Biocomputational identification of microRNAs from indigenous Gaddi dog genome

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that regulate post-transcriptional gene expression in eukaryotes and play a key role in a variety of biological and metabolic functions. Through target-specific cleavage of mRNAs, miRNAs affect the activity of mRNAs important for diverse cellular functions such as signal transduction, cell proliferation, differentiation, and disease control, among others. Bioinformatics algorithms for predicting putative miRNAs from genomic sequence data using typical properties of previously found miRNAs like secondary structures, GC content, and minimum free energy index (MFEI), among others, enable cost-effective identification of putative miRNAs. The goal of this study is to uncover putative miRNAs utilizing a comparative biocomputational technique from an indigenous dog breed, the Gaddi, in which miRNAs have yet to be discovered. Individually, the five contig level whole-genome assemblies of Gaddi dogs in our lab (Bio Project: PRJNA843534) were blasted against miRbase's known non-redundant miRNAs dataset of mammalian origin. Minimum E-value, maximum percent identity, and pre-miRNA sequences were filtered out of the results. Furthermore, protein-coding sequences were removed from these pre-miRNA sequences using BLASTX, and the remaining sequences were screened for secondary structure, GC content, and MFEI value. In total, 22 putative miRNAs were detected in individual five samples, ranging from 03 to 06 in number, which were then compared to previously reported mature miRNAs from Canis lupus familiaris and 39 other mammalian species and novel miRNAs were identified. In the genome of the local Gaddi dog, miRNAs have been computationally predicted for the first time. Six miRNAs were selected based on the miRNA with the lowest E value, and they were then validated using real-time PCR, SYBR green chemistry, and U6 as an internal control. Three of the six miRNAs selected (Bta-mir 1277, Mmu-mir-466 m-5p, and Mmu-mir-669f) show no discernible expression in Gaddi dog PBMCs, according to the results of quantitative PCR (qPCR). This study is the first to use whole genome sequencing data to estimate native Gaddi dog miRNAs, followed by empirical validation. These results may serve as a springboard for further research elucidating the regulatory functions of miRNAs in native dog populations.