Advanced approaches for elucidating structures of large RNAs using NMR spectroscopy and complementary methods

Kotar, Anita; Foley, Hannah N.; Baughman, Kirk M.; Keane, Sarah C.

doi:10.1016/j.ymeth.2020.01.009

Cited by 29 publications

(26 citation statements)

References 186 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The purification and analysis of lncRNAs poses a significant challenge for analyses of lncRNA structure, particularly if one wants to preserve the native, structural elements mediating function: Folding of long RNAs in vivo in many cases is not merely based on thermodynamic properties of the RNA, but requires the function of endogenous factors or RNA chaperones, which are not usually present in in vitro approaches. [46][47][48] Additionally, many RNA purification methods involve heat denaturation and refolding, which can result in misfolding and aggregation of lncRNAs. [49,50] Therefore, several different approaches that avoid RNA denaturation have been developed to overcome these issues in recent years.…”

Section: Purification Of Lncrnas For Structure Determinationmentioning

confidence: 99%

From structure to function: Route to understanding lncRNA mechanism

Graf

Kretz

2020

BioEssays

View full text Add to dashboard Cite

RNAs have emerged as a major target for diagnostics and therapeutics approaches. Regulatory nonprotein-coding RNAs (ncRNAs) in particular display remarkable versatility. They can fold into complex structures and interact with proteins, DNA, and other RNAs, thus modulating activity, localization, or interactome of multi-protein complexes. Thus, ncRNAs confer regulatory plasticity and represent a new layer of regulatory control. Interestingly, long noncoding RNAs (lncRNAs) tend to acquire complex secondary and tertiary structures and their function-in many cases-is dependent on structural conservation rather than primary sequence conservation. Whereas for many proteins, structure and its associated function are closely connected, for lncRNAs, the structural domains that determine functionality and its interactome are still not well understood. Numerous approaches for analyzing the structural configuration of lncRNAs have been developed recently. Here, will provide an overview of major experimental approaches used in the field, and discuss the potential benefit of using combinatorial strategies to analyze lncRNA modes of action based on structural information.

show abstract

Section: Purification Of Lncrnas For Structure Determinationmentioning

confidence: 99%

From structure to function: Route to understanding lncRNA mechanism

Graf

Kretz

2020

BioEssays

View full text Add to dashboard Cite

show abstract

“…In cellulo, Gag recognizes particular motifs such as Rev Response Element (RRE) and the packaging signal (including SL1 and SL3) [ 31 , 69 ]. Recent studies involving a series of impressive NMR studies [ 6 , 7 , 62 , 64 , 70 , 71 , 72 ] have shown outstanding results using smart labeling strategies in order to answer these questions [ 73 , 74 , 75 ]. In these studies, the binding of NCp7, used as a substitute for Gag in the context of gRNA dimerization and its selective packaging, was extensively studied.…”

Section: Nc Domain In Its Immature States (Gag Ncp15 Ncp9)mentioning

confidence: 99%

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States

Mouhand

Pasi

Catala

et al. 2020

Viruses

View full text Add to dashboard Cite

HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with nucleic acids, and p1 and p6, two unstructured regions, p6 containing the motifs to bind ALIX, the cellular ESCRT factor TSG101 and the viral protein Vpr. The processing of Gag by the viral protease subsequently liberates NCp15 (NC-p1-p6), NCp9 (NC-p1) and NCp7, NCp7 displaying the optimal chaperone activity of nucleic acids. This review focuses on the nucleic acid binding properties of the NC domain in the different maturation states during the HIV-1 viral cycle.

show abstract

“…In addition, H–C dipolar coupling can severely limit the sensitivity and resolution of the H–C correlation NMR spectra obtainable for larger RNAs with longer rotational correlation times. For these reasons, high-resolution NMR-based structural studies were generally limited to relatively small RNAs [ 80 , 81 ]. The average size of NMR-derived RNA structures deposited in public databases is 30 nucleotides and only six structures were reported for RNAs comprising more than 100 nucleotides ( ).…”

Section: Introductionmentioning

confidence: 99%

“…The average size of NMR-derived RNA structures deposited in public databases is 30 nucleotides and only six structures were reported for RNAs comprising more than 100 nucleotides ( ). However, new 2 H-edited NMR methods recently enabled structural studies of RNA elements within the intact, dimeric 5′-leaders of two strains of HIV-1 (HIV-1 NL4-3 and HIV-1 MAL ) (>700 nucleotide dimers; >230 kDa) [ 71 , 75 , 81 ]. Hybrid approaches that combine high-resolution local structural information provided by NMR with lower-resolution global structural information from cryogenic electron microscopy (cryo-EM), or small angle X-ray scattering (SAXS) could enable structural studies of even larger RNAs and protein–RNA complexes that are important for packaging.…”

Section: Introductionmentioning

confidence: 99%

NMR Studies of Retroviral Genome Packaging

Boyd

Brown

et al. 2020

Viruses

View full text Add to dashboard Cite

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

show abstract

Advanced approaches for elucidating structures of large RNAs using NMR spectroscopy and complementary methods

Cited by 29 publications

References 186 publications

From structure to function: Route to understanding lncRNA mechanism

From structure to function: Route to understanding lncRNA mechanism

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States

NMR Studies of Retroviral Genome Packaging

Contact Info

Product

Resources

About