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RNA interference (RNAi) for Crops

What is RNA interference (RNAi):
A method of silencing or blocking the function of a gene by introducing the short sequenced RNA resulting in transcriptional inhibition or no protein production.
Classes of RNAs involved in RNAi:
There are different classes of RNA which are involved in method of silencing of genes. these different classes of RNA includes:
dsRNA: double stranded RNA or dsRNA is actively involves in RNAi. Complementary base pairings of two strands of single stranded RNA results in formation of dsRNA. It is present naturally in cells, long dsRNAs are derived from the events such as virus induction or just after the formation of dsRNA, the process of RNAi is initiated.
Micro RNA: micro RNA or mi-RNAs are single stranded RNAs of short nucleotides (19-25 nt) which are present in all eukaryotes. These RNAs are encoded in host genome and then processed by the Dicer which is an endonuclease enzyme. Micro RNAs can block the activity of gene by destructing its homologus mRNA in animals and plants. They are often referred as small temporal rnas due to their importance in regulating the developmental timing.
Shart hairpin RNAs: short hairpin RNAs or shRNAs are 19-29 nucleotides longer RNAs which are synthetically manufactured. They contain an antisense strand. Sense strand and spacer (i.e. small sequence of loop present between antisense and sense strands). Complementary sequences of antisense and sense strand results in formation of hairpin shaped dsRNA.
Proteins or enzymes involved in RNAi:
Dicer: dicer plays an important role in processing of dsRNA during the mechanism of RNAi. It is an endoribonuclease belongs to family of RNase III that functions in cleaving the dsRNA and pre miRNA into short dsRNA of 20-25 bp long fragments of 2-nt overhang on 3’end which are known as small interfering RNA (siRNA). Activity of this enzyme is not dependent on ATP. Dicer contains four functional domains:
a) helicase domain at N-terminal: its function is still unknown.
b) c-terminal dsRBD (double stranded RNA binding domain): it binds the dsRNA but specific site of binding is not defined.
c) Two RNase-III domain: involves in cleaving the strands of dsRNA, results in shortening of dsRNA.
d) PAZ domain (piwi,argonaute,zwille): it involves in interacting with PAZ domain of RISC complex.
RISC (RNA induced silencing complex): RISC is multiprotein-complex which involves in incorporation of single strand of siRNA. It has activity of ribonuclease with ability of cutting RNA. This mechanism is very important in regulation of gene by miRNAs and in defense system against the viral action as they used dsRNA as infectious vector. RISC consists of dicer, siRNA, argounate protein, PAZ domain and some other components. Only antisense strand is incorporated into RISC from double stranded siRNAs, this strand is selected by help of argounate protein which is RNase and is catalytically active. The sense strand is dedraded by RISC. The RISC degraded the mRNA which is complementary to its siRNA results in repression of protein translation and effectively silencing the gene.
RDRPS (RNA dependent RNA polymerase): RDRPS play vital role in RNAi and in PTGS (post transcriptional gene silencing) mechanisms.
Mechanism of RNAi:
Initiator Step: just after the entry of dsRNA; through introduced transgene, an aberrant genetic material or viral infections, initiate the pathway of RNAi of the cells, resulting in activation of Dicer enzyme.
dsRNA cleavage: dicer enzyme produced the fragments of small interfering RNA of about 20-25 nt long by cleaving the dsRNA.
Effector step: RISC then differentiate between two siRNA fragmentseithe antisense or sense fragment. Sense fragments are then degraded by RISC.
Integration of antisense fragment: antisense fragments are then integrated into RISC. They are used in targeting of complementary mRNA.
mRNA silicing: After binding the complementary mRNA with siRNA, argounate protein of RISC performs the activity of “slicing” i.e. it cuts the mRNA, so the mRNA is blocked results in no production of protein.

