Publications

Levengood JD, Potoyan D, Penumutchu S, Kumar A, Zhou Q, Wang Y, Hansen AL, Kutluay S, Roche J, Tolbert BS. Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions. Sci Adv. 2024 Jul 12;10(28):eadk6580. doi: 10.1126/sciadv.adk6580. Epub 2024 Jul 10. PMID: 38985864; PMCID: PMC11235170.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein’s RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

Davila-Calderon J, Li ML, Penumutchu SR, Haddad C, Malcolm L, King J, Hargrove AE, Brewer G, Tolbert BS. Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication. Sci Adv. 2024 Feb 16;10(7):eadg3060. doi: 10.1126/sciadv.adg3060. Epub 2024 Feb 16. PMID: 38363831; PMCID: PMC10871541.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)–RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Abedeera SM, Davila-Calderon J, Haddad C, Henry B, King J, Penumutchu S, Tolbert BS. The Repurposing of Cellular Proteins during Enterovirus A71 Infection. Viruses. 2023 Dec 31;16(1):75. doi: 10.3390/v16010075. PMID: 38257775; PMCID: PMC10821071.

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)–RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

Bhat P, Aksenova V, Gazzara M, Rex EA, Aslam S, Haddad C, Gao S, Esparza M, Cagatay T, Batten K, El Zahed SS, Arnaoutov A, Zhong H, Shay JW, Tolbert BS, Dasso M, Lynch KW, García-Sastre A, Fontoura BMA. Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex. Nat Commun. 2023 Apr 21;14(1):2304. doi: 10.1038/s41467-023-37911-0. PMID: 37085480; PMCID: PMC10121598.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)−2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3’ UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

Mackeown M, Kung YA, Davila-Calderon J, Ford WP, Luo L, Henry B, Li ML, Brewer G, Shih SR, Tolbert BS. The 5’UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation. J Biol Chem. 2023 Apr;299(4):103028. doi: 10.1016/j.jbc.2023.103028. Epub 2023 Feb 15. PMID: 36805339; PMCID: PMC9930382.

Chiu LY, Davila-Calderon J, Cai Z, Tolbert BS. Biophysical Analysis of Small Molecule Binding to Viral RNA Structures. Methods Mol Biol. 2023;2570:205-222. doi: 10.1007/978-1-0716-2695-5_16. PMID: 36156785.

Bevilacqua PC, Tolbert BS. Regulatory Mechanisms through RNA Conformational Switching and Dynamics. J Mol Biol. 2022 Sep 30;434(18):167794. doi: 10.1016/j.jmb.2022.167794. Epub 2022 Aug 18. PMID: 35988750; PMCID: PMC9484478.

Wang BA, Mehta HM, Penumutchu SR, Tolbert BS, Cheng C, Kimmel M, Haferlach T, Maciejewski JP, Corey SJ. Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms. Leukemia. 2022 Oct;36(10):2499-2508. doi: 10.1038/s41375-022-01672-4. Epub 2022 Aug 8. PMID: 35941213.

Chiu LY, Emery A, Jain N, Sugarman A, Kendrick N, Luo L, Ford W, Swanstrom R, Tolbert BS. Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors. J Mol Biol. 2022 Sep 30;434(18):167728. doi: 10.1016/j.jmb.2022.167728. Epub 2022 Jul 21. PMID: 35870649; PMCID: PMC9945881.

Donlic A, Swanson EG, Chiu LY, Wicks SL, Juru AU, Cai Z, Kassam K, Laudeman C, Sanaba BG, Sugarman A, Han E, Tolbert BS, Hargrove AE. R-BIND 2.0: An Updated Database of Bioactive RNA-Targeting Small Molecules and Associated RNA Secondary Structures. ACS Chem Biol. 2022 Jun 17;17(6):1556-1566. doi: 10.1021/acschembio.2c00224. Epub 2022 May 20. PMID: 35594415; PMCID: PMC9343015.

Zafferani M, Haddad C, Luo L, Davila-Calderon J, Chiu LY, Mugisha CS, Monaghan AG, Kennedy AA, Yesselman JD, Gifford RJ, Tai AW, Kutluay SB, Li ML, Brewer G, Tolbert BS, Hargrove AE. Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures. Sci Adv. 2021 Nov 26;7(48):eabl6096. doi: 10.1126/sciadv.abl6096. Epub 2021 Nov 26. PMID: 34826236; PMCID: PMC8626076.

Basu A, Penumutchu S, Nguyen K, Mbonye U, Tolbert BS, Karn J, Komar AA, Mazumder B. A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells. J Virol. 2022 Jan 26;96(2):e0167821. doi: 10.1128/JVI.01678-21. Epub 2021 Nov 10. PMID: 34757848; PMCID: PMC8791291.

