Micro RNA in plants and roots

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

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.
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Engineering

General structure of an
miRNA gene
In Eukaryotes
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Engineering

Functions of miRNAs
Control of gene expression by regulating:
• Transcription factors
• Stress response proteins
• Proteins that impact development, growth
and physiology of plants.
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Engineering

miRNAs may arise from introns of
protein coding genes
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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.
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Engineering

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Engineering

Alternative splicing of
miRNAs
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HATs and HMTs
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Biogenesis and action of miRNAs
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Dicer structure
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Argonaute proteins
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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.
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Engineering

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
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.
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Engineering

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.
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Engineering

miRNA stabilization
and degradation
• miRNAs protected and stabilized by AGO-
associated miRISCs.
• Large number of AGOs decrease miRNA
accumulation.
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Engineering

3/30/2016 Sarbesh D. Dangol, PhD Agricultural Genetic
Engineering

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

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).
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Engineering

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

miRNAs in radish root
thickening
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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
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.
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Engineering

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.
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Engineering

Thank you. 
3/30/2016
Engineering

Micro RNA in plants and roots

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