RNA2DMut: a web tool for the design and analysis of RNA structure mutations

Abstract

With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis- and trans-regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/.

Keywords: EBV; MALAT1; RNA; influenza; ncRNA; structure.

FIGURE 1.

Example screenshots of the RNA2DMut web interface. (Left panel) Shown are the input fields for the RNA2DMut Sequence Mutation tool. Fields for entering in sequence data (pasted in or uploaded), temperature, a constraint mask for making mutations, and a place to define the 2D structure used to generate image files are shown. Users can also enter in an e-mail address and have results sent upon completion of the calculation. At the top are additional tabs for other RNA2DMut tools: the Sequence Evaluation tool (evaluates the energy and ensemble diversity of input sequences) and the Sequence Manipulation tools (used to generate sequential indels or substitutions, scramble or reverse complement sequences). Additional links to an “About” page (which has a user guide for RNA2DMut), contact information; citation information and links to useful sites are included. (Right panel) Shown is an example output page with the two text (.txt) outfile download links. Below these are links to download two 2D structure image (.eps) files and, below these, the images of the 2D structures embedded in the page. Here “minimage” shows the mutated positions with the minimum ED relative to WT, while “maximage” shows those mutated sites that have the maximum ED relative to WT. The blue and red color intensity maps define the magnitude of ED change from WT. Command line input is also generated for users to manipulate 2D models within VARNA.

FIGURE 2.

Summary of results for the MALAT1 mascRNA. (Top) Partial output for the calculation taken from outfile1 (Supplemental File) that shows the wild-type (WT) result, followed by the top five most maximizing and minimizing mutations affecting the ensemble diversity (ED). Maximizing point mutations are annotated in red on the sequence, while minimizing are in blue. Mutant_89, the most maximizing mutant, has minimum free energy (MFE) base pairs predicted to deviate from the WT structure, highlighted in red on the dot-bracket notation structure. (Bottom left) The 2D model of the WT ensemble centroid structure annotated with the maximal predicted change in ED from mutant (Mut) to WT at each base, represented by a red heat map. The most disruptive mutation (Mutant_89) is indicated with a red base and its location is shown by the red arrow. (Bottom right) The same 2D model for the mascRNA, only now annotated with the minimal possible ED mutations, where a blue intensity map (comparing the change in ED from WT to Mut) is used. The minimal ED mutant (Mutant_39) is indicated with a blue base and its location with a blue arrow.

FIGURE 3.

Summary of results for the influenza virus splice site regulatory structure. (Top) Results from outfile1 (Supplemental File) are shown and annotated similarly as in Figure 2 (the MFE energy and structure are omitted for space). The Jiang and colleagues double point-mutation evaluated using the RNA2DMut Evaluation tool is added in the second position. Centroid predicted base pairs that differ from the literature model are annotated in red in the Jiang and colleagues structure, as well as Mutants_111 and 99, which are combined in the Jiang and colleagues double point-mutant. (Bottom left) The most ED maximizing mutations annotated on the literature 2D model. Annotations are similar to Figure 2. In addition, the branch point (BP) and poly(Y) (pyrimidine) tract are annotated in purple and green, respectively. The 3′ splice site (ss), used to generate M2 mRNA, is indicated with a brown arrow. A pseudoknot helix from an alternative conformation is indicted in gray and connected with a dashed line. (Bottom right) The alternative fold stabilized by the double point-mutation (red bases) used by Jiang and colleagues. The pairs that differ from the literature model are colored red and two MFE pairs that are missing from the centroid structure model are indicated with dashed lines. Annotations for functional elements are the same as the structure to the left.

FIGURE 4.

Summary of results for the Epstein–Barr virus (EBV) EBER2 ncRNA. (Top) Results from outfile1 (Supplemental File) are shown and annotated as in the two previous figures, however, the MFE structure and folding energy are omitted for space considerations. (Bottom left) The WT 2D structure model is annotated with the maximal ED changing mutation results, as in Figure 1 (bottom left). The nt that can base-pair with the terminal repeat (TR) transcript are annotated with green; these were not allowed to mutate in the calculation. (Bottom middle) The minimal ED changing mutant results are annotated on the WT 2D model, as in Figure 1 (bottom right). (Bottom right) The 2D centroid structure for Mutant_155, the most ED reducing mutant found. Centroid base pairs that differ from the WT model are annotated in red (as in the top dot bracket structure) and the stabilizing mutation (C83) is indicated in blue.

FIGURE 5.

Xist repA region structure models annotated with the most maximizing and minimizing ED changes annotated in red and blue, respectively, in the left and right panels. The A repeat units are indicated with black outlines.

FIGURE 6.

Optimized insertion sites of an MS2 aptamer into the hY5 ncRNA. The WT hY5 ensemble centroid model is shown to the left. Ensemble centroid models for two low ED predictions for loop insertions follow, where the MS2 aptamer sequence is indicated with orange outlines.