Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes.

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes.

Discovery of Dehydroamino Acid Residues in the Capsid and Matrix Structural Proteins of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) remains a deadly infectious disease despite existing antiretroviral therapies. A comprehensive understanding of the specific mechanisms of viral infectivity remains elusive and currently limits the development of new and effective therapies. Through in-depth proteomic analysis of HIV-1 virions, we discovered the novel post-translational modification of highly conserved residues within the viral matrix and capsid proteins to the dehydroamino acids, dehydroalanine and dehydrobutyrine.

Identifying Protein Interactomes of Target RNAs Using HyPR-MS.

RNA-protein interactions are integral to maintaining proper cellular function and homeostasis, and the disruption of key RNA-protein interactions is central to many disease states. HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) is a highly versatile and efficient technology which enables multiplexed discovery of specific RNA-protein interactomes. This chapter provides extensive guidance for successful application of HyPR-MS to the system and target RNA(s) of interest, as well as a detailed description of the fundamental HyPR-MS procedure, including: (1) experimental design of controls, capture oligonucleotides, and qPCR assays; (2) formaldehyde cross-linking of cell culture; (3) cell lysis and RNA solubilization; (4) isolation of target RNA(s); (5) RNA purification and RT-qPCR analysis; (6) protein preparation and mass spectrometric analysis; and (7) mass spectrometric data analysis.

HyPR-MS for Multiplexed Discovery of MALAT1, NEAT1, and NORAD lncRNA Protein Interactomes.

RNA-protein interactions are integral to the regulation of gene expression. RNAs have diverse functions and the protein interactomes of individual RNAs vary temporally, spatially, and with physiological context. These factors make the global acquisition of individual RNA-protein interactomes an essential endeavor.

Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry.

HIV-1 replication requires myriad interactions between cellular proteins and the viral unspliced RNA. These interactions are important in archetypal RNA processes such as transcription and translation as well as for more specialized functions including alternative splicing and packaging of unspliced genomic RNA into virions. We present here a hybridization capture strategy for purification of unspliced full-length HIV RNA-protein complexes preserved in vivo by formaldehyde crosslinking, and coupled with mass spectrometry to identify HIV RNA-protein interactors in HIV-1 infected cells.

Proteoform Suite: Software for Constructing, Quantifying, and Visualizing Proteoform Families.

We present an open-source, interactive program named Proteoform Suite that uses proteoform mass and intensity measurements from complex biological samples to identify and quantify proteoforms. It constructs families of proteoforms derived from the same gene, assesses proteoform function using gene ontology (GO) analysis, and enables visualization of quantified proteoform families and their changes. It is applied here to reveal systemic proteoform variations in the yeast response to salt stress.

Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements.

Proteomics is presently dominated by the "bottom-up" strategy, in which proteins are enzymatically digested into peptides for mass spectrometric identification. Although this approach is highly effective at identifying large numbers of proteins present in complex samples, the digestion into peptides renders it impossible to identify the proteoforms from which they were derived. We present here a powerful new strategy for the identification of proteoforms and the elucidation of proteoform families (groups of related proteoforms) from the experimental determination of the accurate proteoform mass and number of lysine residues contained.