characterization of microRNA through bioinformatics & sequencing technology

1. Insilico & Genomics Approaches for the
Characterization of Abiotic Stress Responsive
MicroRNA
Rishiraj Raghuvanshi
M.Sc. Previous
Dept. of Plant Molecular Biology & Biotechnology

2. • Non coding RNA
•Only in eukaryotes

4. 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

6.  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

7. 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…

8. Mechanism of action of miRNA in plants

9. 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

10.  Ab-intio tools for
prediction & validation
 miRNA sequencing and
validation
 Conserved miRNA
homology based search
Methodology

11. 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

12. 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

13. Conserved micro RNA’s in plants

14. Conservation based- database
“MIRFINDER”
http://www.bioinformatics.org/mirfinder/

15. “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

16. Bioinformatics tools & software
for micro RNA

17.  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

18.  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

19.  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…

20.  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

21. 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

22. miRBase

23. Steps of search protocol

24. Conti…

25.  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/

26. psRNATarget

27. Interpretation

28. RegRNA 2.0 is an integrated web server for identifying
functional RNA motifs in an input RNA sequence.
RegRNA2.0

29. How to use RegRNA2.0

30. Results explanation

32. miRNA sequencing and validation

33. 1. Sequencing
2. Data Analysis
 Novel miRNA Discovery
 Differential Expression Analysis
 Target Prediction
 Target Validation
Steps in miRNA sequencing and validation

34. Sequencing

35. Data Analysis In Different Way For Different
Objective
miRNA prediction from
known gene sequecnces
Target Prediction
Discover noval
miRNA
Differential
Expression Analysis

36. Data Analysis

37. 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

38. 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…

39.  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 differential expression
Differential Expression Analysis

40. Case study - l

41.  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

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

43.  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

44. Case study -II

45.  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:

46. Plant datasets
Sequence data were extracted from libraries from
the
Gene Index project
(http://compbio.dfci.harvard.edu/tgi/),
 Datasets belonging to angiosperms
We 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…

47. 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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