RNA-PAIRS: RNA probabilistic assignment of imino resonance shifts

Abstract

The significant biological role of RNA has further highlighted the need for improving the accuracy, efficiency and the reach of methods for investigating RNA structure and function. Nuclear magnetic resonance (NMR) spectroscopy is vital to furthering the goals of RNA structural biology because of its distinctive capabilities. However, the dispersion pattern in the NMR spectra of RNA makes automated resonance assignment, a key step in NMR investigation of biomolecules, remarkably challenging. Herein we present RNA Probabilistic Assignment of Imino Resonance Shifts (RNA-PAIRS), a method for the automated assignment of RNA imino resonances with synchronized verification and correction of predicted secondary structure. RNA-PAIRS represents an advance in modeling the assignment paradigm because it seeds the probabilistic network for assignment with experimental NMR data, and predicted RNA secondary structure, simultaneously and from the start. Subsequently, RNA-PAIRS sets in motion a dynamic network that reverberates between predictions and experimental evidence in order to reconcile and rectify resonance assignments and secondary structure information. The procedure is halted when assignments and base-parings are deemed to be most consistent with observed crosspeaks. The current implementation of RNA-PAIRS uses an initial peak list derived from proton-nitrogen heteronuclear multiple quantum correlation ((1)H-(15)N 2D HMQC) and proton-proton nuclear Overhauser enhancement spectroscopy ((1)H-(1)H 2D NOESY) experiments. We have evaluated the performance of RNA-PAIRS by using it to analyze NMR datasets from 26 previously studied RNAs, including a 111-nucleotide complex. For moderately sized RNA molecules, and over a range of comparatively complex structural motifs, the average assignment accuracy exceeds 90%, while the average base pair prediction accuracy exceeded 93%. RNA-PAIRS yielded accurate assignments and base pairings consistent with imino resonances for a majority of the NMR resonances, even when the initial predictions are only modestly accurate. RNA-PAIRS is available as a public web-server at http://pine.nmrfam.wisc.edu/RNA/.

Fig. 1

a Standard steps for solving RNA structures using NMR. The process can be conceptually divided into three main steps: 1 imino proton assignments and secondary structure validation, 2 full resonance assignment and restraint list construction, 3 structure calculation. The vast majority of work in these steps requires manual intervention—although some automation support is available for structure calculation and refinement. Circular arrows indicate some of the possible steps for iterative refinement while the dotted arrow suggests the potential need for construct modifications. Imino regions of 2D NOESY and HMQC NMR spectra are often highly informative for step 1. b Two types of NMR spectra containing information relevant for assigning NMR signals for imino protons and nitrogens in an RNA molecule (BMRB ID 17921)

Fig. 2

Design of RNA-PAIRS network is presented in block form. Blocks with heavier dark outlines represent the more complex portions of the algorithm—both in terms of computational complexity as well as algorithm design. The box for “user input” identifies the current peak list input for the software, which will be extended to include other experimental data. The direction of the arrows identifies the “flow of logic” in the software. The rightmost box (that intersects “curved” arrows) is the portion of the software where probabilities are updated using the “back and forth” (reverberating) iteration