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Insilico & Genomics Approaches for the Characterization of Abiotic Stress Responsive
MicroRNA
Rishiraj Raghuvanshi M.Sc. Previous
Dept. of Plant Molecular Biology & Biotechnology
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• Non coding RNA•Only in eukaryotes
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According to standard nomenclature system Name of any MicroRNA is written as mir-123 miR = Micro RNA (mature form) mir = precursor Micro RNA Number indicates order of discovery, Annotated
with an addional lower case letter e.g.-miR123a & miR 123b, it differs in only one
or two nucleotides.
Nomenclature of micro RNA
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According to recent studies with Arabidopsis,- miR399, miR395, and miR398 are induced in response to phosphate-,
sulfate-, and copper-deprived conditions.
Phosphate homeostasis is partially controlled through miR399
miR399 is upregulated in low phosphate-stressed plants and is not induced by other common stresses.
Micro RNA involved in Abiotic stress response
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Conti…
Wheat miRNAs showed differential expression in response to heat stress by using Solexa high-throughput sequencing.
Among the 32 miRNA families detected in wheat, nine conserved miRNAs were putatively heat responsive.
For example: miR172 was significantly decreased, and miRNAs
(including miR156, miR159, miR160, miR166, miR168, miR169, miR393 and miR827) were upregulated under heat stress
Conti…
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Mechanism of action of miRNA in plants
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Figure: Vertical axis indicates the density of protein-coding genes [TAIR10 (Ath), RGAP6.1 (Osa) and Phytozome7.0 (Aly, Ptc, Bdi and Ppe)] and MIR genes (miRBase17) per million genomic base pairs
Ath (Arabidopsis thaliana) Osa (Oryza sativa) Aly (Arabidopsis lyrata) Ptc (Populus trichocarpa) Bdi (Brachypodium distachyon) Ppe (Prunus persica)
Density of protein-coding and MIR genes in six plant lineages
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Ab-intio tools for prediction & validation
miRNA sequencing and validation
Conserved miRNA homology based search
Methodology
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Conservation– based approaches
Based on sequences conservation across different species
unique characteristic to predict putative miRNA sequences
Twenty miRNA families highly conserved between all the three sequenced plant genomes.
in Arabidopsis, 4 families are conserved down to mosses
20 are shared between Arabidopsis and rice 22 conserved between Arabidopsis & poplar
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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
Conserved micro RNA’s in plants
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Conserved micro RNA’s in plants
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Conservation based- database “MIRFINDER”
http://www.bioinformatics.org/mirfinder/
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“MIRFINDER”
MIRFINDER computational pipelines facilitates:
prediction is based on the conservation between the genomes of Arabidopsis & rice
based on properties of the secondary structure of the miRNA precursor.
MIRFINDER predicted 91 miRNA genes of A. thaliana of those 58 had at least one nearly perfect match with an Arabidopsis
mRNA,based on a comparison of the Arabidopsis and Oryza genomes
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Bioinformatics tools & software for micro RNA
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Public databases provide predictions of miRNA targets
General steps for public databases Determination of miRNA:mRNA binding pairs
Determination of degree of conservation of miRNA: mRNA
binding pairs across species
Observe for evidence of miRNA targeting in mRNA-seq or
protein expression data: where the miRNA expression is high,
the gene and protein expression of its target is low
Target Prediction
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Obtain gene sequence in fasta format
Translate sequence into RNA seq
Put translated seq into the
database/server that can identifying
functional RNA motifs and sites
(RegRNA 2)
We will get the probable motifs and sites
of miRNA into gene seq
miRNA prediction from known gene sequecnces
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For better distinguishing true positive predictions
from false positive predictions, miRNA-seq data
integrated to mRNA-seq data to observe for
miRNA:mRNA functional pairs
RNA22
TargetScan
miRanda
PicTar
Conti…
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Validation of target cleavage in specific mRNAs
performed using
5' Rapid Amplification of cDNA Ends (RACE) with
a gene-specific primer
3.5 Target Validation for Cleaved mRNA
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miRBase is a biological database that acts as an archive of microRNA sequences
• miRBase has three main aim:- To provide a central place collecting all known microRNA sequences
To provide human and computer readable information for each microRNA
To provide primary evidence for each microRNA
Link- www.mirbase.org
miRBase
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miRBase
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Steps of search protocol
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Conti…
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plant small RNA target analysis server which features two important analysis functions: 1) reverse complementary matching between miRNA and target
transcript using a proven scoring schema, and 2) target site accessibility evaluation by calculating unpaired
energy (UPE) required to open secondary structure around miRNA’s target site on mRNA.
