Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

PROBER identifies proteins associated with programmable sequence-specific DNA in living cells.

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study.

BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response. METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naïve individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination. RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P < 0.001), sphingosine 1-phsophate (S1P) receptor modulators (P < 0.001), mycophenolate (P = 0.002), and B cell lymphoma (P = 0.004); those associated with low cellular response rates included S1P receptor modulators (P < 0.001) and mycophenolate (P < 0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination. CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2. AUTHOR SUMMARY: SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.

SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity.

SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.

A Study on Outcome of Myringoplasty in Dry Ear (Quiescent/Inactive CSOM) Without Using Gelfoam in Middle Ear.

Myringoplasty is a common surgery done for patients with tympanic membrane perforation in our clinical practice. This study was done to know the outcome of myringoplasty done without using gelfoam as a scaffold material. Simultaneously other parameters influencing the outcome were analysed. In a total of 80 patients assessed over 25 months were divided into test and control group by quasi randomisation. All patients underwent endoscopic underlay myringoplasty. Control groups had gelfoam bed created with gelfoam, whereas the test group only middle ear air pocket created with a proper seal without using gelfoam. The study revealed that without using gelfoam and creating a good middle ear air pocket the results were comparable to the method of using middle ear gelfoam bed. Also few insights and understanding regarding the parameters we need to asses preoperatively were also observed. With comparable healing and hearing results without using gelfoam in the middle ear, the normal physiology is restored in the postoperative period much earlier. The result of the surgery also becomes evident in the second postoperative week when the use of gelfoam in the middle ear is avoided.

Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

Author Correction: Methods to study RNA-protein interactions.

In the version of this paper originally published, three references were accidentally omitted: Schwartz, J. C. et al. Cell Rep. 5, 918-925 (2013); Tundup, S. et al. FEBS Lett. 580, 1285-1293 (2006); and Itri, F. et al. Biochem. Biophys. Res. Commun. 492, 67-73 (2017). The PDF and HTML versions of the paper now include these as references 58, 59, and 60, respectively, and subsequent references have been renumbered accordingly.

Methods to study RNA-protein interactions.

Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.

Profiling of rotavirus 3'UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication.

Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA-protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3' terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3'UTR consensus sequences. During RV infection, ATP5B bound to the RV 3'UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.

RNA-protein interaction detection in living cells.

RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.