1. 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)
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
2. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
3. General structure of an
miRNA gene
In Eukaryotes
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
4. Functions of miRNAs
Control of gene expression by regulating:
• Transcription factors
• Stress response proteins
• Proteins that impact development, growth
and physiology of plants.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
5. miRNAs may arise from introns of
protein coding genes
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
6. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
14. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
15. 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.3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
16. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
18. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
19. miRNA stabilization
and degradation
• miRNAs protected and stabilized by AGO-
associated miRISCs.
• Large number of AGOs decrease miRNA
accumulation.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
23. miRNA expression
• Tissue- or stage-specific manner.
• Induced by external stimuli.
• Highly variable at distinct developmental
stages.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
24. 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).
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
25. miRNA diffusion
• miRNAs and siRNAs are also implicated in
long-distance transport through phloem
rather than just cell to cell movement.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
27. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
28. miRNAs in radish root
thickening
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
29. 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.3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
30. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering
31. References
1. 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.
3/30/2016
Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering