ImiRP: a computational approach to microRNA target site mutation

Abstract

Background: MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs that function as post-transcriptional regulators of messenger RNA (mRNA) through base-pairing to 6-8 nucleotide long target sites, usually located within the mRNA 3' untranslated region. A common approach to validate and probe microRNA-mRNA interactions is to mutate predicted target sites within the mRNA and determine whether it affects miRNA-mediated activity. The introduction of miRNA target site mutations, however, is potentially problematic as it may generate new, "illegitimate sites" target sites for other miRNAs, which may affect the experimental outcome. While it is possible to manually generate and check single miRNA target site mutations, this process can be time consuming, and becomes particularly onerous and error prone when multiple sites are to be mutated simultaneously. We have developed a modular Java-based system called ImiRP (Illegitimate miRNA Predictor) to solve this problem and to facilitate miRNA target site mutagenesis.

Results: The ImiRP interface allows users to input a sequence of interest, specify the locations of multiple predicted target sites to mutate, and set parameters such as species, mutation strategy, and disallowed illegitimate target site types. As mutant sequences are generated, ImiRP utilizes the miRBase high confidence miRNA dataset to identify illegitimate target sites in each mutant sequence by comparing target site predictions between input and mutant sequences. ImiRP then assembles a final mutant sequence in which all specified target sites have been mutated.

Conclusions: ImiRP is a mutation generator program that enables selective disruption of specified miRNA target sites while ensuring predicted target sites for other miRNAs are not inadvertently created. ImiRP supports mutagenesis of single and multiple miRNA target sites within a given sequence, including sites that overlap. This software will be particularly useful for studies looking at microRNA cooperativity, where mutagenesis of multiple microRNA target sites may be desired. The software is available at imirp.org and is available open source for download through GitHub ( https://github.com/imirp ).

Keywords: Automation; MicroRNA; Mutagenesis; Seed Sequence; Software; Target Site.

Fig. 1

The problem associated with miRNA target site mutagenesis. A wild type 3’UTR sequence contains a predicted 7mer-m8 target site for miR-7 (green). Mutation disrupts the interaction between miR-7 and its predicted target site, but creates an illegitimate 7mer-m8 target site for miR-296-3p in the process (red)

Fig. 2

ImiRP input user interface. a The user is first directed to create a project and specify project parameters. The project can be named, and in this example we have named the project “ImiRP Test”. A DNA or RNA sequence must be input along with information about the species of interest. The input sequence will be displayed for viewing. Finally, at least one “mutation site”, the region into which mutations will be introduced, is specified by typing the sequence position complementary to miRNA position 7 into the textbox. All selected mutation sites, complementary to miRNA positions 2-7, appear bolded in the displayed original input sequence for inspection. In this example, a segment of the mouse Pax6 3’UTR and three hypothetical predicted target sites have been used as input. b Upon creation of a project, the user is asked to specify mutation parameters. In the “Mutation Strategy” tab, nucleotides to use for mutation and number of nucleotide changes per specified mutation site can be selected. In the “Define Invalid Sites” tab, users can specify the types of newly created miRNA target sites they do not want present in their final mutant sequence. In this case, any mutant sequences provided as output will contain two adjacent nucleotide changes per specified target site using all four nucleotides for mutation, and no mutant sequences containing newly created 8mer, 7mer-m8, or 7mer-A1 predicted target sites will be provided as output

Fig. 3

ImiRP Workflow. ImiRP can be divided into three major modules. The Sequence Mutation module a feeds mutated sequences into the Target Site Prediction module b, the results of which are supplied to the Master Mutant Assembly module c to optimize and output a final mutant sequence

Fig. 4

The Sequence Mutation module. a The input sequence is divided into “mutationally independent” regions. User-specified target sites spaced less than 7 nucleotides apart are grouped into a single independent region and are annotated based on the positions of the first and last nucleotides of sites within the given region. b To reduce computational overhead, sites within a given independent region are mutated as a unit, while sites in other regions are unchanged

Fig. 5

The target site prediction module. Predicted miRNA target sites within input and mutant sequences are identified using a custom component that identifies predicted target sites based on complementarity to the miRNA 5’ end. FASTA-format miRNA sequence data was collected from miRBase version 21 [11]. Information about predicted sites present in each mutant sequence that are absent from the input sequence are stored in a database for analysis

Fig. 6

ImiRP output user interface. Input mutation sites spaced less than seven nucleotides apart are grouped into independent regions and each region is mutated independently of other regions. The output user interface displays up to five mutant sequences for each region that satisfy the specified mutation parameters. For example, the output mutations for ImiRP Test (Fig. 2) will not have generated any new 8mer, 7mer-m8, or 7mer-A1 predicted miRNA target sites. The original input sequence for each region is displayed above the mutants for comparison, and brackets denote the bounds of each specified mutation site. The user must select one desired mutant for each region, and the changes are displayed as an assembled mutant sequence. Once the selection process is complete, the user may download a folder containing information about their assembled mutant sequence, and predicted miRNA target sites in the input and mutant sequences