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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Hum Mutat. Author manuscript; available in PMC Nov 1, 2011.
Published in final edited form as:
PMCID: PMC3001138
NIHMSID: NIHMS230141

MicroSNiPer: a web tool for prediction of SNP effects on putative microRNA targets

Abstract

MicroRNAs are short, approximately 22 nucleotide non-coding RNAs binding to partially complementary sites in the 3'UTR of target mRNAs. This process generally results in repression of multiple targets by a particular microRNA. There is substantial interest in methods designed to predict the microRNA targets and effect of single nucleotide polymorphisms (SNPs) on microRNA binding, given the impact of microRNA on posttranscriptional regulation and its potential relation to complex diseases. We developed a web-based application, MicroSNiPer, which predicts the impact of a SNP on putative microRNA targets. This application interrogates the 3'-untranslated region and predicts if a SNP within the target site will disrupt/eliminate or enhance/create a microRNA binding site. MicroSNiPer computes these sites and examines the effects of SNPs in real time. MicroSNiPer is a user-friendly web-based tool. Its advantages include ease of use, flexibility and straightforward graphical representation of the results. It is freely accessible at http://cbdb.nimh.nih.gov/microsniper.

Keywords: microRNA, SNP, FASTA program, 3'UTR, gene expression, computational prediction

Introduction

Short regulatory microRNAs (miRNAs) were originally discovered in plants and thought to be a unique phenomenon for this biological kingdom. However, miRNAs were soon discovered in animals, including mammals. Currently, the number of confirmed miRNAs in human exceeds six hundred [Griffiths-Jones et al., 2008].

Initially, miRNAs are processed from transcript and form hairpin-like loops [Winter et al., 2009]. Then, mature miRNAs, approximately 22 nucleotides long, together with the protein silencing complex (RISC), bind to target sites on the messenger RNA (mRNA). This event induces posttranscriptional repression via mRNA destabilization and/or translational repression.

The elucidation of mRNA targeting mechanism by miRNAs has become an important aim for computational and experimental biologists [Bartel, 2009; Mendes et al., 2009]. Various methods of computational predictions have been developed after it was demonstrated that 3'-untranslated regions (3'UTR) contain miRNA binding sites for those miRNAs which possess a certain degree of complementarity. By including the criterion of evolutionary conservation for these miRNA target sites (miRTSs), a sign of purifying selection, it has been possible to filter out many false positive targets.

It has been demonstrated that perfect complementarity in region 2 through 7 nucleotides starting from the 5'-end of the miRNA, the so-called `seed', significantly enriches true positive predictions of miRTS. Experiments have shown that base pairing disruption in this seed region significantly impairs target mRNA down-regulation. However, the requirement of a conserved, contiguous, stretch of 7 nucleotides in the 3'UTR, complementary to the seed region, would capture most of the mammalian miRNA binding sites. By increasing this length from 7 to 8 nucleotides improves the specificity, yet increases the number of false negatives, since most mRNA binding sites require only 7 nucleotides [Brennecke et al., 2005; Lai, 2002; Lewis et al., 2003]. Using a region of 7 conserved nucleotides, matched to the miRNA seed, Lewis et al. estimated the ratio of correctly predicted targets to false positive as 3.5:1 using a five species alignment of 3'UTRs [Lewis et al., 2005]. It has been estimated that the preservation of 3'-UTR pairing to miRNAs under purifying selection, is a characteristic of the majority of the human protein-coding genes [Chen and Rajewsky, 2006; Saunders et al., 2007].

Currently, approximately 1300 experimentally supported miRNA target sites (miRTS) have been reported [Papadopoulos et al., 2009]. Computational predictions based purely on matching seeds, regardless of other evolutionary and biological considerations such as conservation, spatial, temporal distribution of targets and miRNAs, would yield many false positive or false-negative binding sites. To add to this complexity, a single gene can be targeted by several miRNAs in multiple sites [Brennecke et al., 2005; Grimson et al., 2007; Hon and Zhang, 2007; Krek et al., 2005].

