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Micro RNAs are 22nt long noncoding post transcriptional regulators of gene expression found in all eukaryotic cells. miRNAs were first observed by Victor Ambrose, Rosalind Lee and Rhonda Feinbaum in 1993. While studying lin-14 gene in C.elegans they observed that the abundance of LIN-14 protein was regulated by small RNA molecule encoded by lin-4 gene. lin-4 gene encode a 61 nt long miRNA precursor which is matured in 22nt long mature miRNA having sequence complementary to multiple sequences in 3'UTR of lin-14 mRNA .This complementarities inhibits the translation of lin-14 mRNA into LIN-14 protein (Lee et al.1993). Later many other miRNA were discovered showing their role in gene expression regulation. Till now in plants nearly 872 miRNA belonging to 42 different families has been identified in 71 different plants (Zhang et.al.2005). Nomenclature system gives name to miRNA according to their order of discovery (Ambrus et.al.2003). miRNAs are evolutionary conserved in both animals and plants (Weber 2005, Bonnet et al. 2004, Sunkar and Zhu, 2004). In animals both precursor and mature microRNAs are conserved but in plants only mature microRNAs are conserved (Reinhart et al. 2002, Zhang et al.2005). In animals many miRNA such as miR7 and miR18 are conserved from worm to human (Weber 2005, Zhang et al. 2005). In plants also miRNAs and their targets are conserved between monocots and dicots (Bonnet et al.2004, Zhang et al.2005). Conservation of miRNA and their targets indicates deep origin of miRNA in plant phylogeny. On the basis of conservation there are four classes of miRNA namely
Non conserved miRNAs(Zhang et.al.2005)
Conserved miRNAs play important role in conserved gene regulation while the non conserved miRNAs play some specific role such as fiber formation in cotton plants.
MiRNAs play important role in gene expression in plants. miRNAs bind to complementary sequences on target miRNA transcript and cause translational repression and gene silencing. In plants miRNAs have nearly perfect complementary sequence to its mRNA target with few or no mismatched bases. Plant miRNAs have their target located in 3'UTR or coding region of mRNA. Plant miRNA play important roles in regulating plant growth and development, differentiation, biotic and abiotic stress, responses (Bartel. 2004, Zhang et al. 2005).
Biogenesis of miRNA:-
Biogenesis of miRNAs includes multiple steps to form mature miRNA from miRNA gene.
Transcription of miRNA gene-
Transcription of miRNA genes takes place by RNA polymerase II (Bartel. 2004, Lee et al.2004). RNA pol II generates primary miRNA which contain an imperfect loop structure and a long sequence of several nucleotides.
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Fig. Biogenesis of miRNA in plants
Nuclear processing of Primary miRNA (Pri miRNA)-
Pri miRNA is excised to stem loop intermediate precursor miRNA (Pre miRNA) by the action of a double stranded RNase called Dicer like I (Tang et.al.2003, Kurihara.2004, Briere et.al.2009). DCL I is associated with some other dsRNA binding Proteins like HYPONASTY LEAVES I (HYL I), SERRATE (SER) and DAWDLE (DDL) proteins, for miRNA processing. Pre miRNA is further processed by same DCL I protein complex to generate a 20-22 nucleotide long canonical ds miRNA/miRNA* duplex (Yu et al.2008). This double stranded pre miRNA is having 2 nucleotide overhang at 3' ends on both sides .this ds pre miRNA is methylated at 3' ends by HUA ENHANCER I(HEW I) protein. Double stranded pre miRNA is then dissociated by DCL I protein complex into two strands namely guide and passenger strand. This is called mature miRNA and transported to cytoplasm.
Transport of miRNA to Cytoplasm:-
Transport of mature miRNA to cytoplasm takes place by exportin homolog HASTY (Lund et al.2004, Zeng and Cullen.2004, Park et al.2005).Passenger strand (miRNA*) is generally destroyed and the guide strand is involved into RNA induced Silencing Complex (RISC) (Briere et al.2009).
Mechanism of gene regulation by miRNAs:-
Mature miRNA is a part of an active RNA induced silencing complex. RISC is the heterogeneous molecular complex that can be programmed to target almost all type gene silencing. RISC programming is triggered by the appearance of dsRNA in the cytoplasm of a eukaryotic cell (Pratt and Machae.2009). The dsRNA is processed into small regulatory RNA that assembles into RISC and guide complex to complementary RNA targets. Once programmed with target RNA RISC can silence gene at following levels:-
At genome level through formation of heterochromatin or by DNA elimination
At post transcription level through mRNA degradation
At translation level by translational repression
Every RISC contains a member of argonautes protein family that binds to miRNA. Role of Argonaute protein is to bind the miRNA and position it in a conformation that facilitates target recognition.
Argonaute proteins are well defined in prokaryotes as compared to eukaryotes. In prokaryotes argonautes bind to small guide DNAs and use these guide to locate and cleave target RNAs. Argonaute proteins have a bilobed structure with each lobe responsible for binding opposite end of guide DNA. The N terminal lobe contains a PAZ domain which binds to 3' end small guide DNA. The C terminal lobe contains a middle domain which binds to 5' phosphate of the guide DNA and the PIWI domain whose terminal carboxy group interacts with the 5' phosphate through co ordination of a divalent cation. PIWI domain adopts RNase-H like fold and can hydrolyze target RNAs by RNase-H like mechanism (Song et.al.2004). A flexible hinge composed of two stranded Î² sheet connect the two lobes. Flexibility in the hinge allows the lobes to pivot relative to each other opening a cleft to accommodate the guide DNA and RNA target (Wang et al.2008). 5' phosphate of the guide DNA is held in a pocket between the middle and PIWI domains which acts as an anchoring point for the DNA in the protein. In eukaryote 5' phosphate license small RNA for entry into the RNAi pathway (Nykanen et al.2001). The first nucleotide base on the 5' end also tucks into a small defined binding pocket. This allows Argonaute protein to make base specific contacts with the first nucleotide in the guide strand. Rest of DNA contacts the protein through its phosphodiester backbone. Two terminal bases on 3' end clamp into a hydrophobic cleft in the PAZ domain.11-18 bases of guide are disordered. This intrinsic plasticity is helpful for Argonaute proteins for accommodating the guide strands of different lengths (Martinez et.al.2002).
Slicing of target mRNAs- The simplest consequence of target recognition is mRNA hydrolysis or slicing which break the reading frame of the encoded protein and promote target degradation by cellular exonucleases (Tolia et al.2007).Target slicing requires a catalytically active Argonaute (slicer) and nearly perfect sequence complementarities between guide and target RNAs. The RNase activity of argonaute PIWI domain catalyzes the slicer cleavage reaction of target RNAs. Cleavage always occurs between the target nucleotide that pairs with bases 10-11 of guide strand (Yuan et al.2005, Elbashir et al.2001). Sometimes RISC can perform multiple rounds of target cleavage.
Transcriptional Silencing and heterochromatin formation:-
Some RISC can act directly on genome. Upon target recognition the complex recruits histone methyl transferase which modify histone associated with DNA locus forming heterochromatin ( Buhler et al.2006). PIWI clade appear to function in transcriptional silencing and heterochromatin formation(Aravin et al.2007).
Translational repression can be carried out either by repression of initiation of translation or repressing translation by deadenylation and degradation of target mRNA.
