Micro RNAs

Micro RNAs and Their

Regulatory Roles in Plants

Ambika

Micro RNAs (miRNAs) are endogenous, 22 nucleotide

RNAs.

Play important regulatory roles in animals and plants

by targeting mRNAs for cleavage or translational

repression.

Introduction

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.

Micro RNAs

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

miRNAs and siRNAs

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.

•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.

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

Forward Genetics

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.

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.

Bioinformatics

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

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

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.

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.

Conserved Micro RNAs in Plants

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.

Conserved Micro RNAs in Plants

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

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.

Micro RNA BIOGENESIS

Transcription of Micro RNA Precursors.

Micro RNA Processing and Export.

Micro RNA Incorporation into the Silencing Complex.

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.

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.

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Incorporation in RISC

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.

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.

Plant Micro RNA Expression

MECHANISMS OF MICRO RNAFUNCTION

RNA cleavage

Translational repression

Transcriptional silencing

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.

RNA cleavage

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.

Translational Repression

TranscriptionalSilencing

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

REGULATORY ROLES OFPLANT 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.

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

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

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.

Identification of Plant Micro RNA Targets

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.

Transcription – factor targets

miR family

Target family

A.thaliana

oryza populus Con. 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

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

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

Strategies

Over expression in Arabidopsis

Over expression in Arabidopsis

Transgenic Arabidopsis expressing miRNA-resistant targets

Transgenic Arabidopsis expressing miRNA-resistant

targets

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.

summary

Thank you

A small but mighty

that is

RNA world