The duality of mechanism of down-regulating gene expression adds an additional layer of complexity to the experimental validation of computational predictions since high throughput expression analysis measures degradation of target mRNA, ignoring the translational repression mechanism. Eventual confirmation requires the analysis of the expression of a reporter gene, e.g. luciferase, with a cloned putative miRNA targets and co-expression of miRNA and target mRNA [Cheng and Li, 2008; Liu and Kohane, 2009; Sethupathy and Collins, 2008].

Target-prediction computational tools mostly fall into two categories. The first category includes tools that use the conservation of binding site as the main criteria. Some well known programs in this category are TargetScan [Friedman et al., 2009], which considers stringent seed pairing, number of pairs, and the secondary structure accessibility of sites, and PicTar [Lall et al., 2006], which also takes into account the stability of predicted pairs. The target prediction algorithm used by DIANA-microT [Maragkakis et al., 2009] depends on the principles of binding and conservation, i.e. on conserved and non-conserved number of binding sites. This tool also integrates biological pathways and the analysis of predicted interactions of target genes.

RNAhybrid [Rehmsmeier et al., 2004] identifies multiple miRNA target sites with the most favorable energy of binding. It is able to determine the optimal and the suboptimal binding energies where the user can define the degree of complementarity. Miranda [Betel et al., 2008] and miRBase [Griffiths-Jones et al., 2008] are not as strict about seed pairing though these tools require additional pairing beyond the seed region. Recent programs such as Pita Top [Kertesz et al., 2007] and mirWIP [Hammell et al., 2008] require reasonable seed pairing while considering the site context.

Tools in the second category such as versions of TargetScan [Grimson et al., 2007] and Pita [Kertesz et al., 2007] compute miRNA targets regardless of the target conservation among species. These programs require not only strict seed pairing but also site accessibility. RNA22 [Miranda et al., 2006] uses a pattern-based approach where patterns are generated from sets of known miRNAs. This program bypasses conservation analysis and only considers base pairing stability. Another web-tool, MicroInspector [Rusinov et al., 2005], detects miRNA binding sites in 3'UTRs based on complementarity using two sliding windows of six nucleotides with dynamic hybridization, allowing G:U wobble base pairs, and the subsequent folding of the duplex with Vienna RNA secondary structure routines [Hofacker, 2003].

Currently, it is accepted that disease-associated functional SNPs alter gene expression. With this in mind, the interplay between SNPs and miRNAs has become more important [Sethupathy and Collins, 2008]. Recently, there have been a number of findings of SNPs in 3'UTRs that affect expression due to binding of miRNA; the binding of miRNA was affected by the SNP, i.e. one allele reduced or eliminated the binding [Abelson et al., 2005; Clop et al., 2006; Sethupathy and Collins, 2008; Wang et al., 2008]. The predicted miRNA binding sites, in conjunction with SNPs, are catalogued in PolymiRTS [Bao et al., 2007] and in the Patrocles database [Hiard et al., 2010] which integrates SNPs, phenotype and expression data.

We have developed a user-friendly online tool, MicroSNiPer, which allows researchers to estimate the impact of a SNP on a putative miRNA target. It provides information about the creation or disruption of putative miRNA binding sites in a gene due to the presence of alternative SNP alleles. This web-tool provides a high degree of flexibility at the input stage. A user can load 3'UTR from the University of California Santa Cruz (UCSC) collection, automatically with their associated SNPs from dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) and the HapMap project [Frazer et al., 2007], or manually enter 3'UTR sequences and SNPs.

MicroSNiPer is useful for labs carrying out in-house discovery and characterization of novel transcripts and SNPs. This package makes it possible to combine catalogued and novel SNPs, as well as to manually enter or edit 3'UTRs based on newly available experimental data. Computational prediction of SNP impact on miRTS, followed by the experimental validation of the miRNA binding, can determine whether this is a functional SNP affecting gene expression. We consider MicroSNiPer as a useful tool that can complement other SNP-centered tools such as an the open source SNP-Laboratory Information Management System [Barenboim et al., 2003] for those laboratories that routinely discover and analyze novel transcripts and SNPs.

