A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

HTT-OMNI: A Web-based Platform for Huntingtin Interaction Exploration and Multi-omics Data Integration.

Huntington's disease (HD) is a progressive neurological disorder that is caused by polyglutamine expansion of the huntingtin (HTT) protein. With the hope to uncover key modifiers of disease, a focus of the field of HD research has been on characterizing HTT-interacting proteins (HIPs) and the effect of the HTT polyglutamine expansion on the cellular omics landscape. However, while hundreds of studies have uncovered over 3000 potential HIPs to date, a means to interrogate these complementary interaction and omics datasets does not exist. The lack of a unified platform for exploring this breadth of potential HIPs and associated omics data represents a substantial barrier toward understanding the impact of HTT polyQ expansion and identifying interactions proximal to HD pathogenesis. Here, we describe the development of a web-based platform called HTT-OMNI (HTT OMics and Network Integration). This application facilitates the visualization and exploration of ∼3400 potential HTT interactors (from the HINT database) and their associated polyQ-dependent omics measurements, such as transcriptome and proteome abundances. Additionally, HTT-OMNI allows for the integration of user-generated datasets with existing HIPs and omic measurements. We first demonstrate the utility of HTT-OMNI for filtering existing HTT PPIs based on a variety of experimental metadata parameters, highlighting its capacity to select for HIPs detected in specific model organisms and tissues. Next, we leverage our application to visualize the relationships between HTT PPIs, genetic disease modifiers, and their multiomic landscape. Finally, we generate and analyze a previously unreported dataset of HTT PPIs, aimed at defining tissue-specific HTT interactions and the polyQ-dependent modulation of their relative stabilities in the cortex and striatum of HD mouse models.

The Nuclear DNA Sensor IFI16 Indiscriminately Binds to and Diminishes Accessibility of the HSV-1 Genome to Suppress Infection.

Human cells identify invading pathogens and activate immune signaling pathways through a wide array of pattern recognition receptors, including DNA sensors. The interferon-inducible protein 16 (IFI16) is a nuclear DNA sensor that recognizes double-stranded DNA from a number of viral sources, including genomes of nuclear-replicating viruses. Among these is the prevalent human pathogen herpes simplex virus 1 (HSV-1). Upon binding to the HSV-1 DNA genome, IFI16 both induces antiviral cytokine expression and suppresses virus gene expression. Here, we used a multiomics approach of DNA sequencing techniques paired with targeted mass spectrometry to obtain an extensive view of the interaction between IFI16 and the HSV-1 genome and how this binding affects the viral DNA structure and protein expression. Through chromatin immunoaffinity purification coupled with next-generation DNA sequencing (ChIP-seq), we found that IFI16 binds to the HSV-1 genome in a sequence-independent manner while simultaneously exhibiting broad enrichment at two loci: UL30, the viral DNA polymerase gene, and US1 to US7. The assay for transposase-accessible chromatin with sequencing (ATAC-seq) revealed that these two regions are among the most accessible stretches of DNA on the genome, thereby facilitating IFI16 binding. Accessibility of the entire HSV-1 genome is elevated upon IFI16 knockout, indicating that expression of IFI16 globally induces chromatinization of viral DNA. Deletion of IFI16 also results in a global increase in the expression of HSV-1 proteins, as measured by parallel reaction monitoring-mass spectrometry of viral proteins representing 80% of the HSV-1 genome. Altogether, we demonstrate that IFI16 interacts with the HSV-1 genome in a sequence-independent manner, coordinating epigenetic silencing of the viral genome and decreasing protein expression and virus replication. IMPORTANCE Mammalian host defense against viral infection includes broad-acting cellular restriction factors, as well as effectors of intrinsic and innate immunity. IFI16 is a critical nuclear host defense factor and intrinsic immune protein involved in binding viral DNA genomes, thereby repressing the replication of nucleus-replicating viruses, including the human herpes simplex virus 1. What has remained unclear is where on the viral genome IFI16 binds and how binding affects both viral DNA structural accessibility and viral protein expression. Our study provides a global view of where and how a nuclear restriction factor of DNA viruses associates with viral genomes to exert antiviral functions during early stages of an acute virus infection. Our study can additionally serve as a systems-level model to evaluate nuclear DNA sensor interactions with viral genomes, as well as the antiviral outcomes of transcriptionally silencing pathogen-derived DNA.

A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.

The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies-herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.

Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity.

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

TRANSPIRE: A Computational Pipeline to Elucidate Intracellular Protein Movements from Spatial Proteomics Data Sets.

Protein localization is paramount to protein function, and the intracellular movement of proteins underlies the regulation of numerous cellular processes. Given advances in spatial proteomics, the investigation of protein localization at a global scale has become attainable. Also becoming apparent is the need for dedicated analytical frameworks that allow the discovery of global intracellular protein movement events. Here, we describe TRANSPIRE, a computational pipeline that facilitates TRanslocation ANalysis of SPatIal pRotEomics data sets. TRANSPIRE leverages synthetic translocation profiles generated from organelle marker proteins to train a probabilistic Gaussian process classifier that predicts changes in protein distribution. This output is then integrated with information regarding co-translocating proteins and complexes and enriched gene ontology associations to discern the putative regulation and function of movement. We validate TRANSPIRE performance for predicting nuclear-cytoplasmic shuttling events. Analyzing an existing data set of nuclear and cytoplasmic proteomes during Kaposi Sarcoma-associated herpesvirus (KSHV)-induced cellular mRNA decay, we confirm that TRANSPIRE readily discerns expected translocations of RNA binding proteins. We next investigate protein translocations during infection with human cytomegalovirus (HCMV), a ß-herpesvirus known to induce global organelle remodeling. We find that HCMV infection induces broad changes in protein localization, with over 800 proteins predicted to translocate during virus replication. Evident are protein movements related to HCMV modulation of host defense, metabolism, cellular trafficking, and Wnt signaling. For example, the low-density lipoprotein receptor (LDLR) translocates to the lysosome early in infection in conjunction with its degradation, which we validate by targeted mass spectrometry. Using microscopy, we also validate the translocation of the multifunctional kinase DAPK3, a movement that may contribute to HCMV activation of Wnt signaling.

Mitochondria and Peroxisome Remodeling across Cytomegalovirus Infection Time Viewed through the Lens of Inter-ViSTA.

Nearly all biological processes rely on the finely tuned coordination of protein interactions across cellular space and time. Accordingly, generating protein interactomes has become routine in biological studies, yet interpreting these datasets remains computationally challenging. Here, we introduce Inter-ViSTA (Interaction Visualization in Space and Time Analysis), a web-based platform that quickly builds animated protein interaction networks and automatically synthesizes information on protein abundances, functions, complexes, and subcellular localizations. Using Inter-ViSTA with proteomics and molecular virology, we define virus-host interactions for the human cytomegalovirus (HCMV) anti-apoptotic protein, pUL37x1. We find that spatiotemporal controlled interactions underlie pUL37x1 functions, facilitating the pro-viral remodeling of mitochondria and peroxisomes during infection. Reciprocal isolations, microscopy, and genetic manipulations further characterize these associations, revealing the interplay between pUL37x1 and the MIB complex, which is critical for mitochondrial integrity. At the peroxisome, we show that pUL37x1 activates PEX11ß to regulate fission, a key aspect of virus assembly and spread.