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Article ; Online: 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

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2404, Page(s) 219–244

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Mass Spectrometry ; Nucleic Acid Hybridization ; Oligonucleotides ; Proteomics ; RNA/genetics
Chemical Substances	Oligonucleotides ; RNA (63231-63-0)
Language	English
Publishing date	2021-10-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1851-6_12
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Miller, Rachel M / Knoener, Rachel A / Benner, Bayleigh E / Frey, Brian L / Scalf, Mark / Shortreed, Michael R / Sherer, Nathan M / Smith, Lloyd M

Journal of proteome research

2022 Volume 21, Issue 4, Page(s) 993–1001

Abstract: 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 ... ...

Abstract	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. We further confirmed their presence by labeling the reactive alkene, characteristic of dehydroamino acids, with glutathione via Michael addition. Dehydroamino acids are rare, understudied, and have been observed mainly in select bacterial and fungal species. Until now, they have not been observed in HIV proteins. We hypothesize that these residues are important in viral particle maturation and could provide valuable insight into HIV infectivity mechanisms.
MeSH term(s)	Capsid/chemistry ; Capsid/metabolism ; Capsid Proteins/analysis ; Capsid Proteins/chemistry ; Capsid Proteins/genetics ; HIV-1/genetics ; Humans ; Proteomics ; Virion
Chemical Substances	Capsid Proteins
Language	English
Publishing date	2022-02-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.1c00867
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Article: Discovery of Dehydroamino Acid Residues in the Capsid and Matrix Structural Proteins of HIV-1

Miller, Rachel M. / Knoener, Rachel A. / Benner, Bayleigh E. / Frey, Brian L. / Scalf, Mark / Shortreed, Michael R. / Sherer, Nathan M. / Smith, Lloyd M.

Journal of proteome research. 2022 Feb. 22, v. 21, no. 4

2022

Abstract	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. We further confirmed their presence by labeling the reactive alkene, characteristic of dehydroamino acids, with glutathione via Michael addition. Dehydroamino acids are rare, understudied, and have been observed mainly in select bacterial and fungal species. Until now, they have not been observed in HIV proteins. We hypothesize that these residues are important in viral particle maturation and could provide valuable insight into HIV infectivity mechanisms.
Keywords	Human immunodeficiency virus 1 ; amino acids ; antiretroviral agents ; capsid ; chemical reactions ; fungi ; glutathione ; infectious diseases ; pathogenicity ; post-translational modification ; proteome ; proteomics ; research ; virion
Language	English
Dates of publication	2022-0222
Size	p. 993-1001.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.1c00867
Database	NAL-Catalogue (AGRICOLA)

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

Whitworth, Isabella T / Knoener, Rachel A / Puray-Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M'bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B / Smith, Lloyd M / Sherer, Nathan M

bioRxiv : the preprint server for biology

2023

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2023-05-16
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.05.15.540806
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Article ; Online: Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Journal of proteome research

2023 Volume 23, Issue 1, Page(s) 149–160

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Humans ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Subgenomic RNA ; RNA, Viral/genetics ; RNA, Viral/metabolism ; COVID-19/genetics ; Virus Replication/genetics ; Genomics ; RNA-Binding Proteins/genetics
Chemical Substances	Subgenomic RNA ; RNA, Viral ; MSI2 protein, human ; RNA-Binding Proteins
Language	English
Publishing date	2023-12-03
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.3c00506
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Article ; Online: Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Whitworth, Isabella T. / Knoener, Rachel A. / Puray-Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M’bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B. / Smith, Lloyd M. / Sherer, Nathan M.

bioRxiv

Abstract	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.
Keywords	covid19
Language	English
Publishing date	2023-05-16
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2023.05.15.540806
Database	COVID19

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

Knoener, Rachel A / Becker, Jordan T / Scalf, Mark / Sherer, Nathan M / Smith, Lloyd M

Scientific reports

2017 Volume 7, Issue 1, Page(s) 16965

Abstract: 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 ... ...

