Revitalizing miRNAs mediated agronomical advantageous traits improvement in rice.

One of the key enigmas in conventional and modern crop improvement programmes is how to introduce beneficial traits without any penalty impairment. Rice (Oryza sativa L.), among the essential staple food crops grown and utilized worldwide, needs to improve genotypes in multifaceted ways. With the global view to feed ten billion under the climatic perturbation, only a potent functional master regulator can withstand with hope for the next green revolution and food security. miRNAs are such, miniature, fine tuners for crop improvement and provide a value addition in emerging technologies, namely large-scale genotyping, phenotyping, genome editing, marker-assisted selection, and genomic selection, to make rice production feasible. There has been surplus research output generated since the last decade on miRNAs in rice, however, recent functional knowledge is limited to reaping the benefits for conventional and modern improvements in rice to avoid ambiguity and redundancy in the generated data. Here, we present the latest functional understanding of miRNAs in rice. In addition, their biogenesis, intra- and inter-kingdom signaling and communication, implication of amiRNAs, and consequences upon integration with CRISPR-Cas9. Further, highlights refer to the application of miRNAs for rice agronomical trait improvements, broadly classified into three functional domains. The majority of functionally established miRNAs are responsible for growth and development, followed by biotic and abiotic stresses. Tabular cataloguing reveals and highlights two multifaceted modules that were extensively studied. These belong to miRNA families 156 and 396, orchestrate multifarious aspects of advantageous agronomical traits. Moreover, updated and exhaustive functional aspects of different supplemental miRNA modules that would strengthen rice improvement are also being discussed.

Systems biology approach to study the role of miRNA in promoter targeting during megakaryopoiesis.

The distinct process of megakaryopoiesis requires occurrence of endomitosis for polyploidization of the megakaryocytes. Although, Cyclins, CDKs and have been described to regulate endomitosis, the exact mechanism still remains an enigma. miRNA which were otherwise known as post transcriptional gene silencers are now emerging with various non-canonical functions including gene regulation at pre-transcriptional level by miRNA binding at promoter region. Out of the many processes they regulate, miRNA have been manifested to play a role in megakaryocyte differentiation. In this study an attempt has been made to identify miRNA that could regulate cell cycle genes (Cyclins and CDKs) by targeting their promoters, during megakaryopoiesis. A new computational algorithm was implemented using Perl programming to identify putative targets of miRNA in CDK and Cyclin promoters. Perl script was also used to check nuclear localizing miRNA based on the presence of a consensus sequence. Real-time PCR was performed to analyze the expression of miRNA and their predicted targets in Dami vs. PMA treated Dami cells. Putative targets of miRNAs with longest, high complementarity matches in CDK/Cyclin promoters were obtained. We identified two significant miRNA, miR-1273g-3p and miR-619-5p with longest seed sequence matches. We further identified three main targets (CDK10, CDK11, Cyclin F) through which these two miRNA could regulate cell cycle during megakaryopoiesis. Our results reinforce the role of promoting targeting miRNA in regulation of cell cycle through certain CDK/Cyclins to support the process of endomitosis during megakaryopoiesis.

Generation and characterization of a tomato DCL3-silencing mutant.

DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (lmiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SlDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SlDCL3 levels by ∼64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21- and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in SlDCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SlDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SlDCL4 and SlDCL2b as surrogates for SlDCL3.