Proceedings oI International ConIerence on Computing Sciences

WILKES100 ICCS 2013

ISBN: 978-93-5107-172-3
In silico compositional analysis oI stress related miRNAs in
Arabidopsis thaliana and Oryza sativa
Harpreet Singh
1*
and R. Raheja
2

1
Assistant Professor, Department of Bioinformatics, Hans Raj Mahila Maha Vidyalya, Jalandhar City, Punjab, India, 144008
2
Student, Department of Bioinformatics, Hans Raj Mahila Maha Vidyalya, Jalandhar City, Punjab, India, 144008
Abstract
Plants are oIten subjected to a variety oI biotic and abiotic stress and they have developed diIIerent mechanisms to deal with
these stress elements. Recently with the discovery oI miRNAs and their role in regulation oI various genes involved in growth
and development oI plants, a great interest have been created in investigating the relationship oI miRNAs and stress related
genes. A variety oI biochemical, molecular, and bioinIormatics approaches and technologies are available Ior miRNA
detection and analysis. GC content has been considered as a critical parameter for predicting stress regulated
miRNAs in plants like Arabidopsis thaliana. An investigation was carried out to analyze the composition oI
nucleotides in stress regulated miRNAs in comparison with all miRNAs Irom Arabidopsis thaliana and Oryza sativa. 266
mature and 232 primary miRNAs Irom Arabidopsis thaliana; 548 mature and 496 primary miRNAs Irom Oryza sativa; and 26
stress related miRNAs (both premature and mature miRNAs Irom the two plant species) were analyzed Ior various nucleotide
composition Ieatures (GC per cent, Length and Nucleotide Irequency distribution). The average GC content Ior stress
regulated miRNAs (60) was Iound to signiIicantly greater than all miRNAs in Arabidopsis thaliana (44) and Oryza sativa
(47) Ior both primary and mature miRNAs. Stress regulated miRNAs show more random distribution oI position speciIic
nucleotide Irequency. Also, the length oI miRNA, mature as well as primary was reported to be smaller in stress regulated
miRNAs. These compositional parameters may be used to predict stress related miRNAs in plants like Arabidopsis thaliana
and Oryza sativ
2013 Elsevier Science. All rights reserved.
Keywords: miRNA, Oryza sativa and Arabidopsis thaliana
1. Introduction
Plants are oIten subjected to diIIerent environmental Iactors and pathogens causing signiIicant reduction in
crop yield. The inIluence oI environmental Iactors such as water, temperature, nutrients, light etc is known as
Abiotic stress. However, the eIIect oI pathogens is reIerred to as Biotic stress. MicroRNAs are small, RNA
molecules encoded in the genome oI plants & animals. These highly conserved, ~21-nt RNAs regulate the
expression oI genes by binding to the 3` untranslated region (3`-UTR) oI speciIic mRNA. Each miRNA is
thought to regulate the multiple genes, since hundreds oI miRNA genes are predicted to be present in higher
eukaryotes. miRNAs have emerged as master regulators oI plant growth and development and the evidence oI
involvement oI miR398 in controlling the expression oI stress tolerance genes suggested the role oI miRNAs in
plant stress responses. It has also been observed that the expression proIiles oI most miRNAs involved in plant
growth and development are signiIicantly altered during stress (Sunkar et. al., 2012). MicroRNA primarily
Iunctions as translational repressor (Chen et al., 2003) by binding to complementary target sequence in the
3`UTR oI mRNA. Between 10-30 oI all human genes are targets Ior miRNA regulation. A single target gene
oIten contains putative binding sites Ior multiple miRNA that can bind cooperatively. The pl ant mi RNA
genes are predomi nant l y l ocat ed i n t he Int ergeni c regi ons. However si gni Ii cant number oI
*
Corresponding author. arpreet singh
232 Elsevier Publications, 2013
Harpreet Singh and R. Raheja
miRNA genes in animals located in the introns oI pre-mRNAs. miRBase (http://microrna.sanger.ac.uk/) database
serves as a comprehensive resource Ior microRNA sequence data, annotation and predicted gene targets
(GriIIiths-Jones et. al., 2005). There are currently 24591 entries oI miRNAs that have been deposited in
