Rapid NMR screening of RNA secondary structure and binding

Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald

doi:10.1007/s10858-015-9967-y

Cited by 52 publications

(65 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The combined NOE and chemical shift information provide direct evidence that DIS adopts an extended intermolecular duplex structure in the context of the dimeric 5′-leader. 47 ], that gives rise to a well-resolved A46-H2 NMR signal (∼6.5 ppm) (48). In a homodimeric 5′-L 344 sample with A 2r G r C r labeling, A46-H2 exhibits a typical cross-strand NOE with the H 1 ′ proton of A14, as well as a sequential NOE with A47-H2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

Keane

Van

Frank

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

show abstract

Section: Resultsmentioning

confidence: 99%

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

Keane

Van

Frank

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In vitro transcription was performed using a method that inhibits self-templated run-on (51,52). The 5′-capped RNAs were prepared using a Vaccinia virus capping enzyme prepared in house with plasmid kindly provided by Stephen Cusack, European Molecular Biology Laboratory (EMBL), Grenoble, France (38).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Kharytonchyk

Monti

Smaldino

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

119

View full text Add to dashboard Cite

The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine ( Cap 1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines ( Cap 2G or Cap 3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap 1G, Cap 2G, and Cap 3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap 1G 5′-leader RNA adopts a dimeric structure, the Cap 2G and Cap 3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimerpromoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.HIV-1 | 5′-leader | transcription | RNA | structure

show abstract

“…In combination with the standard T7 φ6.5 promoter, this enzyme prefers a guanine as the first base, whereas in combination with the T7 φ2.5 promoter, transcription starts with an adenine (Coleman et al 2004;Ray et al 2006). As templates for the in vitro transcription reaction, we used plasmid DNA that contains an HDV-ribozyme at the 3 ′ end and DNA primers in a nonmethylated or methylated form (Helmling et al 2015).…”

Section: Rna Body Preparationmentioning

confidence: 99%

“…Therefore, the reaction contained an equimolar amount of antisense strand primers bearing the T7 promoter sequence and sense strand primers encompassing 5 ′ -target RNA sequence (in reverse complement)-CC-T7 promoter (in reverse compliment)-3 ′ . Some of the antisense primers were methylated at the 2 ′ -O position of the last 2 nt (Helmling et al 2015) in order to reduce 3 ′ inhomogeneity. The use of methylated primers is indicated in the RNA construct list below.…”

Section: Purification Of Vaccinia Capping Enzymementioning

confidence: 99%

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

View full text Add to dashboard Cite

The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

show abstract

Rapid NMR screening of RNA secondary structure and binding

Cited by 52 publications

References 43 publications

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Contact Info

Product

Resources

About