Implementation

The design of MicroSNiPer is presented in Figure 1. The first step is to choose between two alternative input methods, manual or automatic. The first method is manually entering in a 3'UTR sequence and the corresponding SNPs. The other method is to load a 3'UTR and SNPs automatically from the database. MicroSNiPer allows the user to search 3'UTRs by either specifying the gene name or RefSeq ID (Figure 2A) or alternatively, by entering in the SNP rs ID (Figure 2B). After pressing the Search button, all known RefSeq IDs associated with this gene will be displayed. If a gene consists of several distinct isoforms, multiple RefSeq IDs will be listed. The user must select one RefSeq ID and a SNP collection from an appropriate population (Figure 2). With this choice, a new page will appear with all known SNPs that reside within the 3'UTR (Figure 3).

Figure 1
The schematic pipeline of MicroSNiPer. The web interface consists of the opening screen (Page 1) where manual or automatic selection of the 3'UTR sequence and of a particular set of SNPs is chosen. The 3'UTR and the SNPs within the sequence is loaded ...
Figure 2
The initial screen of MicroSNiPer. The user can specify the input method (either manual or from database), a set of SNPs (either validated dbSNPs or HapMap SNP sets). If user use MySQL database he/she can search the database either by gene name or by ...
Figure 3
The second screen of MicroSNiPer. The user chooses a miRNA sets (`hsa' - human, `mmu' – mouse full miRNA or seed sets), edits the 3'UTR. The user can add novel SNPs in the format `SNP id, position, alleles', update the SNP list to include SNPs ...

3'UTR sequence and SNP database

To store the 3'UTR sequences and SNPs, a SQL database using the MySQL DBMS has been built. The 3'UTRs and SNPs from dbSNP and HapMap have been downloaded using the UCSC table browser (Table 1). Only SNPs residing within the 3'UTRs have been stored in the database. There are two tables in the database. The first table stores gene names (18,026 genes), and all 3'UTR sequences with RefSeq IDs (26,357 IDs), using the ID as the primary key. The second table stores all RefSeq IDs with their corresponding SNPs. JavaScript code (Ajax) is used to search these tables for the presence of SNPs and 3'UTR sequences without reloading the webpage.

Table 1
Statistics of the SNPs in the MySQL database

miRNA sets

Human and mouse miRNAs have been downloaded and stored as a flat file table from miRBase. The user can select either the set of full length miRNAs or the set of miRNAs truncated from 2 to 8 nucleotides (`the seed region') as the library to query (Figure 3).

A user has the option to choose one of four possible sets of SNPs located within the 3'UTR. These sets have been extracted from dbSNP, version 29, (http://www.ncbi.nlm.nih.gov/projects/SNP/) or from the HapMap project [Frazer et al., 2007]. We use only validated SNPs from dbSNP and SNPs from HapMap with MAF ≥ 0.01, i.e. CEU, CHB, JPT and YRI (Table 1, Figure 2).

The next webpage allows the user to edit the 3'UTR and to add or remove the SNPs loaded in from the database (Figure 3). On the same screen, the user can also choose whether to use the full-length miRNAs or only seed regions (2–8 nucleotides from the 5'-end of miRNA).

By clicking on the Run button, the MicroSNiPer algorithm and FASTA program is invoked on the datasets. The results are graphically presented within a HTML page (Figure 4). This provides unambiguous visualization of the results. The web-page includes links from the miRNAs to miRBase (http://miRNA.sanger.ac.uk/).

Figure 4
The output display showing the microRNA-binding sites. The SNPs in the overlapping region are presented on actual page in red. Information about the SNP, rs numbers, position on the 3'UTR and alleles is shown. The 3'UTR sequence and the allele in the ...