miRBASE database. A variety oI biochemical, molecular, and bioinIormatics approaches and technologies are
available Ior miRNA detection and analysis (Schena et al. 1995; Eisen and Brown 1999; Mishra et al. 2009). GC
content has been considered as a critical parameter Ior predicting stress regulated miRNAs in plants
like Arabidopsis thaliana. A signiIicant increase in the GC content oI stress regulated miRNA sequences was
observed which Iurther extends the view that miRNAs act as ubiquitous regulators under stress conditions.
(Mishra et. al. 2009). In the present investigation analysis oI nucleotide composition including the GC per cent,
length and nucleotide Irequency distribution was carried out to reveal diIIerence in stress related and all miRNAs
in Arabidopsis thaliana and Oryza sativa with respect to the above mentioned compositional parameters.
2. Material and Methods
To carry out computational analysis oI stress related miRNAs, a set oI miRNAs were retreived Ior Arabidopsis
thaliana and Oryza sativa. The microRNA sequence database, miRBase was used to create dataset oI miRNA
sequences. These miRNAs were grouped into 3 categories. First, containing all the Arabidopsis thaliana
sequences, second containing all Oryza sativa sequences and the third containing all sequences induced by stress
(drought stress, salt stress and biotic). The details Ior the number oI sequences retrieved have been mentioned in
table 1. To IdentiIy the GC content and length Ior all selected miRNAs the GEECEE and INFOSEQ programs
Irom the JEMBOSS package were used, respectively.
3. Results and Discussion
The compositional analysis oI mature as well as primary micro RNAs Irom Arabidopsis thaliana and Oryza
sativa (Table. 1) was perIormed Ior GC , sequence length and position speciIic relative nucleotide Irequency.
Table 1. Details oI Sequences retrieved Irom miRBase.
3.1. GC %
Average GC oI mature as well as the primary miRNAs was calculated using GEECEE program in
JEMBOSS package with an aim to know the variation in normal and stress related miRNAs. The minimum and
maximum GC observed in each oI the three miRNA sequence sets was also calculated. The results oI GC
variation have been shown in Table 2.
Sequences retrieved
Irom miRBase
Arabidopsis
thaliana
Oryza sativa Stress related (Arabidopsis
thaliana and Oryza sativa)
Mature miRNA 266 548 26
Primary miRNA 232 491 26
233 Elsevier Publications, 2013
In silico compositional analysis of stress related niRNAs in Arabidopis thaliana and Oryza sativa
Sequence set Mature miRNA Primary miRNA
Average
GC
content
Minimum Maximum Average
GC content
Minimum Maximum
Arabidopsis
thaliana
44 19 67 38 23 54
Oryza sativa 47 14 86 44 23 83
Stress related
(Arabidopsis
thaliana and
Oryza sativa)
60 43 67 52 31 57
Table 2. Variation in GC
The average GC per cent Ior mature miRNA was Iound to be 44, 47 and 60 in Arabidopsis thaliana,
Oryza sativa and Stress set, respectively. This clearly indicates that stress related primary miRNAs are rich in GC
content as compared to the other two sets. The minimum value oI GC in Stress set (43) is signiIicantly higher
than the other two sets. However, the maximum value oI GC Ior Stress set (67) was less than that observed
Ior Oyza sativa and equal to that observed Ior Arabidopsis thaliana set. Same trends oI variation in GC percent
were observed Ior primary miRNAs with the average GC per cent oI 38, 44 and 52 in Arabidopsis thaliana,
Oryza sativa and Stress set, respectively. Similar results were obtained in an analysis oI Arabidopsis thaliana
stress related miRNAs by Mishra et al. 2009. It may be hypothesized that the GC content may play a signiIicant
role in the interaction oI these miRNAs with their target genes Ior regulating gene expression under stress
conditions. The high GC content may be signiIicant because oI two reasons Iirstly the higher GC ratio gives a
higher chance oI complementarities with the coding regions also having a higher GC ratio, secondly since GC
pair is bound by 3 hydrogen bonds, these bonds are more stable thereby making miRNA binding and degradation
oI the target genes more probable.