Establishment of genetic engineered plants using RNAi Step 1: Target gene identification:
We identify target genes which are nutritionally beneficial or nutritionally adverse for the crops. We identify them through:
Application of bioinformatics tool
Through sequencing genome
Transcriptomics, proteomics and metabolomics analysis.
Step 2: Development of vector:
After identification of suitable genes, specific vectors are prepared for constructing transgenic plants using RNAi. It is done by:
Specific promoter selection
Specific vector selection
Screening by using selected markers
Step 3: Transformation of vectors in plants and their screening:
RNAi delivery
Tissue culturing of transgenic plants
Screening of transformed plant and their selection
Step 4: Transgenic lines evaluation for quality improvement:
Evaluation through morphology
Evaluation through transcriptome
Evaluation through biochemical pathway
Applications of RNAi in improving crops
Altering the architecture of plant: Architecture of plant likes its height, elongation of stem, branching of roots, morphology of leaf and inflorescence are important for plant physiology, yield, biochemical process and its to resistance for environmental stresses. Bu now, plant architecture are modified by using technology of RNAi for improving the productivity of plants. e.g. RNAi decreased the expression of gene OsGA20ox2 in rice, this enzyme encodes GA 20-oxidase, involves in the synthesis of gibrellins (GAs) which are biologically active in the plants. so, transgenic plants with suppressed OsGA20ox2 produced low content of GA1, decreasing the height of plant, resulting the phenotype which is semi-dwarf. Semi-dwarf plants shows more productivity, shorter stems and resistant than wild plants.
Tolerance against abiotic stresses: abiotic factors (temp, water, salinity etc) greatly affect the productivity of plants. now, it is discovered that RNAi is involved in plant responses against abiotic stresses. E.g. miR393 inhibit TIRI gene expression, which reduced the growth of seedling and signaling of auxin in abiotic stress and also play role in antibacterial resistance.
Tolerance against biotic stresses: biotic factors (viruses, nematodes, fungi etc) contribute to the maximum loss of plant productivity especially viruses, and their control is also difficult. But antiviral activity of RNAi plays important there. E.g. papaya fruit plant which is a major source of enzyme papin is affected by the attack of viruses. Transgenic plants are produced which have gene of coat protein, resistance to attack of virus.
Removal of allergens: hypersensitivity in response to normal harmless food due to role of IgE is known as food allergy. Some allergies due to food are very severe and even threatened for life. Food allergies must be avoided as there are no medicines for them. RNAi also plays an important role there. E.g. soybean is legume plant and is allergic due to Gly m Bd 30 K protein. Transgenic soybeans have been produced in which this gene is silenced, with no harms of allergy.
Secondary metabolites enhancement: secondary metabolites of plants are major sources of pigments, drugs, food additives, fragrances and pesticides. RNAi technology is now used in order to silence the gene which decreases the production of secondary metabolites. E.g. through RNAi potato ptatins which reduces the production of potato tubers (efficient expression system of protein i.e. therapeutic glycoprotein) were eliminated.
Flower scent and color modulation: horticultural trait such as scent and color of plants contribute to economic as well as aesthetic value. Garden plant flower color was modulated by using the technique of RNAi against CHs (chalcon synthase) gene.
Male sterile plants development: to ensure purity male sterility is very important in breeding. It act as an alternative process for producing hybrids when naturally male sterility is absent.
Shelf-life prolongation: vegetables and fruits shelf life is increased to reduce the deterioration and the senescence of the fruit to reduced spoilage and quality of food in order to store and transport safely. e.g. shelf life of tomato plants was increased by using RNAi
Nutritionally improved plants: RNAu technology is being used to produced plants with optimized nutrients (such as flavonids, vitamins, antioxidants, amino acids, fatty acids etc) in order to produced biofortified crops. e.g. by using technology of RNAi transgenic plants of Brassica napus were produced in which sinapate esters level in seeds were minimized upto 75% by blocking sinapateglucosyltransferase: UDP-Glc gene.