DNA elimination requires the PIWI clade proteins TWI1 (Mochizuki et al.2004).DNA elimination process is best studied in a ciliate Tetrahymena thermophila. The entire content of newly formed micronucleus is transcribed into small RNAs which are then loaded into TWI1. The resulting RISC then scans the entire genome of old parental macronucleus. Small RNAs complimentary to the old parental macronucleus are discarded. This results in a filtered set of RISCs that target only DNA sequence new to cell from sexual conjugation. These RISCs locate DNA sequences in new macronucleus and tag them for elimination. The result is the elimination of new DNA sequence from transcriptionally active macronucleus which is acquired through sexual conjugation. This process is helpful as a defense mechanism against foreign parasitic DNA sequences (Mochizuki et al.2004)
Identification of miRNAs:-
After the discovery of lin-4 miRNA in 2001, at present thousand of miRNAs have been discovered in various organisms like C.elegans, fishes, mouse, humans and plants. (Lai 2003). Plant miRNAs were identified nearly 10 years later then animal miRNAs.
Currently following approaches are applied for identifying miRNAs:-
Direct Cloning after isolation of small RNAs
Expressed Sequence Tag Analysis
Initially genetic screening is used for identification of miRNAs. Like the identification of traditional genes miRNA genes were also identified using genetic screening (Lee et al.1993, Zhang et al.2005).This method was used for the identification of few miRNAs like lin-4, lin-7 etc. this method suffer from some limitations.
It is an expensive method.
It is time consuming and results are dominated by chance.
Direct Cloning After Isolation of Small RNAs:-
In this method small RNAs are first isolated by size fractionation. Isolated RNAs are ligated to RNA adapters at their 5' and 3' ends. After ligation they are reverse transcribed into cDNA. cDNA is amplified and sequenced (Lu et.al.2005, Zhang et.al.2005). This method is used for identification of miRNAs in various plants and animals. This method is further improved by Lu et.al by combining it with massively parallel signature sequencing (MPSS) to study Arabidopsis miRNAs. This method identifies as well as quantifies miRNA abundance in plants (Lu et.al.2005).
Computational Approach:-This method is based on genome sequences. Various laboratories have designed computational programs to predict miRNA genes (Lin et.al.2003 (a,b)Wang et.al.2005, Unver et.al.2009).
Example of such programs are-
MiRAlign (http://bioinfo.au.tsighua.edu cn/miralign).
This method is not so efficient because predicted miRNAs need to be confirmed by experiments.
EST approach is used to identify miRNA homologies because miRNAs are evolutionary conserved from species to species (Weber.2005). But this method can only identify conserved miRNAs.
Real Time RT-PCR:-
Too overcome poor sensitivity and low throughput of conventional technique now a days a sensitive RT-PCR method is used for miRNA detection.
Recent real time RT-PCR uses stem loop primer followed by a Tagmann assay to quantify and identify miRNAs. Stemloop primers provide better specificity and sensitivity than linear primers (Chen et.al.2005, Varkonyi-Gasic et.al.2007). in plants RT-PCR procedure is carried out for detection of miRNAs from 20pg tissue total RNA and from 0.1 Âµl phloem sap.( Varkonyi-Gasic et.al.2007)
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Fig. Real time RT-PCR method for miRNA identification and quantification. (Varkonyi-Gasic et.al.2007)
Identification of miRNA Targets:-
In plants miRNA targets are predicted easily because miRNA-mRNA targets have significant complementarity. Many tools are used to predict miRNA targets.
Computational Method:- Several algorithms are used for predicting miRNA-mRNA targets in plants.MirU, Mircheck are the softwares which are used for prediction of target mRNAs for miRNA (Rhoades and Bartel.2004, Unver et.al.2009)
Experimental Method: - Genome wide expression profiling is used for predicting miRNA targets.