Specifications

This application is platform independent. MicroSNiPer was developed using Perl, PHP, and JavaScript. The script is embedded within a PHP-based web-interface and it uses the MySQL relational database as the backend database. MicroSNiPer is publicly available from the internet (http://cbdb.nimh.nih.gov/microsniper/).

MicroSNiPer has been developed using the Perl language, a MySQL database system for data storage. The web interface was created using standard development tools (HTML, PHP, Ajax, and JavaScript), and a CGI Perl module running on a web-server Apache 2 (http://www.apache.org) using Red Hat Enterprise Linux 5.3, handled the exchange of information between the user and the database. FASTA (version 35) package was downloaded from the University of Virginia (http://fasta.bioch.virginia.edu).

The current release of mature human and mouse the miRNAs (release 13.0) has been downloaded from miRBase (http://miRNA.sanger.ac.uk/) [Griffiths-Jones et al., 2008]. Sets of mature human and mouse miRNAs and sets of seeds miRNAs (heptamers: from 2 to 8 nucleotides from the 5'-end) were assembled and stored as flat files. The current version of MicroSNiPer allows the user to choose between sets of mature human or mouse miRNAs or just the seed region.

Results

Description of the algorithm

Genomic features used to identify miRNA Target Sites (miRTS)

We used the seed region binding in the miRNA as the major criterion in our algorithm. We applied the FASTA alignment program to determine if a change in an allele would shift the miRNA along the 3'UTR sequence, indicating a creation/disruption event in the miRNA target site. The alignment between the 3'UTR and the miRNA requires an uninterrupted match of at least 6 nucleotides from the 5'-end of miRNA which would mimic what is observed experimentally.

Processing of the 3'UTR sequence and the SNPs

On the initial screen, a user is able to select the SNPs of particular interests, e.g., specific SNPs associated with a given disease or SNPs within a region known for its association with a disease. MicroSNiPer constructs 3'UTR sequence variants with all possible combinations of alleles (haplotypes) in separate files. These 2k sets (haplotypes) of 3'UTR sequence variants are used as input to FASTA, where k is the number of SNPs included in analysis. Running haplotypes through FASTA is particularly important when two SNPs are positioned within a range of 22 nucleotides so their combination might improve or weaken the binding of the miRNAs. Also a user can conveniently run several SNPs in one run. Currently, the limit is set to 6 SNPs running simultaneously.

Modifications of the FASTA algorithm in MicroSNiPer

FASTA is a heuristic algorithm for finding significant matches between a query string (e.g. 3'UTR) and a database string (e.g. miRNAs) [Pearson and Lipman, 1988]. It finds the most significant diagonals in the dot-plot or dynamic programming matrix. A word-size parameter, k (ktup), is set to 6 by default for DNA which is also the minimum size of a miRNA seed. FASTA combines high scoring sub-alignments into a single larger alignment, allowing gaps into the alignment. The miRNA length is ~22 nucleotides leading to high E-values. A banded Smith-Waterman dynamic program is used along the best matched regions. If the banded Smith-Waterman score is equal to zero, the dynamic programming algorithm did not yield any alignment and no alignment will appear in the output.

The FASTA algorithm imposes a penalty for opening a gap and for extending the gap. FASTA outputs the single best alignment and this might occur before the SNP region in the 3'UTR. To force the FASTA algorithm to choose the best match in the SNP region, MicroSNiPer masks all the nucleotides more than 50 nucleotides downstream from the SNP with a character `N's usually reserved for repetitive elements (Figure 5A). FASTA ignores this stretch of `N's.

Figure 5
MicroSNiPer pre-processing of FGF20 input sequence before running with FASTA program and its FASTA output. (A) Two FGF20 input sequences in separate files generated with SNP rs12720208 in position 182[C/T]. (B) Two FASTA program output files corresponding ...

