Computational protocol: Genome-Wide Analysis of mir-548 Gene Family Reveals Evolutionary and Functional Implications

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Protocol publication

[…] All the miRNA members in the mir-548 gene family (http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000317), including their annotations, miRNA/miRNA* and pre-miRNA sequences from different animal species, were obtained from the miRBase database (Release 18.0, http://www.mirbase.org/) []. Multiple sequence alignment of miRNA and pre-miRNA sequences were aligned with Clustal X 2.0 []. Phylogenetic trees of pre-miRNAs based on Neighbor-Joining (NJ) method were reconstructed with MEGA 5.0 [] by 1,000 bootstrap resampling. Nucleotide diversity and average number of nucleotide differences of miR-548-5p and miR-548-3p populations were estimated in DnaSP version 5 software []. Percentage of nucleotide substitution and insertion/deletion at each position was estimated for miR-548-5p and miR-548-3p without considering gaps/missing sites in the terminus regions. The most abundant nucleotide at each position was selected as the reference nucleotide, which would help estimate substitution trend more precisely. Further, phylogenetic network of pre-miRNA members was reconstructed in SplitsTree 4.10 [] by using the Neighbor-Net method [] based on Jukes-Cantor model. Based on the network, we attempted to reconstruct the evolutionary history and discover potential evolutionary pattern. All the gaps/missing data were deleted in the phylogenetic tree. In order to infer ancestral miRNA members to understand origin of miRNAs, we also reconstructed evolutionary network [] with Network 4.6.1.0 (http://www.fluxus-engineering.com/) based on the mature miRNA sequences. Due to various 5′/3′ ends, miR-548 sequences (including miR-548-5p and miR-548-3p) were dealt according to their pre-miRNAs based on the core sequences. Some miRNAs that largely deviated from the core miRNA sequences were removed from the analysis. Based on a great amount of miRNA members in the gene family, we also searched for the phenomenon of interaction between miRNAs according to miRNA and pre-miRNA sequences. If miRNA sequence could be accurately mapped to other pre-miRNAs, potential miRNA:miRNA interaction could be detected. Generally, the miRNA pairs might be located on sense/antisense strands in the same genomic region, or located on different genomic regions. Similar to miRNA:miRNA* duplex, they could form miRNA:miRNA duplex with 5′-/3′-overhangs.We integrated the predicted Target mRNAs of the prediction software programs Pictar [], TargetScan [] and miRanda programs []. These genes were then queried for gene ontology (GO) enrichments by using CapitalBio Molecule Annotation System V4.0 (MAS, http://bioinfo.capitalbio.com/mas3/). We also constructed functional interaction networks using Cytoscape v2.8.2 Platform []. […]

Pipeline specifications

Software tools Clustal W, MEGA, DnaSP, SplitsTree, PicTar, TargetScan, MiRanda
Databases miRBase
Application Phylogenetics
Organisms Homo sapiens
Diseases Neoplasms