Identifying Protein Interactomes of Target RNAs Using HyPR-MS

Henke, Katherine B.; Miller, Rachel M; Knoener, Rachel A.; Scalf, Mark; Spiniello, Michele; Smith, Lloyd M.

doi:10.1007/978-1-0716-1851-6_12

Cited by 5 publications

(8 citation statements)

References 43 publications

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“…The hybridization, capture, and release protocols used in this work are adapted from the HyPR-MS protocol described in Henke et al , summarized here including any changes . Buffer compositions are included in Supplementary Methods, SI.…”

Section: Methodsmentioning

confidence: 99%

“…The hybridization, capture, and release protocols used in this work are adapted from the HyPR-MS protocol described in Henke et al, summarized here including any changes. 24 Buffer compositions are included in Supplementary Methods, SI. All capture oligonucleotides (COs) contained a biotin-triethylene glycol (TEG) tag on the 3′ end to enable capture on streptavidincoated beads and an 8-nt toehold sequence on the 5′ end to allow for toehold-mediated release and multiplexing.…”

Section: ■ Introductionmentioning

confidence: 99%

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Elucidating the RNA–Protein Interactomes of Target RNAs in Tissue

et al. 2023

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RNA–protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA–protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA–protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA–protein complexes.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Elucidating the RNA–Protein Interactomes of Target RNAs in Tissue

et al. 2023

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“…Cells were washed twice in 1× PBS, pelleted by centrifugation, and frozen at −80 °C. The lysis, hybridization, capture, and release protocols are adapted from the HyPR-MS protocol in the study of Henke et al, summarized here to include any changes . Cells were lysed and sonicated as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…The lysis, hybridization, capture, and release protocols are adapted from the HyPR-MS protocol in the study of Henke et al, summarized here to include any changes. 19 Cells were lysed and sonicated as previously described. Capture oligonucleotides (COs) targeting gRNA, S sgRNA, N sgRNA, and ORF8 sgRNA were added to the cell lysates along with negative control scrambled sequence oligonucleotides (SOs) ( Supporting Information Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

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

Whitworth,

Knoener,

Puray-Chavez

et al. 2023

J. Proteome Res.

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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. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

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“…Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID- 19) which has been associated with over 6.8 million deaths since its emergence in 2019. 1,2 Like all viruses, SARS-CoV-2 encodes a limited number of proteins and relies on interactions with host factors for efficient replication.…”

Section: Introductionmentioning

confidence: 99%

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

Whitworth

Knoener

Puray-Chavez

et al. 2023

Preprint

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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. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

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Identifying Protein Interactomes of Target RNAs Using HyPR-MS

Cited by 5 publications

References 43 publications

Elucidating the RNA–Protein Interactomes of Target RNAs in Tissue

Elucidating the RNA–Protein Interactomes of Target RNAs in Tissue

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

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

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