Using the MicroSNiPer interface, the user has the ability to handle miRNAs or seed sequences as RNA sequences by setting the -U flag. Thus, in addition to the canonical base-pairing, FASTA allows G:U base pairs by scoring “G-A” and “T-C” as “G-G”.

MiRTS-SNP detection and filtering

The FASTA output indicates the coordinates of the base-pair binding interval (overlap) in the 3'UTR sequence. A change in either the overlap, indicating a shift in the 3'UTR target site, or a mismatch arising from the alternative allele, is used to filter the FASTA result file. Six contiguous matches starting from the first to the third nucleotide on the 5'-end of the miRNA is required for each record in the output file (Figure 6). There will be many 6-mers that are fully complementary to a 3'UTR sequence. However, MicroSNiPer selects only those miRNAs which have a SNP located within the target site. This constraint significantly reduces the number of candidate miRNAs in the output. If the alternative allele does not change the base pairing of the miRNA/seed to the 3'UTR, this SNP will have no effect on the alignment (Figure 6A). Using this condition, MicroSNiPer outputs only those miRNAs/seeds where the alignment has been shifted due to a change in the alleles. Another condition MicroSNiPer uses to process the raw FASTA output is to require that at least one SNP occurs between the initial and final nucleotide bond (Figure 6A).

Figure 6
Process of selection of miRNAs and miRTS for MicroSNiPer output. (A) Flowchart of the selection procedure by MicroSNiPer. The FASTA output indicates the coordinates of the base-pair binding interval (overlap) in the 3'UTR sequence. A change in either ...

MicroSNiPer builds a unique lookup key composed of the miRNA name, e.g. hsa-miR-340, the banded Smith-Waterman score, e.g. score `46' or `38', and the coordinates of the overlap, e.g. (198-180:2-21) or (198-187:2-14) (Figure 5B, Figure 6B). MicroSNiPer processes the FASTA output files looking for a shift in the overlap region. It will report only those cases where at least one SNP is present within the miRNA overlap.

By default, the output from MicroSNiPer shows matches with a minimum overlap of 6 base pairs. In order to increase the specificity, a user is able to increase the minimum seed length. This action filters out targets with overlaps less than a given threshold.

Limitations of the MicroSNiPer algorithm

The processing of the FASTA output requires a seed region of at least 6 nucleotides (Figure 6A). FASTA reports only one sequence per run. When selecting multiple SNPs for a single run, MicroSNiPer can miss reporting additional target sites if the additional target sites have identical SNPs in them and the FASTA algorithm calculates the identical Smith-Waterman score for a given miRNA. This rare event can be prevented by selecting a single SNP per run.

Validation

Even though there are approximately 1300 known experimentally supported miRTS [Papadopoulos et al., 2009], there are only 7 experimentally confirmed cases where the disease-associated SNPs positioned in the miRTS have an effect on the miRNA binding [Abelson et al., 2005; Adams et al., 2007; Jensen et al., 2009; Kapeller et al., 2008; Mishra et al., 2007; Sethupathy et al., 2007; Tan et al., 2007; Wang et al., 2008]. We have used [Sethupathy and Collins, 2008] which compiled and reviewed most of the known cases where miRTS SNPs affected the miRNA binding (Table 2). From [Sethupathy and Collins, 2008], we choose only in vitro/in vivo validated interactions. From 7 cases, 5 were corroborated using MicroSNiPer while the remaining 2 either had a seed region of less than 6 nucleotides while in the other case the SNP was outside the miRTS.

Table 2
MicroSNiPer approach validated with experimental data modified from table in [Sethupathy and Collins, 2008] based on 3'UTR SNPs

There exits two other databases with similar purpose to MicroSNiPer: PolymiRTS [Bao et al., 2007] and Patrocles [Hiard et al., 2010]. Comparing their performance with MicroSNiPer, in the absence of large sets of experimentally confirmed miRTS-SNPs, is difficult. We ran validated examples from Table 2 on PolymiRTS and Patrocles. From 7 cases, only 2 were corroborated with either PolymiRTS or Patrocles `Polymorphic Targets' precomputed database (Table 2). In the case of gene SLITRK1, there was no option in PolymiRTS to enter a SNP lacking a dbSNP rs number. Patrocles has a `Finder' utility where a user can manually enter polymorphic 3'UTR sequences. Applying this utility, we obtained results similar to MicroSNiPer's output, though additional matches were included that have not been experimentally validated. The `Finder' utility requires the user to manually enter and alter a DNA sequence which makes it difficult to apply.