PsRNATarget incorporates recent discoveries in plant miRNA target recognition
PsRNATarget is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster.
psRNATarget
http://plantgrn.noble.org/psRNATarget/
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psRNATarget
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Interpretation
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RegRNA 2.0 is an integrated web server for identifying functional RNA motifs in an input RNA sequence.
RegRNA2.0
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How to use RegRNA2.0
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Results explanation
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miRNA sequencing and validation
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1. Sequencing
2. Data Analysis Novel miRNA Discovery Di erential Expression Analysisff Target Prediction Target Validation
Steps in miRNA sequencing and validation
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Sequencing
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Data Analysis In Different Way For Different Objective
miRNA prediction from known gene sequecnces
Target PredictionDiscover noval miRNA
Di erential ffExpression Analysis
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Data Analysis
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1. Obtain reads that did not align to known miRNA
sequences, and map them to the genome
2. RNA Folding Method For the miRNA sequences were an exact match is found, obtain the
genomic sequence including ~100bp of flanking sequence on either
side, and run the RNA through RNA folding software
Folded sequences that lie on one arm of the miRNA hairpin and
have a minimum free energy of less than ~25kcal/mol are short
listed as putative miRNA
The resulting folded sequences are considered novel miRNAs
Novel miRNA Discovery
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3. Star Strand Expression Method (miRdeep)
Novel miRNA sequences are identified based on the
characteristic expression pattern that they display due to
DICER processing: higher expression of the mature
miRNA over the star strand and loop sequences
Conti…
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Comparison of expression levels between samples
miRNA expression is typically examined by microarray
analysis or cloning & sequencing
To identify miRNA that are preferentially expressed Particular time points
Particular tissues or disease states
Use of statistical tests to determine di erential expressionff
Differential Expression Analysis
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Case study - l
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Dissecting the orchestration of regulatory control in a plant cell responding to drought stress.
To identify differentially expressed genes that are regulated by DNA methylation and play a role in drought response.
Methodology
Gene ontology anaysis proteome analysis of 5-azaC treated and drought stressed rice
identifies epigenetically regulated DRG
Objective & Methodology
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Classification of Drought Responsive Genes (DRGs)into nine clusters based on epigenetic/miRNA features and
differential expression
Classification of Drought Responsive Genes (DRGs)
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list of 5468 drought responsive genes (DRGs) of rice identified in multiple microarray studies and mapped the DNA methylation regions found ina genome
identified the chromatin remodeling genes and the genes that are targets of miRNAs.
About 75% of the DRGs annotated to be involved in chromatin remodeling were downregulated.
one-third of the DRGs are targeted by two-thirds of all known/predicted miRNAs in rice which include many transcription factors targeted by more than five miRNAs.
Clustering analysis of the DRGs with epigenetic and miRNA features revealed, upregulated clustern was enriched in drought tolerance mechanisms while the downregulated cluster was enriched in drought resistance
mechanisms evident by their unique gene ontologies (GOs), protein-protein interactions (PPIs), specific transcription factors
CONCLUSIONS
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Case study -II
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MiRNA consensus The 22 conserved miRNA families in angiosperms
were considered for our studies MiR319 and miR159,which encode similar miRNAs, were considered as differentfamilies because they regulate different targets (33).
We considered all members of these families, obtained from miRBASE
• MiRNA target prediction
Methodology used:
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Plant datasetsSequence data were extracted from libraries from the Gene Index project
(http://compbio.dfci.harvard.edu/tgi/), Datasets belonging to angiospermsWe also used the mRNA sequences of A. thaliana
(http://arabidopsis.org) and O. sativa (http://rice.plantbiology.msu.edu/).
Target search was performed using PatMatch (34),
Conti…
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designed a strategy to identify miRNA-regulated genes that is mainly focused on the conservation of
the potential targeting.
Using this strategy we identified and experimentally validated new targets in A. thaliana, even though
this system has already been studied in detail by several different genome-wide approaches
CONCLUSIONS
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References1. 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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49THANK YOU