Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

Gao, Yunrong; Cao, Dong; Pawnikar, Shristi; John, Karen Priyadarshini; Ahn, Hyunjun; Hill, Shaylan; Ha, Ju Mi; Parikh, Priyal; Ogilvie, Claire; Swain, Anshuman; Yang, Amy; Bell, Amber; Salazar, Angela; Miao, Yinglong; Liang, Bo

doi:10.1016/j.str.2020.07.001

Cited by 18 publications

(28 citation statements)

References 60 publications

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“…M2-1 has been extensively characterized using biochemical and structural studies. The protein has three domains separated with flexible linkers [33,34]. Near the N-terminus is a cysteine-cysteine-cysteine-histidine (CCCH) Zn 2+ finger motif necessary for RNA binding and elongation function [32][33][34][35][36].…”

Section: Introductionmentioning

confidence: 99%

“…The protein has three domains separated with flexible linkers [33,34]. Near the N-terminus is a cysteine-cysteine-cysteine-histidine (CCCH) Zn 2+ finger motif necessary for RNA binding and elongation function [32][33][34][35][36]. The central region contains an oligomerization domain that allows the protein to form a symmetrical tetramer, followed by a core domain that also has RNA binding properties [33,34,[37][38][39].…”

Section: Introductionmentioning

confidence: 99%

“…Near the N-terminus is a cysteine-cysteine-cysteine-histidine (CCCH) Zn 2+ finger motif necessary for RNA binding and elongation function [32][33][34][35][36]. The central region contains an oligomerization domain that allows the protein to form a symmetrical tetramer, followed by a core domain that also has RNA binding properties [33,34,[37][38][39]. It is thought that M2-1 is recruited to the transcribing polymerase through tetramer-tetramer interactions with P [28,35,[38][39][40].…”

Section: Introductionmentioning

confidence: 99%

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Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts

et al. 2021

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Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts

et al. 2021

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“…28,[33][34] GaMD has been applied to successfully simulate protein folding and conformational changes [33][34][35][36][37][38] , ligand binding [33][34][35][39][40][41][42][43][44] , protein-protein/membrane/nucleic acid interactions [45][46][47][48][49][50] . More recently, GaMD simulations have successfully captured spontaneous binding of RNA to the human respiratory syncytial virus M2-1 protein 51 .…”

Section: Introductionmentioning

confidence: 99%

Mechanism of RNA recognition by a Musashi RNA-binding protein

Wang

Lan

et al. 2020

Preprint

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The Musashi RNA-binding proteins (RBPs) regulate translation of target mRNAs and maintenance of cell stemness and tumorigenesis. Musashi-1 (MSI1), long considered as an intestinal and neural stem cell marker, has been more recently found to be overexpressed in many cancers. It has served as an important drug target for treating acute myeloid leukemia and solid tumors such as ovarian, colorectal and bladder cancer. One of the reported binding targets of MSI1 is Numb, a negative regulator of the Notch signaling. However, the dynamic mechanism of Numb RNA binding to MSI1 remains unknown, largely hindering effective drug design targeting this critical interaction. Here, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which successfully captured spontaneous and highly accurate binding of the Numb RNA from bulk solvent to the MSI1 protein target site. GaMD simulations revealed that Numb binding to MSI1 involved largely induced fit in both the RNA and protein. The simulations also identified important low-energy intermediate conformational states during RNA binding, in which Numb interacted mainly with the β2-β3 loop and C terminus of MSI1. The mechanistic understanding of RNA binding obtained from our GaMD simulations is expected to facilitate rational structure-based drug design targeting MSI1 and other RBPs.

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“…Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020) .…”

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confidence: 99%

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations

et al. 2021

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Summary Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020) .

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Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

Cited by 18 publications

References 60 publications

Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts

Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts

Mechanism of RNA recognition by a Musashi RNA-binding protein

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations

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