1. Micro RNAs and Their
Regulatory Roles in Plants
Ambika

2. Introduction
Micro RNAs (miRNAs) are endogenous, 22 nucleotide
RNAs.
Play important regulatory roles in animals and plants
by targeting mRNAs for cleavage or translational
repression.

3. Micro RNAs
• Micro RNAs - non-translated RNAs processed by Dicer
from stem-loop regions of longer RNA precursors.
Chemically and functionally similar to siRNAs –
Mediate
• RNA interference (RNAi)
• Post Transcriptional gene Silencing (PTGS)
• Transcriptional gene Silencing (TGS)
siRNAs are processed from long, double-stranded
precursors.

4. miRNAs and siRNAs
miRNAs and siRNAs incorporated in silencing
complexes contain Argonaute proteins, guide repression
of target genes.
Plant miRNAs are complementary to conserved target
mRNAs.
• Arabidopsis - genetic pathways underlie miRNA
mediated regulation and the phenotypic consequences

5. MicroRNA Gene Discovery:
Cloning
Direct method to isolate and clone small RNAs
• First used to identify large numbers of animal miRNAs
• Some protocols used to enrich for Dicer cleavage products
• Cloning experiments in Arabidopsis identified 19 miRNAs,
which fell into 15 families.

6. •Forward genetic screens in round worms.
• mi RNA involvement in plant mutant phenotypes was not
inferred.
• early extra petals1 caused by a transposon insertion of
MIR164c stem-loop and results in flowers with extra petals
Catherine et al., 2005.

7. Forward Genetics
Mutagenesis coupled with redundancy.
Family members have overlapping functions,
buffering against loss at any single miRNA locus.
Overexpression screens can circumvent redundancy
limitations.
• At least three plant miRNAs, miR319 ,miR172 and
miR166 were with developmental abnormalities

8. Micro RNA Gene Discovery:
Bioinformatics
• Cloning is biased toward RNAs that are expressed highly
and broadly.
Sequence-based biases in cloning procedures might also
cause certain miRNAs to be missed.
Bioinformatic approaches to identify miRNAs have provided
a useful complement to cloning.

9. Bioinformatics
• Find homologs of known miRNAs, both within the same
genome and in the genomes of other species.
First accomplished for vertebrate, nematode, and fly
miRNAs
Numerous potential animal miRNAs - confirmed
experimentally, not been directly useful in finding
plant miRNAs.

10. Conserved Micro RNAs
• Cloning, genetics, and bioinformatics resulted in the
annotation of 118 potential miRNA genes grouped into
42 families.
Each family composed of stem-loops with the potential
to produce identical or highly similar mature miRNAs.
• miR-430 family represented by a cluster of 80 loci in
zebrafish,43 loci in human

11. Conserved Micro RNAs
in Plants
miRNA family Arabidopsis Oryza Populus
miR156 12 12 11
miR166 9 12 17
miR169 14 17 32
miR 162 2 2 3
miR 168 2 2 2
miR 394 2 1 2

12. Conserved Micro RNAs
in Plants
• Twenty miRNA families highly conserved between all
the three sequenced plant genomes.
Several additional miRNA families are conserved only
within specific lineages
• Eg.miR403
• Three families identified in Oryza are conserved in
maize.

13. Conserved Micro RNAs in
Plants
• Pairing and non pairing nucleotides is conserved between
homologous miRNA stem-loops from different species.
Guide DCL1 to cleave at the appropriate positions along
the stem-loop.
Bioinformatic methods have focused on miRNAs conserved
between Arabidopsis and Oryza.

14. Conserved Micro RNAs in
Plants

15. Conserved Micro RNAs
in Plants
• Micro array technology - 11 miRNA families
in gymnosperms, miR160 and miR390 in moss.
Direct cloning of small RNAs from moss identified
additional homologs of Arabidopsis miRNAs.
Conserved miRNA families regulate development in
Arabidopsis and proper specification of floral organ
identity or leaf polarity.
Regulate homologous mRNAs in basal plants –
reproductive structures and leaf morphology.

16. Nonconserved
Micro RNAs
• Homology between some non-conserved miRNA precursors
and target genes provides strong evidence potentially
“young” miRNAs arose from duplications.
Eg.miR161, miR163, miR173, miR447, miR475, and
miR476, are known to direct cleavage of target transcripts

17. Non-conserved
Micro RNAs
• Minimal standard for miRNA annotation
• “Small RNA with detectable expression and the potential
to form a stem-loop when joined to flanking genomic
sequence”
Without conservation of both sequence and secondary
structure, it is difficult to be confident that a given cloned
RNA originated from a stem-loop.

18. Micro RNA
BIOGENESIS
Transcription of Micro RNA Precursors.
Micro RNA Processing and Export.
Micro RNA Incorporation into the Silencing
Complex.

19. Transcription of Micro
RNA Precursors
• Plant miRNAs are produced from their own transcriptional
units.
Animal miRNAs - processed from introns of protein coding
genes.
Plant miRNA genes are occasionally clustered - suggesting
transcription of multiple miRNAs from a single primary
transcript.