Wacker A, Weigand JE, Akabayov SR, Altincekic N, Bains JK, Banijamali E, Binas O, Castillo-Martinez J, Cetiner E, Ceylan B, Chiu LY, Davila Calderon J, Dhamotharan K, Duchardt-Ferner E, Ferner J, Frydman L, Fürtig B, Gallego J, Grün JT, Hacker C, Haddad C, Hähnke M, Hengesbach M, Hiller F, Hohmann KF, Hymon D, de Jesus V, Jonker H, Keller H, Knezic B, Landgraf T, Löhr F, Luo L, Mertinkus KR, Muhs C, Novakovic M, Oxenfarth A, Palomino-Schätzlein M, Petzold K, Peter SA, Pyper DJ, Qureshi NS, Riad M, Richter C, Saxena K, Schamber T, Scherf T, Schlagnitweit J, Schlundt A, Schnieders R, Schwalbe H, Simba-Lahuasi A, Sreeramulu S, Stirnal E, Sudakov A, Tants JN, Tolbert BS, Vögele J, Weiß L, Wirmer-Bartoschek J, Wirtz Martin MA, Wöhnert J, Zetzsche H. Correction to ‘Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy’. Nucleic Acids Res. 2021 Jul 9;49(12):7204-7205. doi: 10.1093/nar/gkab568. Erratum for: Nucleic Acids Res. 2020 Dec 16;48(22):12415-12435. doi: 10.1093/nar/gkaa1013. PMID: 34161581; PMCID: PMC8266613.

Luo L, Chiu LY, Sugarman A, Gupta P, Rouskin S, Tolbert BS. HnRNP A1/A2 Proteins Assemble onto 7SK snRNA via Context Dependent Interactions. J Mol Biol. 2021 Apr 30;433(9):166885. doi: 10.1016/j.jmb.2021.166885. Epub 2021 Mar 5. PMID: 33684393; PMCID: PMC8091503.

Li ML, Shih SR, Tolbert BS, Brewer G. Enterovirus A71 Vaccines. Vaccines (Basel). 2021 Feb 27;9(3):199. doi: 10.3390/vaccines9030199. PMID: 33673595; PMCID: PMC7997495.

Levengood JD, Peterson J, Tolbert BS, Roche J. Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR. Proteins. 2021 Jul;89(7):781-791. doi: 10.1002/prot.26058. Epub 2021 Feb 15. PMID: 33550645; PMCID: PMC9122033.

Martina Z, Christina H, Le L, Jesse DC, Liang YC, Christian SM, Monaghan AG, Kennedy AA, Yesselman JD, Gifford RR, Tai AW, Kutluay SB, Li ML, Brewer G, Tolbert BS, Hargrove AE. Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures. bioRxiv [Preprint]. 2020 Dec 6:2020.12.05.409821. doi: 10.1101/2020.12.05.409821. Update in: Sci Adv. 2021 Nov 26;7(48):eabl6096. doi: 10.1126/sciadv.abl6096. PMID: 33299997; PMCID: PMC7724665.

Wacker A, Weigand JE, Akabayov SR, Altincekic N, Bains JK, Banijamali E, Binas O, Castillo-Martinez J, Cetiner E, Ceylan B, Chiu LY, Davila-Calderon J, Dhamotharan K, Duchardt-Ferner E, Ferner J, Frydman L, Fürtig B, Gallego J, Grün JT, Hacker C, Haddad C, Hähnke M, Hengesbach M, Hiller F, Hohmann KF, Hymon D, de Jesus V, Jonker H, Keller H, Knezic B, Landgraf T, Löhr F, Luo L, Mertinkus KR, Muhs C, Novakovic M, Oxenfarth A, Palomino-Schätzlein M, Petzold K, Peter SA, Pyper DJ, Qureshi NS, Riad M, Richter C, Saxena K, Schamber T, Scherf T, Schlagnitweit J, Schlundt A, Schnieders R, Schwalbe H, Simba-Lahuasi A, Sreeramulu S, Stirnal E, Sudakov A, Tants JN, Tolbert BS, Vögele J, Weiß L, Wirmer-Bartoschek J, Wirtz Martin MA, Wöhnert J, Zetzsche H. Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy. Nucleic Acids Res. 2020 Dec 16;48(22):12415-12435. doi: 10.1093/nar/gkaa1013. Erratum in: Nucleic Acids Res. 2021 Jul 9;49(12):7204-7205. doi: 10.1093/nar/gkab568.
PMID: 33167030; PMCID: PMC7736788.

Brewer G, Li ML, Tolbert BS. Editorial for “Methods to characterize virus small RNAs and RNA structures”. Methods. 2020 Nov 1;183:1-3. doi: 10.1016/j.ymeth.2020.10.007. Epub 2020 Oct 16. PMID: 33069799; PMCID: PMC7561520.

Davila-Calderon J, Patwardhan NN, Chiu LY, Sugarman A, Cai Z, Penutmutchu SR, Li ML, Brewer G, Hargrove AE, Tolbert BS. IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex. Nat Commun. 2020 Sep 22;11(1):4775. doi: 10.1038/s41467-020-18594-3. PMID: 32963221; PMCID: PMC7508794.

Haddad C, Davila-Calderon J, Tolbert BS. Integrated approaches to reveal mechanisms by which RNA viruses reprogram the cellular environment. Methods. 2020 Nov 1;183:50-56. doi: 10.1016/j.ymeth.2020.06.013. Epub 2020 Jul 2. PMID: 32622045; PMCID: PMC7329689.