3.2. Length (nucleotide bases)
With the objective oI exploring variation in the length oI miRNAs in stress regulated and normal miRNAs, the
average length as well as minimum and maximum lengths Ior all the three sets was calculated using INFOSEQ
program in JEMBOSS. Mature as well as primary miRNAs were used in this analysis. The results are shown in
Table 3.
Generally, the length oI miRNAs varies between 20-24 nucleotides. The average length Ior mature miRNA
was Iound to be 21.12, 21.72 and 20.8, in Arabidopsis thaliana, Oryza sativa and Stress set, respectively. The
minimum length was almost same in all the three sets where as maximum length was observed to be lower (21)
Ior the Stress set as compared to the other two sets. Similar variation was also observed in case oI primary
miRNAs. The average length Ior primary miRNA was Iound to be 169.7, 152.8 and 70.2 in Arabidopsis thaliana,
Oyza sativa and Stress set, respectively. This indicates that stress related miRNAs are relatively shorter than the
normal miRNAs.
3.3. Nucleotide frequency distribution
The relative Irequency oI each nucleotide at each position oI the sequence was calculated by dividing the
absolute Irequencies (position speciIic nucleotide Irequencies) by the no. oI sequences having a nucleotide base
at that speciIic position. Line graphs were plotted Ior each oI the three sequence sets Ior both types oI miRNAs
(mature as well as primary) to compare the variations in the pattern oI position wise nucleotide Irequency
234 Elsevier Publications, 2013
Harpreet Singh and R. Raheja
distribution between stress regulated miRNAs and all miRNAs. The plots Ior mature miRNAs are shown in
Figure 1 to 3.
It can be seen in the above plots that the degree oI randomness in the position wise nucleotide Irequency
distribution is more in the stress regulated miRNAs (Fig 1). While Ior other two sets (Fig 2 and Fig 3) the
position wise nucleotide Irequency distribution is seems to be much more conserved Ior all the nucleotides. These
results are in line with the Iindings oI Mishra et al., 2009.
Sequence set
Mature miRNA Primary miRNA
Average
Length
Minimum Maximum Length Minimum Maximum
Arabidopsis
thaliana
21.12 20 24 169.7 63 689
Oryza sativa 21.72 19 24 152.8 58 541
Stress 20.8 20 21 70.2 98 272
Table 3. Variation in Length
(LeIt) Fig. 1. The nucleotide position wise AUGC distribution Ior stress regulated miRNAs
(Right) Fig. 2. The nucleotide position wise AUGC distribution oI all mature miRNAs (Arabidopsis thaliana)
With respect to the degree oI randomness, similar trends were observed Ior primary miRNAs as observed Ior
mature miRNAs. The degree oI randomness in the position wise nucleotide Irequency distribution is more in the
stress regulated primary miRNAs. The plots Ior the position wise nucleotide Irequency distribution oI primary
miRNAs are shown in Fig 4 to Fig 6 given below.
4. Conclusion
Nucleotide composition Ieatures, particularly the GC per cent appears to be an important parameter
distinguishing stress related miRNAs Irom other miRNAs in Arabidopsis thaliana and Oryza sativa. These
Ieatures can be used to derive a set oI parameters to investigate and predict more miRNAs Irom Arabidopsis
thaliana, Oryza sativa and related plant species.
235 Elsevier Publications, 2013
In silico compositional analysis of stress related niRNAs in Arabidopis thaliana and Oryza sativa








Fig. 3. The nucleotide position wise AUGC distribution oI all mature miRNAs (Oryza sativa)








Fig. 4. The nucleotide position wise AUGC distribution Ior stress regulated primary miRNAs.












(LeIt) Fig. 5. The nucleotide position wise AUGC distribution Ior stress regulated primary miRNAs.
(Right) Fig. 6. The nucleotide position wise AUGC distribution oI all miRNAs (Oryza sativa)
236 Elsevier Publications, 2013
Harpreet Singh and R. Raheja
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237 Elsevier Publications, 2013
Index

A
Arabidopsis thaliana, 233235

M
miRNA in arabidopsis thaliana and oryza sativa, 232
materials and methods, 233
nucleotide position wise AUGC, 235236
results and disscusion, 233235

O
Oryza sativa, 233235