Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

lactose permease ͉ major facilitator superfamily ͉ single-molecule spectroscopy ͉ transporters T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily, utilizes free energy stored in an electrochemical H ϩ gradient (⌬ Hϩ ) to drive active transport by coupling the downhill, stoichiometric translocation of H ϩ with ⌬ Hϩ to the uphill accumulation of galactopyranosides. Conversely, in the absence of ⌬ Hϩ , LacY utilizes free energy released from downhill translocation of galactosides in either direction to drive uphill translocation of H ϩ with generation of ⌬ Hϩ (reviewed in ref.

We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. the microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

Mehta et al. uncover a novel role for miR-212/132 as a regulator of early B cell development.

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs. (A) Outline of the RAP-MS procedure used to identify RNA-binding proteins on transcripts of interest. (B) TMT ratio (Irf7/ActB) for proteins identified as enriched on either Irf7 (TMT ratio >1) or ActB (TMT ratio <1) transcripts. (C) qRT-PCR analysis of transcripts captured via RAP for Irf7 (blue) and ActB (gold) probes. (D) RIP followed by qRT-PCR for Irf7 and Rpl32 in TNF-a-stimulated BMDMs. Shown is the relative enrichment of transcripts captured in BUD13 RIP as compared to rabbit IgG RIP. (E) Same as (D), except stimulation with poly(I:C). Data are representative of two independent experiments (C-E) and presented as mean (error bars indicate SD). *p < 0.05, **p < 0.01 and ***p < 0.001, Student's t test.

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4+ and CD8+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity.

Single-molecule fluorescence resonance energy transfer (smFRET) has been widely applied to the study of fluorescently labeled biomolecules on surfaces and in solution. Sorting single molecules based on fluorescent dye stoichiometry provides one with further layers of information and also enables "filtering" of unwanted molecules from the analysis. We accomplish this sorting by using alternating laser excitation (ALEX) in combination with smFRET measurements; here we describe the implementation of these methodologies for the study of biomolecules in solution.

In the present studies we have made the novel observation that protease nexin 1 (PN1), a member of the serine protease inhibitor (SERPIN) superfamily, is a potent inhibitor of the blood coagulation Factor XIa (FXIa). The inhibitory complexes formed between PN1 and FXIa are stable when subjected to reducing agents, SDS, and boiling, a characteristic of the acyl linkage formed between SERPINs and their cognate proteases. Using a sensitive fluorescence-quenched peptide substrate, the K assoc of PN1 for FXIa was determined to be 7.9 ؋ 10 4 M ؊1 s ؊1 in the absence of heparin. In the presence of heparin, this rate was accelerated to 1.7 ؋ 10 6 , M ؊1 s ؊1 , making PN1 a far better inhibitor of FXIa than C1 inhibitor, which is the only other SERPIN known to significantly inhibit FXIa. FXIa-PN1 complexes are shown to be internalized and degraded by human fibroblasts, most likely via the low density lipoprotein receptor-related protein (LRP), since degradation was strongly inhibited by the LRP agonist, receptor-associated protein. Since FXIa proteolytically modifies the amyloid precursor protein, this observation may suggest an accessory role for PN1 in the pathobiogenesis of Alzheimer's disease.