It is difficult to quantitatively compare the performance of all these tools using only 7 miRTS-SNPs. PolymiRTS and Patrocles are less flexible than MicroSniPer in giving the user the ability to choose sets of miRNAs based on their experimental data. In our opinion, a researcher studying individual genes would prefer more matches, including even some false positive hits, rather than an empty set. MicroSNiPer has an option to improve the specificity by selecting a longer overlap in the seed region. However, this option can result in fewer true positives because the consensus is that most mRNA binding sites requires only 7 base pairs.

Case study - FGF20

Using MicroSNiPer, we analyzed the human FGF20 3'UTR containing four validated SNPs. We identified hsa-miR-433 having a seed entirely complementary to the target site when containing SNP rs12720208 C-allele (Figure 6B) but not with the T-allele. The T-allele disrupted this seed region for has-miR-433. Binding of hsa-miR-433 to this region has been experimentally confirmed and rs12720208 has been shown to be a functional SNP affecting gene expression [Wang et al., 2008].

Surprisingly, neither Patrocles precomputed dataset nor PolymiRTS resulted in any target sites overlapping rs12720208 in the FGF20 3'UTR. This is one of the best characterized and experimentally validated cases of a SNP effect on miRTS where hsa-miR-433 binds to the C-allele but not to the T-allele. Only by manually entering in the C and T allele variants of the FGF20 3'UTR into the Patrocles's `Finder' utility, did the program yield positive results, albeit including false positives. It is true that MicroSNiPer found 9 miRTS for the same SNP. However, increasing the threshold to an 8-mer seed overlap resulted in the correct prediction. In addition, having knowledge about the directionality of expression of FGF20 relative to an allele, gives a researcher the ability to remove extra miRNAs which putative impact do not conform to this directionality. A user can further eliminate the miRNAs by focusing only on miRNAs expressed in tissues under investigation.

Discussion

Mature miRNAs are non-coding RNAs approximately 22 nucleotides long and derived from stem-loop structures. They are delivered to the silencing protein complex, RISC, repressing the expression of target mRNA at the post-transcriptional level.

There are two important steps in the computational identification of potential miRNA binding sites: 1) complementarity analysis with a miRNA seed, requiring 6 or more contiguous matches 2) cross-species conservation of a seed [Lewis et al., 2005]. However, if the potential binding site is in a novel transcript and the 3'UTR is not conserved, existing programs might not detect a target site. It is also possible that the allele on the reference sequence does not create a target site while the alternative allele does.

The new studies show that one miRNA could have multiple binding sites on a single 3'UTR. G:U wobbles in the seed region, bulges and 3'-compensatory sites also adds to the complexity of site prediction of miRNA binding [Didiano and Hobert, 2006; Vella et al., 2004; Yekta et al., 2004]. So far, the major criterion increasing the specificity of finding binding-sites is a complementarity between the 5'end of miRNA and its 3'UTR target site [Lewis et al., 2005] and MicroSNiPer is able to utilize this characteristic of miRNA binding.

MicroSNiPer can be applied to evaluate not only 3'UTR as a target sequence but also any RNA/DNA sequence of interest which can be manually entered on the second webpage. This could be useful as new experimental data emerges that miRTSs are present in 5'UTRs or even in the gene ORFs [Kloosterman et al., 2004; Lytle et al., 2007]. This is one of the most important distinctions of MicroSNiPer from the PolymiRTS and Patrocles. These tools has a precomputed output while MicroSNiPer is doing computation on-the-fly allowing to alter sequences and add a novel SNPs in the list of existing dbSNPs. MicroSNiPer is able to compute an output using a haplotypes which are the combinations of all alleles on 3'UTR. It is conceivable that two SNPs positioned within target site range could create a novel miRNA target site with particular combination of their alleles. MicroSNiPer will detect this newly created miRTS automatically.

