it contains a nuclear transfer factor 2 (NTF2)-like protein recognition module that is essential for Ubp3 activity. In this study, we report the x-ray crystal structure of the Bre5 NTF2-like domain and show that it forms a homodimeric structure that is similar to other NTF2-like domains, except for the presence of an intermolecular disulfide bond in the crystals. Sedimentation equilibrium studies reveal that under non-reducing conditions, the Bre5 NTF2-like domain is exclusively dimeric, whereas a disulfide bond-deficient mutant undergoes a monomer-dimer equilibrium with a dissociation constant in the midnanomolar range, suggesting that dimer formation and possibly also disulfide bond formation may modulate Bre5 function in vivo. Using deletion analysis, we also identify a novel N-terminal domain of Ubp3 that is necessary and sufficient for interaction with Bre5 and use isothermal titration calorimetry to show that Bre5 and Ubp3 form a 2:1 complex, in contrast to other reported NTF2-like domain/protein interactions that form 1:1 complexes. Finally, we employ structure-based mutagenesis to map the Ubp3 binding surface of Bre5 to a region near the Bre5 dimer interface and show that this binding surface of Bre5 is important for Ubp3 function in vivo. Together, these studies provide novel insights into protein recognition by NTF2-like domains and provide a molecular scaffold for understanding how Ubp3 function is regulated by Bre5 cofactor binding.

The mRNA nuclear export factor Hpr1 is regulated by Rsp5-mediated ubiquitylation.

Ubiquitin conjugation and in particular two distinct HECT ubiquitin ligases, Rsp5p and Tom1p, have been shown to participate in the regulation of mRNA export in Saccharomyces cerevisiae. The identification of the ubiquitin ligase substrates represents a major challenge in understanding how this modification may modulate mRNA export. Here, we identified Hpr1p, a member of the THO/TREX (transcription/export) complex that couples mRNA transcription to nuclear export as a target of the ubiquitin-proteasome pathway. Hpr1p degradation is enhanced at high temperature and appears linked to on-going RNA-polymeraseII-mediated transcription. Interestingly, the stability of the other THO complex components is not affected under these conditions indicating that Hpr1p turnover could control the formation of the THO/TREX complex and consequently mRNA export. Using in vivo and in vitro approaches we demonstrate that Rsp5p is responsible for the ubiquitylation of Hpr1p that also involves the ubiquitin-conjugating enzyme Ubc4p. Thus, Hpr1p represents the first nuclear export factor regulated by ubiquitylation, strongly suggesting that this post-translational modification participates in the coordination of transcription and mRNA export processes.

Minihelix-containing RNAs mediate exportin-5-dependent nuclear export of the double-stranded RNA-binding protein ILF3.

The karyopherin-related nuclear transport factor exportin-5 preferentially recognizes and transports RNAs containing minihelix motif, a structural cis-acting export element that comprises a double-stranded stem (>14 nucleotides) with a base-paired 5' end and a 3-8-nucleotide protruding 3' end. This structural motif is present in various small cellular and viral polymerase III transcripts such as the adenovirus VA1 RNA (VA1). Here we show that the double-stranded RNA-binding protein, ILF3 (interleukin enhancer binding factor 3) preferentially binds minihelix motif. Gel retardation assays and glutathione S-transferase pull-down experiments revealed that ILF3, exportin-5, RanGTP, and VA1 RNA assembled in a quaternary complex in which the RNA moiety bridges the interaction between ILF3 and exportin-5. Formation of this complex is facilitated by the ability of both exportin-5 and ILF3 to mutually increase their apparent affinity for VA1 RNA. Using microinjection in the nucleus of HeLa cells and transfection experiments, we show here that formation of the cooperative RanGTP-dependent RNA/ILF3/exportin-5 complex promotes the co-transport of VA1 and ILF3 from the nucleus to the cytoplasm. Exportin-5 thus appears as the first example of a nuclear export receptor that mediates RNA export but also promotes transport of proteinaceous cargo through appropriate and specific RNA adaptors.

Exportin-5 mediates nuclear export of minihelix-containing RNAs.

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt protruding 3' end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.