Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions.

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation.

A single species, Candida albicans, causes half of all invasive fungal infections in humans. Unlike other fungal pathogens, this organism switches between growth as budding yeast and as pseudohyphal and hyphal filaments in host organs and in vitro. Both cell types play a role in invasive disease: while hyphal and pseudohyphal filaments penetrate host cells and tissues, yeast cells are likely to facilitate dissemination through the bloodstream and establishment of distant foci of infection. Many regulators of the yeast-to-hypha switch have emerged from intensive investigations of this morphogenetic step, but the hypha-to-yeast switch remains poorly understood. Using a forward genetic approach, a novel putative regulator involved in the hypha-to-yeast switch was identified, the C. albicans pescadillo homolog, PES1. In eukaryotes from yeast to human, pescadillo homologs are involved in cell cycle control and ribosome biogenesis, and are essential. We find a pescadillo homolog to act in fungal morphogenesis, specifically in lateral yeast growth on filamentous cells. We also find essentiality of PES1 in C. albicans to be dependent on cell type, because hyphal cells, but not yeast cells, tolerate its loss. PES1 is therefore critical for completion of the C. albicans life cycle, in which the fungus switches between filamentous and yeast growth. Consistent with these in vitro findings, PES1 is required for C. albicans virulence in an in vivo insect model of infection.

Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity.

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.