Insilico & Genomics Approaches for the Characterization of Abiotic Stress Responsive

MicroRNA

Rishiraj Raghuvanshi M.Sc. Previous

Dept. of Plant Molecular Biology & Biotechnology

• Non coding RNA•Only in eukaryotes

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

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

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…

Mechanism of action of miRNA in plants

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

Ab-intio tools for prediction & validation

miRNA sequencing and validation

Conserved miRNA homology based search

Methodology

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

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

Conserved micro RNA’s in plants

Conservation based- database “MIRFINDER”

http://www.bioinformatics.org/mirfinder/

“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

Bioinformatics tools & software for micro RNA

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

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

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…

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

 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

miRBase

Steps of search protocol

Conti…

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/

psRNATarget

Interpretation

RegRNA 2.0 is an integrated web server for identifying functional RNA motifs in an input RNA sequence.

RegRNA2.0

How to use RegRNA2.0

Results explanation

miRNA sequencing and validation

1. Sequencing

2. Data Analysis Novel miRNA Discovery Di erential Expression Analysisff Target Prediction Target Validation

Steps in miRNA sequencing and validation

Sequencing

Data Analysis In Different Way For Different Objective

miRNA prediction from known gene sequecnces

Target PredictionDiscover noval miRNA

Di erential ffExpression Analysis

Data Analysis

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

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…

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

Case study - l

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

Classification of Drought Responsive Genes (DRGs)into nine clusters based on epigenetic/miRNA features and

differential expression

Classification of Drought Responsive Genes (DRGs)

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

Case study -II

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:

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…

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

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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