What to do next with MicroSNiPer results?

There are several approaches that a scientist can use to apply additional experimental data and databases in order to evaluate the feasibility of the MicroSNiPer prediction to focus on the most plausible miRTS. First of all, it is possible to classify miRTS types according to [Bartel, 2009] as 7mer-A1, 8mer, 6mer sites, etc (Figure 7). The [Bartel, 2009] provides a pie chart with frequencies of each type. Analysis of different modes of miRNA targeting reveals that the 3'-compensatory binding, a pairing on the 3'-end of miRNA lacking a `seed' at 5'-end, comprises less than 1% of in the binding at the conserved sites in mammals [Bartel, 2009].

Figure 7
Generalized types of miRNA target sites. (A) Canonical and marginal sites together with (B) 3'-supplementary sites comprise approximately 99% of experimentally validated target sites; (C) 3'-compensatory sites lacking 5'-miRNA seed comprise ~1% of known ...

After using MicroSNiPer, one can estimate the degree of conservation of a predicted miRTS by entering the 3'UTR target sequence into the UCSC genome browser (http://genome.ucsc.edu/) with the conservation track engaged. A high degree of conservation provides additional confidence of the validity of the miRTS. Obviously, the expression of the miRNA and its target has to coincide both spatially and temporally, i.e. be expressed in the same tissue and at the same developmental stage. Several additional databases can facilitate this analysis, namely, miRGator, miRNAmap, smiRNAdb [Hsu et al., 2008; Landgraf et al., 2007; Nam et al., 2008]. Also, it is worthwhile to apply RNA secondary structure programs which estimate the occlusion of miRTS in the secondary structure. Also, alternative alleles could expose miRTS which can facilitate binding of miRNAs and that could be an additional point to study this miRTS further. Commonly used programs for secondary structure predictions are Mfold, RNAfold [Hofacker, 2003; Hofacker and Stadler, 2006; Zuker, 2003]. The program, STarMir, estimates energetic characteristics of hybridization between a target forming secondary structure and a miRNA [Long et al., 2008].

In our opinion, in light of the increasing amount of data regarding the involvement of miRNAs in a wide range of developmental and regulatory processes, miRNA computational tools, including MicroSNiPer, will be more in demand, particular as experiments validate new miRNA- target interactions and elucidate the principles of binding beyond the `seed region' rule.

Conclusion

A web-based application, MicroSNiPer, predicts the impact of a SNP on putative microRNA targets. From linkage and whole genome association studies of disease, the importance of SNPs positioned in the 3'UTR regions is becoming evident. MicroSNiPer straightforward, and adaptable tool, will be useful for a wide range of studies that are characterizing novel transcripts and SNPs linked to disease. This makes MicroSNiPer's output more relevant to the specific research goals. This approach distinguishes MicroSNiPer from precomputed databases predicting impact of SNPs on miRNA targeting. Its benefits also include ease of use, flexibility and simple graphical representation of the results.

Availability and requirements

MicroSNiPer is freely accessible via http://cbdb.nimh.nih.gov/microsniper. It is platform independent and compatible with web-browsers Firefox 3.5, Internet Explorer 7 and higher. MicroSNiPer was developed using Perl, PHP, JavaScript and MySQL DBMS.

Acknowledgments

MB conceived and designed the software. MB, BJZ, YZ implemented the software. DRW developed requirements, planned and directed the project. All authors participated in writing the paper and approved the final version.

The work was supported by the Intramural Research Program of the National Institute of Mental Health, NIH.

Footnotes

Competing interests The authors declare that they have no competing interests.

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