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In the past decade, the microRNAs (miRNAs) have emerged to be important regulators of gene expression across various species. Several studies have confirmed different types of post-transcriptional modifications at terminal ends of miRNAs. The reports indicate that miRNA modifications are conserved and functionally significant as it may affect miRNA stability and ability to bind mRNA targets, hence affecting target gene repression. Next Generation Sequencing (NGS) of the small RNA (sRNA) provides an efficient and reliable method to explore miRNA modifications. The need for dedicated software, especially for users with little knowledge of computers, to determine and analyze miRNA modifications in sRNA NGS data, motivated us to develop miRMOD. miRMOD is a user-friendly, Microsoft Windows and Graphical User Interface (GUI) based tool for identification and analysis of 5′ and 3′ miRNA modifications (non-templated nucleotide additions and trimming) in sRNA NGS data. In addition to identification of miRNA modifications, the tool also predicts and compares the targets of query and modified miRNAs. In order to compare binding affinities for the same target, miRMOD utilizes minimum free energies of the miRNA:target and modified-miRNA:target interactions. Comparisons of the binding energies may guide experimental exploration of miRNA post-transcriptional modifications. The tool is available as a stand-alone package to overcome large data transfer problems commonly faced in web-based high-throughput (HT) sequencing data analysis tools. miRMOD package is freely available at .

Plant microRNAs (miRNAs) and their target genes have important functional roles in nutrition deficiency and stress response. However, the underlying mechanisms relating relative expression of miRNAs and target mRNAs to morphological adjustments are not well defined. By combining miRNA expression profiles, corresponding target genes and transcription factors that bind to computationally identified over-represented cis-regulatory elements (CREs) common in miRNAs and target gene promoters, we implement a strategy that identifies a set of differentially expressed regulatory interactions which, in turn, relate underlying cellular mechanisms to some of the phenotypic changes observed. Integration of experimentally reported individual interactions with identified regulatory interactions explains how (i) during mineral deficiency osa-miR167 inhibits shoot growth but activates adventitious root growth by influencing free auxin content; (ii) during sulfur deficiency osa-miR394 is involved in adventitious root growth inhibition, sulfur and iron homeostasis, and auxin-mediated regulation of sulfur homeostasis; (iii) osa-miR399 contributes to cross-talk between cytokinin and phosphorus deficiency signaling; and (iv) a feed-forward loop involving the osa-miR166, trihelix and HD-ZIP III transcription factors may regulate leaf senescence during drought. This strategy not only identifies various regulatory interactions connecting phenotypic changes with cellular or molecular events triggered by stress, but also provides a framework to deepen our understanding of stress cellular physiology.

Trans-acting small interfering RNAs (ta-siRNAs) are a class of endogenous small RNA, associated with posttranscriptional gene silencing. Their biogenesis requires an initial microRNA (miRNA)-mediated cleavage of precursor RNA. Around 20 different ta-siRNA-producing loci (TASs), whose sequences are conserved, are reported in plants. In tomato, two TAS gene families have been identified, which are found to target auxin response factor gene and bacterial spot disease resistance protein Bs4 gene. Using high-throughput computational and experimental approach, we identified a new locus-producing ta-siRNA in tomato. We have also identified the putative miRNA regulating the production of ta-siRNA from this locus. The ta-siRNAs generated from TAS4 were up-regulated upon infection with a DNA virus. The potential targets of ta-siRNAs were predicted to be variety of proteins including MYB transcription factors and cell cycle regulators for some of the ta-siRNAs produced.

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