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Azadirachta indica MicroRNAs: Genome-Wide Identification, Target Transcript Prediction, and Expression Analyses

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Abstract

MicroRNAs are short, endogenous, non-coding RNAs, liable for essential regulatory function. Numerous miRNAs have been identified and studied in plants with known genomic or small RNA resources. Despite the availability of genomic and transcriptomic resources, the miRNAs have not been reported in the medicinal tree Azadirachta indica (Neem) till date. Here for the first time, we report extensive identification of miRNAs and their possible targets in A. indica which might help to unravel their therapeutic potential. A comprehensive search of miRNAs in the A. indica genome by C-mii tool was performed. Overall, 123 miRNAs classified into 63 families and their stem-loop hairpin structures were predicted. The size of the A. indica (ain)-miRNAs ranged between 19 and 23 nt in length, and their corresponding ain-miRNA precursor sequence MFEI value averaged as −1.147 kcal/mol. The targets of ain-miRNAs were predicted in A. indica as well as Arabidopsis thaliana plant. The gene ontology (GO) annotation revealed the involvement of ain-miRNA targets in developmental processes, transport, stress, and metabolic processes including secondary metabolism. Stem-loop qRT-PCR was carried out for 25 randomly selected ain-miRNAs and differential expression patterns were observed in different A. indica tissues. Expression of miRNAs and its targets shows negative correlation in a dependent manner.

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Abbreviations

ain-miRNA:

Azadirachta indica microRNA

DCL1:

Dicer-like-1

EST:

expressed sequence tag

GRFs :

growth regulating factors

HEN1:

Hua-Enhancer1

MFE:

minimum folding free energy

MFEI:

minimal folding energy index

NGS:

next-generation sequencing

NF-YA :

nuclear transcription factor Y subunit alpha

pri-miRNAs:

primary miRNA transcripts

pre-miRNAs:

precursor miRNA transcripts

RISC:

RNA-induced silencing complex

SPL :

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE

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Acknowledgments

This research work was financially supported under BSC0203 and Institutional major laboratory project (MLP04) by Council of Scientific and Industrial Research (CSIR), Govt. of India. Research fellowship from University Grants Commission, Govt. of India, is duly acknowledged. AcSIR is acknowledged for the academic support. The authors are sincerely thankful to the Director, CSIR-CIMAP, for encouragement, support and providing the required laboratory facilities.

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Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Near Kukrail Picnic Spot, Lucknow, 226015, India

    Raja Rajakani, Pravin Prakash, Dolly Ghosliya, Ranjana Soni, Arpita Singh & Vikrant Gupta

  2. Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Kamla Nehru Nagar, Sector-19, Ghaziabad, UP, 201002, India

    Raja Rajakani, Ranjana Soni & Vikrant Gupta

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Contributions

RR and VG conceived and designed the experimental plans. RR, PP, and DG applied the plan and carried out experiments. RS and AS helped in data collection and analysis of experiments. RR carried out the stem-loop qRT-PCR, qPCR analysis, and wrote the manuscript. VG guided the experimental designs and finalized the manuscript. All the authors have read and agree to the content of the finalized manuscript.

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Correspondence to Vikrant Gupta.

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Supplementary Information

Supplementary Fig. 1

Schematic representation of in silico analysis strategy used for the identification of ain-miRNAs and their potential mRNA transcript targets (PPTX 82 kb)

Supplementary Fig. 2

Stem-loop hairpin structures of C-mii predicted 123 A. indica miRNAs. Mature ain-miRNA nucleotides are marked in red. The precursor’s nucleotide positions in primary ain-miRNAs are indicated in brackets (PPTX 1878 kb)

Supplementary Figure 2

(PDF 911 kb)

Supplementary Fig. 3

Nucleotide dominance in each base position and nucleotide probability analysis in identified mature ain-miRNA sequences (PPTX 512 kb)

Supplementary Fig. 4

a Blast2GO examination of microRNA targets predicted in A. indica leaf transcriptome SRX096301, b Blast2GO analysis of miRNA targets predicted in A. indica seed transcriptome SRR342216 (PPTX 388 kb)

Supplementary Fig. 5

Expression analyses of ain-miRNAs in different neem tissues (immature fruit, leaf, fruit mesocarp, and fruit endocarp) by using semi-quantitative stem-loop RT-PCR. Randomly selected ain-miRNAs were checked for transcript/expression level (miR156, miR169g, miR169h, miR172, miR396b, miR399, miR403c, miR5021, miR5139, miR5522, miR1533f, miR1533g, miR5512, miR157, miR529, miR776, miR1088-5p, miR1511, miR1525, miR5025, miR1533a, miR1533c, miR1533i, miR1533d, and miR1533h) (XLSX 13 kb)

Supplementary Table 1

Primers used in stem-loop qRT-PCR and qRT-PCR studies (XLS 70 kb)

Supplementary Table 2

A. indica mature miRNA sequences with number of mismatches between miRNA and miRNA*, primary miRNA MFE (kcal/mol), precursor miRNA MFE, and precursor miRNA MFEI (kcal/mol) (DOCX 33 kb)

Supplementary Table 3

A. indica mature miRNA sequences identified by C-mii-based computer prediction and their characteristic features (XLS 69 kb)

Supplementary Table 4

psRNATarget analysis of miRNA targets predicted in A. thaliana transcriptome by using the identified A. indica mature miRNA sequences as query (XLS 114 kb)

Supplementary Table 5

Blast2GO analysis of miRNA targets predicted in A. indica leaf transcriptome SRX096301 by using the identified A. indica mature miRNA sequences as query (XLS 63 kb)

Supplementary Table 6

Blast2GO analysis of miRNA targets predicted in A. indica seed transcriptome SRR342216 by using the identified A. indica mature miRNA sequences as query (XLS 63 kb)

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Rajakani, R., Prakash, P., Ghosliya, D. et al. Azadirachta indica MicroRNAs: Genome-Wide Identification, Target Transcript Prediction, and Expression Analyses. Appl Biochem Biotechnol 193, 1924–1944 (2021). https://doi.org/10.1007/s12010-021-03500-4

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  • DOI: https://doi.org/10.1007/s12010-021-03500-4

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