Micro RNA in plants and roots

05/02/2023 Sarbesh D. Dangol, PhD Agricultural Genetic Engineering

An introduction to miRNAs and a brief overview of roles

of miRNAs in root development in plants

Presented by:Sarbesh D. Dangol

(PhD student, Agricultural Genetic Engineering)


What is miRNA?

• A microRNA (miRNA) is a 21–24 nucleotide (nt) dsRNA.

• Small RNA that is the final product of a non-coding RNA gene.

• miRNA genes contain introns.• miRNA genes are capped, spliced and

polyadenylated.


General structure of an miRNA gene

In Eukaryotes


Functions of miRNAs

Control of gene expression by regulating: • Transcription factors• Stress response proteins• Proteins that impact development, growth

and physiology of plants.

05/02/2023

miRNAs may arise from introns of protein coding genes

Sarbesh D. Dangol, PhD Agricultural Genetic Engineering


MIR transcription• Most plants possess over 100 MIR genes.• Located mainly in intergenic regions

throughout the genome.• MIR genes transcribed by RNAP II. • Pri-miRNAs are stabilized by addition of 5’ 7-

methyalguanosine cap and 3’ polyadenate tail.



Alternative splicing of miRNAs



HATs and HMTs


Biogenesis and action of miRNAs


Dicer structure


Argonaute proteins


Pri-miRNA processing• pri-miRNA stem loops are processed into

miRNA:miRNA* strands.• 2-nts 3’ overhangs created by DCL RNase III

endonucleases. • Initial cleavage near the base of the stem.• Subsequent cleavages at ~21-nts intervals

along the stem.

05/02/2023

Sizes of miRNAs and its roles• Predominately 21-nts.• But DCL members can generate sRNAs with

distinct sizes: a) 21-nts for DCL1 and DCL4 b) 22-nts for DCL2 c) 24-nts for DCL3• Intramolecular spacing between RNaseIII

active site and 3’overhang binding pocket of PAZ domain determine length.

• 22-nts miRNAs can trigger production of siRNAs from target mRNAs.

Sarbesh D. Dangol, PhD Agricultural Genetic Engineering


NOT2b in miRNA regulation

• In Arabidopsis, NOT2b interacts with pol II CTD for effcient transcription of MIR and protein coding genes.

• NOT2b interacts with several pri-miRNA processing factors.

• Acts as a scaffold for assembly of larger transcription/splicing/processing complexes.



miRNA stabilization and degradation

• 3’ nts of plant miRNA/miRNA* duplexes are 2’-O-methylated by methyltransferase HEN1.

• SDN1 has 3’-5’ exoribonuclease activity which can degrade 2’-O-methylated substrates.

• SDN1 is inhibited by 3’ oligouridylation.• HESO1 adds 3’ oligouridylate tails to

unmethylated miRNAs.


miRNA stabilization and degradation

• miRNAs protected and stabilized by AGO-associated miRISCs.

• Large number of AGOs decrease miRNA accumulation.





miRNA expression

• Tissue- or stage-specific manner.• Induced by external stimuli.• Highly variable at distinct developmental

stages.


Regulation of miRNAs

• siRNA antisense to miRNA precursor able to deplete generation of mature miRNAs.

• miRNAs* could bind to their complementary sites on their precursors to exert cleavage.

• Two or more AGOs compete for one miRNA and other sRNA thrive to incorporate into specific AGO complex.

• Many targets of endogenous miRNA upregulated on siRNA transfection (again competition of siRNA with miRNA).


miRNA diffusion

• miRNAs and siRNAs are also implicated in long-distance transport through phloem rather than just cell to cell movement.



miRNAs in taproot thickening of radish

• 98 differentially expressed miRNAs identified in radish taproot (Yu et al., 2015).

• Differentiallly expressed miRNAs might play crucial regulatory roles during taproot thickening.


miRNAs in radish root thickening

05/02/2023

miRNAs in root development• miR160: root cap formation in Arabidopsis by

targeting ARFs (Auxin Response Factor).• miR164: Normal lateral root development in

Arabidopsis by targeting NAC1.• miR167: In adventitious rooting by targeting ARFs. • miR390: Involved in auxin signaling pathways.• miR393: In anti-bacterial resistance by repressing

auxin signaling. • miR398: Cu/Zn homeostasis.• miR399: In response to phosphate starvation.• miR169: In response to drought. Sarbesh D. Dangol, PhD Agricultural

Genetic Engineering


miRNA roles during symbiosis

• Repression of plant defense during symbiosis. • miRNAs trigger formation of mycorrhized

roots and nitogen-fixing nodules. • miR160, miR164, miR167 and miR393 were

regulated when inoculated with rhizobia.• miR166 and miR169 involved in controlling

nodulation.


References1. Yan Z. et al. (2016). Identification and functional characterization of

soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection. Plant Biotechnology Journal. 14: 332–341.

2. Ruang Y. et al. (2015). Transcriptome profiling of root microRNAs reveals novel insights into taproot thickening in radish (Raphanus sativus L.). BMC Plant Biol. 15:30.

3. Rogers K. and Chen X. (2013). Biogenesis, turnover, and mode of action of plant MicroRNAs. The Plant Cell. 25: 2383-2399.

4. Bazin J. et al. (2012). Complexity of miRNA-dependent regulation in root symbiosis. Phil Trans R Soc B. 367: 1570-1579.

5. Meng Y. et al. (2011). The regulatory activities of Plant MicroRNAs: A More Dynamic Perspective. Plant Physiology. 157: 1583-1595.

6. Meng Y. et al. (2010). MicroRNA-mediated signaling involved in plant root development. Biochemical and Biophysical Research Communications. 393: 345-349.


Thank you.

Science

Micro RNA in plants and roots