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.

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 RNAprotein 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. RNA and proteins are interconnected biomolecules that can promote each other's life cycles and functions through physical interactions 1. The coding sequence of mRNA carries the instructions for protein synthesis and some regulatory sequences, and the untranslated regions of mRNA influence the fate of the encoded protein by regulating its protein translation, localization, and interactions with other proteins 2. Proteins, in turn, can bind and modulate RNA expression and function from RNA synthesis to degradation 3. RNA-protein interactions are key to cellular homeostasis, and perturbations of RNA-RBP interactions can lead to cellular dysfunction and disease 4,5. Recent work has substantially expanded the number of putative RNA-protein associations in eukaryotes, underscoring the need for a versatile array of methods to identify and characterize their interactions 6,7. Methods for studying the physical interactions between RNA and protein can be classified by the type of molecule they start with. RNA-centric methods start with an RNA of interest

SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity Genomic surveillance efforts have uncovered SARS-CoV-2 variants with mutations in the viral spike glycoprotein, which binds the human angiotensin-converting enzyme 2 (ACE2) receptor to facilitate viral entry. 1 Such variants represent a public health challenge during the COVID-19 pandemic because they increase viral transmission and disease severity. 2 The B.1.351 variant, first identified in South Africa, has three notable mutations in the spike receptorbinding domain (RBD)-namely, K417N, E484K, and N501Y 3 -whereas the B.1.1.7 variant, first identified in the UK, carries the N501Y mutation (appendix pp 2-4). B.1.351 is of particular concern for its potential resistance to antibodies elicited by previous SARS-CoV-2 infection and vaccination. 4 Several mechanisms might account for increased variant transmissibility, such as increased spike protein density, greater furin cleavage accessibility, and enhanced spike protein binding affinity for the ACE2 receptor. 5 To test whether the B.1.351 and B.1.1.7 variants bind ACE2 with

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 Thermophoresis (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.

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.