Upload
tnaugenomics-lab
View
1.393
Download
2
Embed Size (px)
Citation preview
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.
P
R
O
C
E
S
S
I
N
G
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