A genome-wide CRISPR screen reconciles the role of N-linked glycosylation in galectin-3 transport to the cell surface.

Galectins are a family of lectin binding proteins expressed both intracellularly and extracellularly. Galectin-3 (Gal-3, also known as LGALS3) is expressed at the cell surface; however, Gal-3 lacks a signal sequence, and the mechanism of Gal-3 transport to the cell surface remains poorly understood. Here, using a genome-wide CRISPR/Cas9 forward genetic screen for regulators of Gal-3 cell surface localization, we identified genes encoding glycoproteins, enzymes involved in N-linked glycosylation, regulators of ER-Golgi trafficking and proteins involved in immunity. The results of this screening approach led us to address the controversial role of N-linked glycosylation in the transport of Gal-3 to the cell surface. We find that N-linked glycoprotein maturation is not required for Gal-3 transport from the cytosol to the extracellular space, but is important for cell surface binding. Additionally, secreted Gal-3 is predominantly free and not packaged into extracellular vesicles. These data support a secretion pathway independent of N-linked glycoproteins and extracellular vesicles.

A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway.

A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3+ reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.

The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Membrane-Proximal Epitope Facilitates Efficient T Cell Synapse Formation by Anti-FcRH5/CD3 and Is a Requirement for Myeloma Cell Killing.

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens.

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.