Abstract	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. One hundred eighty-nine proteins were identified to interact with unspliced HIV RNA including Rev and Gag/Gag-Pol, 24 host proteins previously shown to bind segments of HIV RNA, and over 90 proteins previously shown to impact HIV replication. Further analysis using siRNA knockdown techniques against several of these proteins revealed significant changes to HIV expression. These results demonstrate the utility of the approach for the discovery of host proteins involved in HIV replication. Additionally, because this strategy only requires availability of 30 nucleotides of the HIV-RNA for hybridization with a capture oligonucleotide, it is readily applicable to any HIV system of interest regardless of cell type, HIV-1 virus strain, or experimental perturbation.
MeSH term(s)	Cytoplasm/genetics ; Cytoplasm/metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; HIV Infections/metabolism ; HIV Infections/virology ; HIV-1/genetics ; Humans ; Mass Spectrometry/methods ; Mass Spectrometry/statistics & numerical data ; Microscopy, Fluorescence ; Nucleic Acid Hybridization/methods ; Proteins/genetics ; Proteins/metabolism ; RNA Splicing ; RNA, Small Interfering ; RNA, Viral/metabolism ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism ; Reproducibility of Results ; Viral Proteins/genetics ; Viral Proteins/metabolism
Chemical Substances	Proteins ; RNA, Small Interfering ; RNA, Viral ; RNA-Binding Proteins ; Viral Proteins
Language	English
Publishing date	2017-12-05
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-017-16793-5
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Spiniello, Michele / Knoener, Rachel A / Steinbrink, Maisie I / Yang, Bing / Cesnik, Anthony J / Buxton, Katherine E / Scalf, Mark / Jarrard, David F / Smith, Lloyd M

Journal of proteome research. 2018 July 04, v. 17, no. 9

2018

Abstract: 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 ... ...

Abstract	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. Although techniques have been reported for discovery of the protein interactomes of specific RNAs they are largely laborious, costly, and accomplished singly in individual experiments. We developed HyPR-MS for the discovery and analysis of the protein interactomes of multiple RNAs in a single experiment while also reducing design time and improving efficiencies. Presented here is the application of HyPR-MS to simultaneously and selectively isolate the interactomes of lncRNAs MALAT1, NEAT1, and NORAD. Our analysis features the proteins that potentially contribute to both known and previously undiscovered roles of each lncRNA. This platform provides a powerful new multiplexing tool for the efficient and cost-effective elucidation of specific RNA–protein interactomes.
Keywords	RNA ; cost effectiveness ; gene expression regulation ; protein-protein interactions ; proteins ; proteome
Language	English
Dates of publication	2018-0704
Size	p. 3022-3038.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.8b00189
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Article: Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

Shortreed, MichaelR / Frey Brian L / Scalf Mark / Knoener Rachel A / Cesnik Anthony J / Smith Lloyd M

Journal of Proteome Research. 2016 Apr. 01, v. 15, no. 4

2016

Abstract: 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 ... ...

Abstract	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. Accurate proteoform masses are determined by standard LC–MS analysis of undigested protein mixtures in an Orbitrap mass spectrometer, and the lysine count is determined using the NeuCode isotopic tagging method. We demonstrate the approach in analysis of the yeast proteome, revealing 8637 unique proteoforms and 1178 proteoform families. The elucidation of proteoforms and proteoform families afforded here provides an unprecedented new perspective upon proteome complexity and dynamics.
Keywords	digestion ; liquid chromatography ; lysine ; mass spectrometry ; peptides ; proteins ; proteome ; proteomics ; spectrometers ; yeasts
Language	English
Dates of publication	2016-0401
Size	p. 1213-1221.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021%2Facs.jproteome.5b01090
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Article ; Online: Correction to "Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements".

Shortreed, Michael R / Frey, Brian L / Scalf, Mark / Knoener, Rachel A / Cesnik, Anthony J / Smith, Lloyd M

Journal of proteome research

2017 Volume 16, Issue 7, Page(s) 2660

Language	English
Publishing date	2017-07-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.7b00333
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