Development of seedless fruits: character of seedless fruits is greatly appreciated for their freshly consumption and also for processing the fruit products. It increase the quality of food as hard seeds gave bad taste. RNAi technology is now used for production of seedless fruits (especially watermelon, guava tomato and apples). e.g. by using RNAi seedless tomatoes are produced by suppressing the ARF7 gene.
Toxic compounds removal: almost all plants contain different types of toxins which causes hindrance during process of pure product extraction. RNAi technology is very powerful for producing toxin free plants. e.g. For production of “tearless onion” production of a ‘lachrymatory factor synthase’ gene was reduced.
Why RNAi?
Efficient: 70-100% of transgenic plants exhibit silencing phenotype.
Precise: no side effects were reported.
User friendly: RNAi in plants is performed by using many “user friendly tools”.
Flexible: with only a single constructs multiple genes could be silenced.
High throughput: vectors for making hpRNAi constructs are design for high throughput.
Stable: hpRNAi shows the stable inheritance among the number of generations.
Better than anti-sense: duplex RNAi silencing is better than either anti-sense or sense silencing.

Paget’s Disease of Bone – Causes and Treatments

Paget’s disease of the bone, also known as Osteitis Deformans (Paget 1877) is a chronic inflammatory condition that results in the proliferation and softening of the bone that may affect any or all parts in the skeleton (Mann, 1990). It is characterised by increased bone remodelling, bone hypertrophy and abnormal bone structure. It is rarely present before the age of 55, therefore is found in increasing prevalence with advancing age. Overall mortility is low. Paget’s disease is the second most common bone disease in the Anglo-Saxon descent (Kuriara et al, 2007) affecting about 3% of the above 55 in the UK.
Before any ability in recognising the disease is acquired, the knowledge of normal should be understood. The normal human skeleton comprises of 206 bones. Bone is a living tissue consisting of 92% mineral or solids and 8% water (Mann, 1990). The solid matter is mainly collagen matrix hardened by impregnation with calcium salts (Thomas, 1985). Bones develop from either small cartilage models in the eight-week-old embryo or from condensed embryonic tissue known as mesenchyme that forms a dense membrane.The exquisite assembly of functionally distinct cell population is required to support both the structural, biochemical and mechanical integrity of this mineralised tissue and its central role in mineral homeostasis. Mechanical forces and metabolic regulatory signals that accommodate the requirement for maintaining serum calcium and phosphate levels are functioning throughout life (Mann et al, 1990). Bone forms along the path of invading blood vessels. Chondrocytes enlarge (hypertrophy) and proliferate (hyperplasia) about the blood vessels becoming osteocytes as the cartilaginous matrix becomes mineralised. Enchondral ossification is a well ordered sequential process of converting the cartilaginous model into bone. It is present under the perichondrium which develops within the bone. This process is referred to as modelling (Mann et al, 1990). And any subsequent changes requiring resorption of pre-existent bone followed by deposition of new bone is remodelling. Thus modelling is an early process while remodelling occurs during normal growth and continues until death. Remodelling of bone involves the actions of two principle cells; the osteoclasts and osteoblasts. During remodelling, osteoclasts are recruited to a site on bone surface and the removal of bone mineral and matrix, creating a resorption matrix. As the osteoclasts moves away osteoblasts move in and fill in the pit osteoid, which is then mineralised. In other words, bone is laid down by osteoblasts and resorption occurs as a result of osteoclast action. Bone remodelling is essential in the maintenance of healthy bone and to repair fractures (Grubb, B, 2010)
Paget’s disease can be monostic or polystotic. In most cases, it is monostotic and asymptomatic occurring in the skull, femur, tibia, vertebra or pelvis.This can lead to pain, deformality, fratures, osteoarthritis, nerve compression syndromes and neoplastic transformations. Paget’s disease is a particularly common condition characterised by focal areas of greatly increased bone turnover. Hyperphosphatasia is a comparatively rare but possibly closely related condition. Osteitis Deformans is a disease of patchy distribution throughout the skeleton and is characterised by a great increase in the activity of osteoclasts, which frequently have many more nuclei(up to 100) than do the osteoclasts of e.g hyperparathyroidism. This leads to great increase in bone resorption, thus resulting in corresponding increase in osteoblastic activity, contributing to an enormous local increase in bone turnover. Consequentially, leading to disorganized (haphazard) laying down of new bones (National Association for the Relief of Paget’s disease). The outcome is that the lamellar bone is replaced by woven bone, there is a loss of Haversian systems and bone architecture is uncoordinated. Due to great increase in bone turnover, increased volume of osteiod is frequently noted, with a normal calcification front (metabolic).