20. Transcription of Micro
RNA Precursors
• Northern, EST, and mapping evidence indicate plant
primary transcript are longer.
Splicing is a prerequisite for Dicer recognition.
Plant pri-miRNAs can be over 1 kb in length, undergo
splicing, polyadenylation, and capping.
Relatively little is known about the regulation of miRNA
transcription.

21. P
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22. Incorporation in RISC

23. Plant Micro RNA
Expression
• Microarray technology adapted to rapidly survey expression
profiles of plant miRNAs.
Some are broadly expressed, others in particular organs or
developmental stages.
Expression patterns of miRNA promoter reporter constructs
described for miR160 and miR171.

24. Plant Micro RNA
Expression
Responsive to phyto hormones or growth conditions;
Eg.miR159 - gibberellins , miR164 - auxin treatments
miR393 levels - stresses.
miR395 is undetectable in plants grown on standard
medium, but induced over 100-fold in sulfate-starved plants
miR399 is specifically induced in plants grown on low
phosphate medium.

25. MECHANISMS OF MICRO RNA
FUNCTION
• RNA cleavage
Translational repression
Transcriptional silencing

26. RNA cleavage
• Small silencing RNAs guide Argonaute component of
RISC to cleave a single phosphodiester bond within
complementary RNA molecules.
The cleavage fragments are then released, freeing the
RISC to recognize and cleave another transcript.
Micro RNA-guided slicer activity is present in wheat germ
and Arabidopsis lysates.

27. MicroRNA-Directed Repression
• First miRNAs identified, the lin-4 and let-7 RNAs,
regulate the expression of heterochronic genes
• The original experiments with lin-4 RNA and two of its
targets, lin-14 and lin-28, indicated that lin-4 RNA
repressed the target proteins.
Bagga et al., 2005.

28. Translational Repression

29. Transcriptional
Silencing
Evidence from several organisms that small RNAs are
important for establishing and/or maintaining these
heterochromatic modifications.
Eg. yeast, Dicer produces small RNAs corresponding to
heterochromatic repeats

30. REGULATORY ROLES OF
PLANT Micro RNAs
Identification of Plant Micro RNA Targets
• High degree of complementarity between Arabidopsis
miRNAs and their target mRNAs allowed the confident
prediction of targets.
• First clue to the general paradigm for miRNA target
recognition in plants came from mapping miR171 to the
genome.

31. Identification of Plant Micro
RNA Targets
• miRNA171 has four matches in the Arabidopsis genome: one
is located between protein coding genes and has a predicted
stem-loop structure,
• Other three are all anti-sense to SCARECROW-LIKE (SCL)
genes and lack stem-loop structures

32. Identification of Plant Micro
RNA Targets
Genome-wide screen identified mRNAs containing
ungapped, anti-sense alignments to miRNAs with 0–3
mismatches.
• EST information to annotated genes yielded additional
targets, ta-siRNA precursors.
• Expression arrays useful in identifying miRNA targets
missed by bioinformatic approaches

33. Identification of Plant Micro
RNA Targets
• Non-transcription factor targets (6%) encode F-box
proteins ,indicating a role for miRNAs in regulating
protein stability.
• DCL1 and AGO1 are also miRNA targets, suggesting
that plant miRNAs play a role in tuning their own
biogenesis and function.

34. Experimental Confirmation of
Plant Micro RNA Targets
• Agrobacterium infiltration to observe miRNA – mediated
cleavage of targets in Nicotiana.
Most useful method of miRNA target validation uses
5’RACE to detect in vivo products of miRNA mediated
cleavage.
• 5’ RACE detection - a necessary prerequisite for biological
relevance.

35. Transcription – factor
targets
miR Target A.thali oryza populus Con.
family family ana method
miR156 SBP 11 9 16 5’RACE
miR160 ARF 3 5 9 5’RACE
miR171 SCL 3 5 9 5’RACE
miR396 GRF 7 9 9 5’RACE

36. REGULATORY ROLES
• Multiple groups isolated dcl1 mutants severe mutations
result in early embryonic arrest, partial loss-of function
mutants result in pleiotropic defects.
Eg.ago1, hen1, hyl1, and hst mutants

37. Strategies
• Mutations that impair a fundamental step in miRNA
biogenesis result in misregulation of numerous miRNA
targets.
• Transgenic Arabidopsis can be generated for investigation
of particular miRNA/target interactions through two reverse
genetic strategies.
Make transgenic plants that overexpress a miRNA.
Make transgenic plants that express a miRNA-
resistant version of a miRNA target

38. Over expression in
Arabidopsis

39. Over expression in Arabidopsis

40. Transgenic Arabidopsis
expressing miRNA-resistant
targets

41. Transgenic Arabidopsis
expressing miRNA-resistant
targets

42. summary
• Bio-informatic approaches have identified targets for nearly
all plant miRNAs.
Several experimental methods have been used to confirm
miRNA-target interactions and explore the biological
significance of miRNA-mediated regulation.

43. A small but mighty
that is
RNA world
Thank you