5' RACE experiment:- RACE means Randomly Amplification of cDNA Ends. This is the most powerful method to confirm miRNA-mRNA targets. Cleaved mRNA product in plants have two diagnostic properties ,one is that the 5" phosphate of a cleaved mRNA product can be ligated to an RNA adapter with T4 RNA ligase. Second is that the precise target cleavage position is that mRNA target nucleotides pairs with the 10th nucleotide of miRNA ( Sunkar et.al.2005, Unver et.al.2009). in 5' RACE experiment total RNA is isolated and poly A RNA is prepared and ligated to a RNA oligo adapter. Oligo dT is used to synthesize the first strand of cDNA with reverse transcriptase. This strand is amplified with 5' and 3' primers for nongene specific amplification. Then 5' RACE PCR and 5' nested PCR are performed using specific primers supplied with kits. RACE products are gel purified, cloned and sequenced.
Plant miRNA Database (PMRD)
The plant miRNA database (PMRD) integrates available plant miRNA data deposited in public databases. This database contains sequence information, secondary structure,
target genes, expression profiles and a genome browser. The PMRD database was constructed by open source technology utilizing a user-friendly web interface, and multiple search tools. It is freely available at http://bioinformatics.cau.edu.cn/PMRD (Zhang et al.2009).
Contents of PMRD:-
miRNA Data collection-
in PMRD data is collected from miRBase ,Rfam and recent literatures. Recently in PMRD there are 1427 entries for Arabidopsis , 2540 entries for rice and 2780 entries for poplar.
miRNA expression profiling -
PMRD database contains miRNA expression profiling for several public miRNA expression datasets including maize miRNA sequence results, poplar cold stress responsive miRNAs, tomato miRNA, expression in leaf tissue etc.( Lu et al.2008, Wang et al.2009)
miRNA target gene, genome browser and stem-loop information-
The target genes collection is curated from literature published in recent years. Location of interaction sites between miRNAs and target genes in Arabidopsis and rice predicted by psRNATarget server are also listed in PMRD database. Information concerning miRNA action on target genes in Arabidopsis, rice and soybean are added in PMRD local gene browser. It also include chromosomal location, secondary structure and dot matrix of miRNA precursor sequence (Zhang et al.2009)
Accessibility of PMRD-
PMRD website is in Hypertext Markup Language (HTML), Perl CGI and MySQL4.0 data engine.
PMRD database web interface enable users to view and analyze each miRNA through browse and search page. Supplementary data is available at NAR online (Zhang et al.2009).
Roles of miRNAs in plants:-
miRNAs are small regulatory molecules with a big impact. In animals miRNAs play important role in many functional processes like development (Weinhold et al.2005), stress responses (Ambros.2003), carcinogenesis (Iorio et al.2005, Croce and Calin.2005).
In plants also miRNAs perform a variety of roles. Some of these are discussed as follows.
miRNAs and plant development-
In miRNAs biogenesis dcl I and hasty genes play important role in synthesis and accumulation of mature miRNAs (Papp et al.2003). Loss of function of the dcl I gene cause many developmental abnormalities including alteration in leaf shape, delayed floral transition, arrested embryos at early stages and female sterility (Park et al.2002, Reinhart et al.2002).loss of function of hasty gene causes pleiotropic phenotypes such as disrupted leaf shapes , flower morphology, reduced fertility, disrupted phyllotaxis of inflorescence (Bollman et al.2003).
Along with these functions miRNAs regulate many development processes in plants like floral differentiation and development, root initiation and development (Mallory et.al.2004b, Guo et al.2005).
miRNA and Leaf Development-
Plant leaves exhibit asymmetric pattern along adaxial/abaxial axis. This pattern is controlled by the polarized expression of class III HD-ZIP transcription factor genes (Juarez et.al.2004). these transcription factors are the targets of miR165 and miR 166 (Emerg et.al.2003, Kim et.al.2005).
miRNA and Floral Development-
In the development of floral organs three classes of transcription factors A, B, C play important role (Lohmann and Weigel.2002) .AP2 is a gene of class A which have an important role in flowering time and floral morphology. AP2 and AP2 like proteins are required for flowering and floral identity. AP2 gene is one if the target of miR172 (Aukerman and Sakai.2003, Chen.2004). Another miRNA, miR171 is also predominately expressed in inflorescence and flower tissues.miR159 and miR156 are also expressed during floral development (Achard et al.2004, Schwab et al.2005).
miRNA in Shoot and Root Development-
Cup shaped cotyledon 1 (CUC1) and CUC2 are two important transcription factors of NAC domain transcription factor family (Riechmann et al.2000). these play important role in embryonic and floral development. These genes are the target of miR164. Over expression of miR164 results in fusion of vegetative and floral parts. miR164 controls organ boundaries and root formation by regulating NAC1 and CUC2 expression (Mallory et al.2004b)
miRNA and Vascular Development-
ATHB15 is a member of HD-ZIP family which is predominately expressed in vascular tissues. ATHB15 is a target of miR166 (Rhoades et al.2002). Over expression of miR166 results in accelerated vascular cell differentiation from cambial or procambial cells and produces an altered vascular system with expanded xylem (Kim et al.2005)
miRNA and Signal Transduction-
Auxin, gibberelic acid and abbsicic acid play important role in plant development. Many miRNAs respond to plant hormones and help in signal transduction by hormones. These include miR159, miR160, miR164, miR167, miR393 etc. (Sunkar and Zhu.2004, Zhang et al.2005, Wang et al.2007).
miRNA and Plant Diseases-
Pathogens infection is an important factor that affects plant growth and development. Pathogens infection causes about 30% yield loss for a majority of crops and fruits. In the long term, plants have developed complex mechanisms to resist infection. One of such mechanisms is pathogen induced post transcriptional gene silencing (PTGS) (Ding 2000, Zhang et al.2005). miRNAs are involved in plant diseases caused by different pathogens and also involved in virus induced gene silencing.
Helper component proteases (HC-Pro), P19, P21 and P69 are viral suppressors of gene silencing (Plisson et al.2003, Zhang et al.2005). these suppressors are called pathogenicity factors. These cause diseases and developmental abnormalities. HC-Pro affect miR171 activity and causes miR171 related developmental defects (Kasschau et.al.2003, Zhang et al.2005, Chapman et al.2004) and cause tumors and other virus induced diseases.
miRNA and Fruit Ripening-
Deep sequencing of tomato genome shows the role of miRNAs in fruit ripening. Studies showed that several miRNAs including miR156, miR 157, miR 164, miR408, miRmiR858and miR894 etc are more abundant in leaves and closed flowers. miR169 is highly expressed in all fruit stages. miR171 is expressed in very small fruits and miR390 has higher accumulation in small fruits (Moxon et.al.2008).
miRNA and Drought Stress-
Recent studies show that expression of miRNAs is also altered in response to drought stress (Li et.al.2008). In Arabidopsis, in response to dehydration expression of miR393, miR319 and miR397 is increased significantly (Sunkar et.al.2004). Similarly,
miR393 expression has also been found to be up-regulated during drought stress in rice (Zhao et.al.2007). Several other miRNAs including miR157, miR167, miR168, miR171, miR408, miR393 and miR396 are also up-regulated in drought-stressed Arabidopsis. In Populus trichocarpa, the expression levels of miR1446a-e, miR1444a, miR1447
and miR1450 were found to be significantly reduced and miR1711-a, miR482.2, miR530a, miR827, miR1445, and miR1448 were slightly down-regulated during drought stress (Sunkar.2010).
miRNA and Salt Stress-
Salt stress can reduce crop yields and threatens the survival of crop plants. Along with numerous genes and pathways, expression of miRNAs is also altered by salt stress.
Expression of miR396, miR168, miR167, miR165, miR319, miR159, miR394, miR156, miR393, miR171, miR158 and miR169 was up-regulated in Arabidopsis in response to salt stress while in P. trichocarpa, miR530a, miR1445, miR1446a-e, miR1447 and miR171l-n were down-regulated, whereas miR482.2 and miR1450 were up-regulated during salt stress (Bartel et.al.2005, Lui et.al.2008).in the same way salt stress effect miRNA expression in various plants including rice, maize etc. (Sunkar.2010).
miRNA and Cold Stress-
Organisms grow optimally at optimum temperature. Fluctuations in optimum temperature above a threshold results in temperature stress. miRNA expression is altered during temperature stress. In Arabidopsis, several miRNAs including miR165/166, miR169, miR172, miR393, miR396, miR397, miR408 are significantly up-regulated, while other miRNAs like miR156/157, miR159/319, miR164, miR394, miR398 show either transient or mild regulation under cold stress (Zhou et.al.2008, Sunkar.2010). In other plants also expression of miRNAs is altered during cold stress.
miRNA and Oxidative Stress-
Accumulation of reactive oxygen species (ROS) leads to oxidative stress in plants. During oxidative stress in Arabidopsis miR398 transcription is suppressed which leads
to the suppression of miR398-directed cleavage of CSD1 and CSD2 transcripts (Sunkar.2006).
miRNA and Hypoxia stress-
Hypoxia stress interferes with mitochondrial respiration. It induces massive changes in the transcriptome and a switch from aerobic respiration to anaerobic respiration (Biley-Serres et.al.2008).miRNAs also plays an important
role in hypoxia stress Several miRNA levels were differentially regulated in maize and Arabidopsis exposed to submergence .miR167, miR166, miR171 and miR396 were induced during the early phase of submergence in maize. On the other hand, miR159,
miR395, miR474 and miR528 are induced after long-term exposure (Zhang et.al.2008, Sunkar.2010).
miRNA and UV radiations-Ultraviolet radiation on the earth's surface has been increasing due to increasing depletion of the stratospheric ozone layer. Elevated UV level have negative effect on plant growth and development by accelerating the generation and accumulation of excessive ROS .Due to excessive exposure of uv radiations six miRNAs including miR169, miR395,miR472, miR168, miR398 and miR408are found to be altered in P.tremula (Jia et.al.2009). MicroRNAs miR156, miR160, miR165/166, miR167 and miR398 are predicted to be UV-B-responsive in Arabidopsis (Zhou et.al.2007).
miRNA and Nutrient Homeostasis-
Plants require optimal levels of nutrients in the soil for normal growth and development. Along with the protein-coding genes, several miRNAs like miR395, miR399, miR398, miR397 and miR408etc also shown to be up regulated when specific nutrients are limiting in the growth media. Hence miRNAs play important role in nutrient homeostasis (Chiou.2007, Sunkar.2010).
miRNA and ion homeostasis-
Sulfate and phosphate are the very important ions of plants. miR395 is involved in sulfate homeostasis. AST68 (a low-affinity sulfate transporter) and
three ATP sulfurylases (APS1, APS3 and APS4) which are involved
in sulfate translocation and sulfate metabolism are the targets of miR395 (Takahashi et.al.1997). Experimental analysis confirmed that miR395 was induced in response to
sulfate-deprivation in Arabidopsis. Likewise PHO1 (a phosphate transporter) and UBC24 (ubiquitin E2 conjugase24), are predicted targets for miR399. This shows involvement of miR399 in phosphate homeostasis. miR399 suppress UBC24 transcripts under phosphate deficiency in Arabidopsis (Fujii et.al.2005, Sunkar.2010).
miRNAs are post transcriptional regulators of gene expression. They regulate various important events in plants including developmental processes; signal transduction various diseases, homeostasis and various stress responses. For several years, genetic and transgenic analysis have greatly helped to understand regulatory roles of miRNAs. But the functional roles of many identified miRNAs are still unknown. Current challenge to plant biologists is the functional characterization of the continuously expanding numbers of miRNAs. Knowledge of roles of miRNAs in plant development will provide
deep insight into the life cycle of plants. A more refined picture of growth, development and evolution will result from a superimposition of the transcriptional and miRNA