Osteitis Deformans is a common disorder of bone and often familial. Based on demographics of Paget’s disease and awareness of the presence of Paget’s disease in multiple members of the families, Grauer et al suggests that “genetic, infectious or environmental factors play an important role in its aetiology”. Previously proposed by Hocking et al, mutations in the sequestosome1 (SQSTM1) was confirmed as the main cause of familial and sporadic Paget’s disease. Three different mutations were identified that affected the ubiquitin-binding domain., the most important mutation being at loci p392L. Sequestosome 1 encodes a component of the RANK-NF{kappa}B signalling pathway (Hocking et al). The member of the TNF receptor family RANK is a receptor activator of the NF{kappa}B ligand is involved in osteoclast differentiation. The binding of ligand to RANK also known as osteoprotegerin-ligand activates downstream signalling pathways that suppresses osteoclast activity and therefore helps to control bone remodelling. Kurihara and collegues(2007) proposed that mutation of sequestosome 1 (p62) gene alone is insufficient to induce Paget’s disease. It is also possible that the ubiquitin-binding domain is a mediator of p62 which causes protein-protein interaction to control the NF{kappa}B signalling in osteoclasts production in reaction to the release of cytokines during inflammation. Any loss of this interaction may cause an amplification of the signalling pathway that leads to increased activation of this pathway (Kurihara et al, 2007). Furthermore in every case of Paget’s disease an additional component “measles virus nucleocapsid protein (MVNP)” was present in bone biopsy. In contrast to these findings, it can be deduced that p62 mutation alone does not alone to cause Paget’s disease but additional factors are also required for the full phenotype to be expressed (Kurihara et al,2007).
Typical features of Paget’s disease can be divided into three categories; bone pain, enlarging bones and impaired hearing (Seibel et al, 1999):
Bone pain is caused by hypervascularity due to disease activity while joint dysfunction due to secondary osteoarthritis evokes the pain pathways. In some patients bone pain can also be associated with bowing that is the result of mechanical incompetence.
Enlarging of bones can be a sign of danger of nerve compression symptoms, if the base of the skull or vertebral column is affected.
Impaired hearing is predominately due to sensineural hearing loss and can be rarely due to the involvement of internal auditory canal.
Other features include patient becoming prone to fractures due to decrease in bone strength in the affected regions. These clinical features are present only in small subset of patients and patients can suffer from this disease for years without being diagnosed (Roodman et al).
Serum calcium and phosphate levels usually remain normal in Paget’s disease. Therefore calcium homeostasis is mostly achieved as a result of high bone turnover. This can be monitored through the detection of high urinary calcium. When there is a removal of part of the stimulus to bone formation, hypercalcaemia can develop. Calcitonin can influence significantly by increasing bone turnover ( in normal adults it is hypocalcaemic).
A more accurate diagnosis is based on either radiographic abnormalities observed in bone scans and by identifying the elevation of bone formation marker alkaline phosphatase (AP). Serum alkaline phosphatase is elevated as a result of increased osteoblastic activity and therefore serves as an index of bone formation in Paget’s disease (Roodman et al). Nevertheless, high serum AP can also be associated with other symptoms and not all sufferers will have raised serum AP (NIAMS). Radiography is therefore the best diagnosis tool for this disease. Bone areas will show localised enlargement of the bone, cortical thickening, sclerotic changes, osteolytic areas such as V-shaped lesions in long bones and osteoporosis circumscripta in the skull (Grauer et al, 1996). The “mosaic” structure appearance in bone biopsy can also confirm Paget’s disease when all other methods are not clear indicators (Langston et al).
Unfortunately, Paget’s disease is incurable and therefore once affected; the patients can only alleviate its symptoms by administration of drugs or surgery. Bisphosphonates a class of pyrophosphate are potent inhibitors of bone remodelling. Two types exists that act on osteoclasts via two mechanisms. One is by elevating programmed cell death of osteoclasts by disturbing the production of ATP and the other is by directly binding to osteoclasts and disrupts its ability to bind to the bone. (Dale et al, 2004). Administration is orally, ideally empty stomach as these drugs have a low absorption rate. Calcitonin is a potent hormonal inhibitor of bone resorption. It has the ability to inhibit osteoclastic bone resorption in patients resulting in a return of bone turnover to normal. On the other hand calcitonin is also good at reliving pain in Paget’s disease. In most cases bisphosphonates are used as they provide a better overall decrease in bone turnover. Where a patient experiences high levels of pain, in addition to the above mentioned drugs, aspirin and Ibuprofen can be prescribed (Langston et al). Dickkopf-1 (DKK-1) that inhibits the Wnt signalling pathways has recently been identified as the therapeutic target for Paget’s disease (McCarthy et al, 2010). Wnt signalling is important in the regulation of healthy bone mass. Over-expression of DKK-1 can lead to bone loss as it inhibits the Wnt pathway. Thus patients with Paget’s disease therapeutically can be treated to elevate DKK-1 levels. This is the potential treatment for PDB, while the research into this is ongoing. Occasionally, patient’s may undergo surgeries to treat internal fractures caused by this disease. Once developed, the management of this disease is very important. Drugs taken in combination can suppress the pathologically increased bone turnover for prolonged period of time
Due to its unknown aetiology, the root cause of this disease remains to be discovered and thus management relies solely on drugs. While ongoing research is being carried out, optimism still arises for the ideal therapy for Paget’s disease of bone.
References:
Alliston T, Derynck R. Medicine: interfering with bone remodelling. Nature. 2002 Apr 18:416(6882):686-7
Dale.M et al (2004) Rang and Dales’ Pharmacology, 6th edition, Churchill Livingstone Elsevier, p.300 -470
Grauer. A and Wuster.C (1996), Clinical Endocrinology, Urban and Schwarzenberg, p336-341
Hocking. LJ et al (2002) Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget’s disease, Human Molecular Genetics, 22: 2735-2739
Kurihara. N et al (2007), Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget’s disease, The Journal of Clinical Investigations, 117:133-142
Langston. A et al (2008), Pathogenesis and management of Paget’s disease of bone, Journal, the lancet, 372(9633):155-63
Mann.R and Murphy.S (1990) Regional Atlas of Bone Disease, Charles C Thomas, p34-60
Martin.T.J et al (2000) Diagnosis of metabolic bone diseases, Chapman and Hall Medical, p159-200
McCarthay. HS and Marshall. MJ (2010), Dickkopf-1 as a potential therapeutic target in Paget’s disease of bone, ISI Web of Knowledge, 14:221-230
National Association for the Relief of Paget’s Disease (NIAMS), (2009), http://www.niams.nih.gov/Health_Info/Bone/Pagets/default.asp, 19/02/2010
Roodman.G and Windle.J (2005), Paget’s Disease of Bone, The Journal of clinical Investigation, 155: 200-205
Seibel.J et al (1999) Dynamics of Bone and Cartilage Metabolism, Academic Press, p165-400

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