The RNA exosome is a key 3’−5’ exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

The balance between cell survival and cell death is often lost in human pathologies such as inflammation and cancer. Autophagy plays a critical role in cell survival: essential nutrients are generated by autophagy-dependent degradation and recycling of cellular garbage. On the other hand, cell death is induced by different programs, such as apoptosis, pyroptosis, and necroptosis. Emerging evidence is revealing how cell survival and cell death pathways are coordinated to determine cell fate. For instance, posttranslational modification of proteins with ubiquitin regulates many steps of autophagy and cell death pathways. In this review article, we will discuss how the ubiquitin system influences cell death and autophagy.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-jB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated Hoip K778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, Hoip K778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-jB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in Hoip K778R/K778R mice, which is rescued by knockout of TNFR1. We propose that sitespecific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.

◥HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here, we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRas G12D -driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1 À/À lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1-deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRas G12D -driven lung tumors in Hace1 À/À mice. In patients with lung cancer, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, whereas elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development.

21The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure 22The Ski complex consists of the MTR4-related RNA helicase SKI2, the tetratricopeptide 1 repeat protein SKI3 and two copies of the WD40-repeat protein SKI8 [31][32][33] . Association of the 2 Ski complex with the exosome core complex requires an additional protein, SKI7 34 . In yeast, 3 the genes encoding Ski7 and Hbs1, which binds to Dom34 (PELOTA in humans and plants) 4for the release of stalled ribosomes 35 , arose from a whole genome duplication event. In other 5 eukaryotes including mammals and plants, the SKI7 and HBS1 proteins are produced by 6 alternative splicing from a single locus [36][37][38] . 7The Ski complex is conserved in Arabidopsis thaliana 39 , but its physical association with 8 the exosome core has not been investigated yet. An initial experiment to affinity-capture 9 factors associated with the Arabidopsis exosome identified the homologue of DIS3 and two 10 nuclear RNA helicases, AtMTR4 and its closely related homologue HEN2 23 . In addition, 11Arabidopsis Exo9 systematically co-purified with a 1840 amino acid ARM repeat protein of 12 unknown molecular function named RESURRECTION 1 (RST1) 23 . RST1 was originally 13identified in a genetic screen for factors involved in the biosynthesis of epicuticular waxes 40 . 14 Epicuticular waxes are a protective layer of aliphatic very long chain (VLC) hydrocarbons 15 that cover the outer surface of land plants 41,42 . rst1 mutants have less wax on floral stems than 16 wild-type plants, and about 70% of the seeds produced by rst1 mutants are shrunken due to 17 aborted embryogenesis 40 . The molecular function of RST1 remains unknown. Interestingly, 18 one of the two RRP45 exosome core subunits encoded in the Arabidopsis genome, named 19 RRP45B or CER7 (for ECERIFERUM 7) was also identified in a genetic screen aimed at 20 identifying enzymes or regulators of wax biosynthesis 43 . The wax-deficient phenotype of 21 rrp45b/cer7 mutants (cer7 from now on) is suppressed by mutations in genes encoding RNA 22

Summary The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIP, HOIL-1L, and SHARPIN, promotes tumor necrosis factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice ( Hoil-1l T201A;R208A/T201A;R208A ) expressing a HOIL-1L NZF mutant and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production, and liver damage markers. Cells derived from Hoil-1l T201A;R208A/T201A;R208A mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that HOIL-1L NZF cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo .

The Linear Ubiquitin Assembly Complex (LUBAC), composed of HOIP, HOIL-1L and SHARPIN, promotes Tumor Necrosis Factor (TNF)-dependent NF-kB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological role in vivo has remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT20;A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant, and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production and liver damage markers. Cells derived from the HOIL-1L knockin mice show reduced TNF-dependent NF-kB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that the HOIL-1L-NZF domain cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammation in vivo.