Decreased mechanotransduction prevents nuclear collapse in a Caenorhabditis elegans laminopathy.

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

Control of oocyte meiotic maturation in C. elegans.

In virtually all sexually reproducing animals, oocytes arrest in meiotic prophase and resume meiosis in a conserved biological process called meiotic maturation. Meiotic arrest enables oocytes, which are amongst the largest cells in an organism, to grow and accumulate the necessary cellular constituents required to support embryonic development. Oocyte arrest can be maintained for a prolonged period, up to 50 years in humans, and defects in the meiotic maturation process interfere with the faithful segregation of meiotic chromosomes, representing the leading cause of human birth defects and female infertility. Hormonal signaling and interactions with somatic cells of the gonad control the timing of oocyte meiotic maturation. Signaling activates the CDK1/cyclin B kinase, which plays a central role in regulating the nuclear and cytoplasmic events of meiotic maturation. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation whereas cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is less well understood. Classically, meiotic maturation has been studied in organisms with large oocytes to facilitate biochemical analysis. Recently, the nematode Caenorhabditis elegans is emerging as a genetic paradigm for studying the regulation of oocyte meiotic maturation. Studies in this system have revealed conceptual, anatomical, and molecular links to oocytes in all animals including humans. This review focuses on the signaling mechanisms required to control oocyte growth and meiotic maturation in C. elegans and discusses how the downstream regulation of protein translation coordinates the completion of meiosis and the oocyte-to-embryo transition.

DPFF-1 transcription factor deficiency causes the aberrant activation of MPK-1 and meiotic defects in the Caenorhabditis elegans germline.

The d4 family of transcription factors consists of three members in mammals. DPF1/neuro-d4 is expressed mainly in neurons and the peripheral nervous system, and is important for brain development. DPF2/requiem/ubi-d4 is expressed ubiquitously and presumably functions as an apoptotic factor, especially during the deprivation of trophic factors. DPF3/cer-d4 is expressed in neurons and in the heart, and is important for heart development and function in zebrafish. In Drosophila, there is only one member, dd4, whose function is still unknown, but it is expressed in many tissues and is particularly abundant in the brain of developing embryos and in adults. Here, we present DPFF-1, the only member of this family of proteins in the nematode C. elegans. DPFF-1 is similar to its mammalian homolog DPF2/requiem/ubi-d4 because it is ubiquitously expressed during embryogenesis and in adult tissues, and because it is important for the induction of germ cell apoptosis during stress. Here, we show that dpff-1 null mutant animals produce less progeny than wild-type nematodes, presumably due to meiotic defects. Gonads of dpff-1 deficient animals showed more germ cells in pachytene and overexpressed the P-MPK-1 signal. Additionally, these animals presented higher levels of p53-induced germ cell apoptosis than wild-type animals. Furthermore, we observed that dpff-1 deficient animals are more sensitive to heat shock. This is the first report showing that the d4 family of transcription factors could be involved in meiosis and stress protection.

IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes.

In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

Multiple Mechanisms Inactivate the LIN-41 RNA-Binding Protein To Ensure a Robust Oocyte-to-Embryo Transition in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1 Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF (Skp1, Cul1, and F-box protein)-type E3 ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two nonoverlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sequences is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte messenger RNA targets. Based on these observations, we propose a model for the elimination of LIN-41 by the SEL-10 E3 ubiquitin ligase and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

LIN-41 and OMA Ribonucleoprotein Complexes Mediate a Translational Repression-to-Activation Switch Controlling Oocyte Meiotic Maturation and the Oocyte-to-Embryo Transition in Caenorhabditis elegans.

An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined messenger RNAs (mRNAs) associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1 Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 which encode the RNA regulators SPN-4 and MEG-1 We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.

The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures.