“GENE KNOCKOFF” by RNAi Technology

TANVI Ph.D I

MBB 605

Gene silencing is a technique that aims to reduce or eliminate the production of a protein from it’s corresponding gene.

It generally describe the “switching off” of a gene by a mechanism other than genetic modification i.e., a gene which would be expressed (“turned on”) under normal circumstances is switched off by machinery in the cell.

It occurs when RNA is unable to make a protein during translation.

Gene silencing is same as gene knock off but is totally different from gene knock out.

When genes are knock off ,there expression is reduced , where in contrast when genes are knocked out, they are completely erased from organism’s genome and thus have no expression.

INTRODUCTION

1990 Jorgensen: To deepen the pigmentation in petunias, introduction of transgenes

homologous to endogenous genes often resulted in plants with both gene suppressed called co suppression.

Resulted in degradation of the endogenous and transgene mRNA.1995 Guo and Kemphues: injection of either antisense or sense RNAs in the germline of C.

elegans was equally effective at silencing at homologous target genes.

1998 Mello and Fire: Extension of above experiments, combination of sense and

antisense RNA(=dsRNA) was 10 times more effective than single strand RNA.

Short History of gene silencing

The discovery of the mechanism of RNA interference by ds RNA by Prof. Andrew Fire and Prof. Craig Mello in 1998, gave them the Nobel prize in 2006.

2001 Tuschl T and colleagues  First described RNAi in mammalian cells2003 Song  EFirst reported that siRNAs can be used therapeutically in whole animals

HOW does it works?1. This is accomplished by binding a specific strand of RNA

to an existing m-RNA strand.2. The m-RNA creates a copy of DNA strand.3. By binding the RNA to the m-RNA, m-RNA is prevented

from replicating that portion of the DNA.4. Specific genes can be targeted and prevented from

replicating in to new DNA strands.

Gene silencing methods used in research

Antisense oligonucleotides

discovered by Paul Zamecnik and Mary Stephenson in 1978

Oligonucleotides are short nucleic acid fragments, that bind to complementary target mRNA molecules when added to the cell.

Ribozymes

•  Sidney Altman and Thomas Cech first discovered catalytic RNA molecules

• These are catalytic RNA molecules used to inhibit gene expression.

• cleaves mRNA molecules, essentially silencing the genes that produced them.

RNAi

Transcriptional Post transcriptional

Types of gene silencing

Genome imprinting Paramutations RNA directed DNA

methylation Transposon

silencing(Histone modification)

Transgene silencing Position effect

RNA Interference RNA silencing Non sense

mediated decay

Genome imprinting

RNA interference ( RNAi), is a technique in which exogenous, double-stranded RNAs ( dsRNAs ) that are complimentary to known mRNAs, are introduced into a cell to specifically destroy that particular mRNA, thereby diminishing or abolishing gene expression.

RNA interference was known by other names, including post transcriptional gene silencing (Plants)and quelling(Fungi) .

RNAi mediated gene knockoffRNA Interference

Defense Mechanism Defense against Infection by viruses. As a defense mechanism to protect against

transposons and other insertional elements

Genome Wide Regulation RNAi plays a role in regulating development

and genome maintenance. 30% of human genome regulated

Need For Interference

RNAi was found to work in many diverse species

Fungi

Trypanosomes

Insects

Zebrafish

Mice

Provides valuable insight into evolution found in all eukaryotes except some fungi= ancient

biochemical mechanism

“indicates that gene expression is the key to evolving complex organisms”

Invaluable tool for functional genomics Knockdowns are easier than knockouts= saves time

and money

" Allows for study of function with a variety of controls: positive, negative, rescue”

Why RNAi is important

RNAi pathway guided by,Si RNA (small interfering RNA): Small interfering RNAs that have an integral role in the

phenomenon of RNA interference (RNA i), a form of post- transcriptional gene silencing

RNAi: 21-25 nt. fragments, which bind to the complementary portion of the target mRNA and tag it for degradation

Mi RNA (micro RNA):  mi RNA Originate from capped & polyadenylated full

length precursors (pri-miRNA) Hairpin precursor ~70 nt (pre-mi RNA) Mature mi RNA

~22 nt (mi RNA)  mi RNA originates from ssRNA that forms a hairpin

secondary structure. Mi RNA regulates post-transcriptional gene expression.

Dicer:  RNAse III-like dsRNA-specific ribonuclease • Enzyme involved in the initiation of RNA i. • It is able to digest dsRNA into uniformly sized small RNAs (si RNA) Dicer family proteins are ATP- dependent nucleases. Loss of dicer→loss of silencing process in vitro RISC (RNA Inducing Silencing Complex): RISC is a large (~500-kDa) RNA-multi protein

complex, which triggers mRNA degradation in response to Si RNA

Unwinding of double- stranded Si RNA by ATP independent helicase.

The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand.

The long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway.

First, the dsRNAs get processed into 20-25 nucleotide

(nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer.

Then, the siRNAs assemble into endoribonuclease-

containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process.

The siRNA strands subsequently guide the RISCs to

complementary RNA molecules, where they cleave and destroy the cognate RNA (

Cleavage of cognate RNA takes place near the middle

of the region bound by the siRNA strand.

The Mechanism of RNA Interference

Drosha

Exportin 5

RNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural plant toxins.

For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption.

RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests.

Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants

Role of RNAi in Biotechnology…

It may be possible to exploit RNA interference in therapy.

A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy

Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease.

RNA interference is also often seen as a promising way to treat cancer by silencing genes differentially upregulated in tumor cells or genes involved in cell division.

Modulation of HIV-I replication by RNA i.

Role of RNAi in Medicine…

Current trends in clinical trials using RNAi Many pharmaceutical companies devoted to RNAi research are coming up with interesting

insights into how human ailments can be cured using the RNAi mechanism. Although the trials are in their early phases. For example, Nucleonics Inc., a pharmaceutical company, has initiated a phase l clinical trial of the systemically administrated RNAi-based therapeutic NucB-1000 for the potential treatment of HBV infection. NucB-1000 consists of a plasmid DNA construct designed to produce four different shRNAs, targeting different sequences of the HBV genome, under control of an RNA polymerase III promoter. The plasmid DNA was formulated with a cationic-lipid delivery system.

HIV was the first infectious agent targeted by RNAi perhaps owing to the fact that the life cycle of HIV is well understood as its pattern of gene expression. Synthetic and expressed siRNAs have been used to target a number of early and late HIV-encoded RNAs including the TAR element, tat, rev, gag, env, vif, nef and reverse transcriptase. Cellular cofactors, such as NFkb, the HIV receptor CD4 and co-receptors CXCR4 and CCR5 have also been successfully down regulated by RNAi resulting in an inhibition of HIV replication

RNA interference: A futuristic tool and itstherapeutic applications

Anghesom Ambesajir et al, Saudi Journal of Biological Sciences(2012)

Animal models are widely used to investigate the therapeutic efficiency of RNAi. in vivo utilization of siRNA was effectively performed by targeting the colorectal cancer-associated gene β-catenin. Decreased proliferation and diminished invasiveness were observed following siRNA-mediated silencing of this gene in human colon cancer cells. Additionally, when treated cancer cells were placed in a nude mouse, prolonged survival was seen compared with mice receiving unmanipulated tumors.

The future of RNAi researches is exciting and there are many applications to be considered in this field. An instance where RNAi technology can be used to silence the gene(s) responsible for the production of boxalylaminoalanine-L-alanine (BOAA), a neurotoxin found in a leafy vegetable known as Lathyrus sativus, which is commonly used as a food by the people in the lower socioeconomic class of the poor nations.

The ripening hormone, ethylene is known to initiate, modulate and co-ordinate the expression of various genes involved in the ripening process. Therefore ethylene is held accountable for the tons of post-harvest losses due to over-ripening and subsequently resulting in fruit rotting. In the present investigation, delayed ripening tomatoes were generated by silencing three homologs of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene during the course of ripening using RNAi technology. The chimeric RNAi-ACS construct designed to target ACS homologs, effectively repressed the ethylene production in tomato fruits. Fruits from such lines exhibited delayed ripening and extended shelf life for 45 days, with improved juice quality. ∼The ethylene suppression brought about compositional changes in these fruits by enhancing polyamine (PA) levels. Further, decreased levels of ethylene in RNAi-ACS fruits has led to the altered levels of various ripening-specific transcripts, especially the up-regulation of PA biosynthesis and ascorbic acid (AsA) metabolism genes and down-regulation of cell wall hydrolyzing enzyme genes. These results suggest that the down-regulation of ACS homologs using RNAi can be an effective approach for obtaining delayed ripening with longer shelf life and an enhanced processing quality of tomato fruits. Also, the chimeric gene fusion can be used as an effective design for simultaneous silencing of more than one gene. These observations would be useful in better understanding of the ethylene and PA signalling during fruit ripening and molecular mechanisms underlying the interaction of these two molecules in affecting fruit quality traits.

Delayed ripening and improved fruit processing quality in tomato by RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate synthase geneAarti Gupta, Ram Krishna Pal, Manchikatla Venkat Rajam

received:4

October 2012

Accepte

d:18

February

2013Published:16 March 2013

Generation of tomato transformants With RNAi-ACS (chimera)construct

Isolation of small RNA for detection of siRNA in RNAi-ACS tomato line

Estmiation of rate of ethylene production in tomato fruits

Polyamine analysis

Determination of fruit shelf life

Determination of ascorbic acid content

Fruits from all thirty RNAi-ACS tomato lines along with unrelated control and WT plants were analysed for rate of ethylene evolution. Results shows that fruits from RNAi-ACS lines liberated reduced levels of ethylene. Ethylene liberation was found to be least in RNAi-ACS60 and RNAi-ACS81, releasing only 4–5% when compared with controls (WT and UR). Fruits of other RNAi-ACS lines showed 10–70%of ethylene evolution to that of control fruits. This difference in ethylene evolution among RNAi-ACS lines could be attributed to variable suppression of the target gene.On the basis of RT-PCR and siRNA detection results, RNAi-ACS lines were categorized into highly silenced lines (RNAi-ACS60 and RNAi-ACS81) and moderately silenced lines (RNAi-ACS123 and RNAi-ACS125).Reduction in ethylene levels has led to significant reduction in CO2 evolution (marker for respiration) in RNAi-ACS tomato lines over controls (WT and UR). Among the various lines analyzed For rate of respiration, RNAi-ACS60 and RNAi-ACS81 were found to exhibit

50% reduction in respiratory activity in harvested .Fruits over control fruits, while rest of ∼the RNAi-ACS lines showed up to30% reduction in rate of Respiration. The variation in respiratory activity of different fruits corresponds to the different levelsof ethylene liberated by such fruits. Increased PA accumulation in fruits of RNAi-ACS lines may have Also influenced the enhanced shelf life possibly by stabilizing the membranes.

RESULTS

It is the regulation of gene expression and widely used in agriculture and biotechnology.

Besides the all types of gene silencing the RNAi is the important post transcriptional gene silencing.

It is an important part of the cellular machinery that provides viral immunity and a mechanism for the control of gene expression.

A variety of RNA triggers function in the RNAi mechanism result in gene suppression that can be both temporary and permanent.

RNAi approaches are economical and provide a tremendous amount of flexibility by allowing time- and tissue-specific knockdown of genes or specific splice forms, in addition to avoiding the common problem of embryonic lethality from gene knockout. The field is relatively young and much remains to be discovered

Conclusion

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