RNAi : a revolutionarized

technology in Agri-biotech

Utpal Bhadra

Homology dependent gene silencing

Cosuppression: Dispersed

homologous gene silencing.

Homology alone is necessary but not sufficient.

First identified in plants (1987-89)

Ten years later, came in animals (1997)

(Pal-Bhadra M., Bhadra U and JA Birchler (1997). Cell 90: 479-490

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RNAi

RNA Interference (RNAi) - Double-stranded regulatory RNA triggers the degradation of a homologous messages by a RNA dependent RNA degradation process.

Dynamic association of RISC and siRNA

Regulatory RNAs

Antisense RNANon- coding RNA

UnRNAUracil riched RNAs

siRNA miRNA RasiRNA Trans-acting siRNA

Recently discovered but distinct from each other. Do not follow same parameters

PiRNA

small regulatory RNA

400,000 small endogenous control our genes vs 30,000 DNA Unit control of gene

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RNAi-mediated pathwaysTGS PTGS

Application of Gene silencing in nature

Development and differentiation.

Viral resistance in plants and animals

Silencing of transposable elements

Bringing chromosomes together in Polycomb repression

Controlling Heterochromatin formation and Histone methylation

Regulation of dominant suppressors

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High packaging of chromosomes

required for normal cell division

Disruption of high packaging by the

disruption of RNAi

Active RNAiIntact RNAi

Defective RNAi

me me me me

Normal expression of mini-w in packaging chromosome

Increased eye color due to loss of RNAi machinery

Pal-Bhadra et al. Science 303:669 (2004)

Distribution of histone lysine 9 methylation on chromosomes

Loss of H3me2K9 by the disruption of RNAi

me

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Two identical Fab-7 can interact in the nucleus, leading to an increase of PcG-dependent silencing

RNAi Components are Required for Nuclear Clustering of Polycomb Group Response Elements.

Grimaud et al. Cell 124:957-971 (2006)

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Establishment and Maintenance of long-distancechromosomal interactions

Grimaud et al. Cell 124:957-971 (2006)

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NucleusGene-1Gene-2Merge

Active RNAi

Restoration RNAi

Defective RNAi

(Loss of Interaction)

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is RNAi so simple ?

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Screening for RNAi machinery components

RNAi sensor worms

Science. 308, 1164 (2007)

In vitro transcription

In-vitro dsRNA

90 new components

in C. elegans

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Two main approaches

Genetic approach : To isolate major RNAi factors by genetic screening.

biochemical approach : To isolate and characterize associate protein partners involved in nuclear and cytplasmic RNAi machineries.

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Screening for RNAi machinery components

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promoter

RNAi sensor

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Gene

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20 MB

500 KB

FirstScreening

SecondScreening

Mutation detection

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Genome wide genetical screening (from our lab) identified

73 components of RNAi in Drosophila

23 highly homologou

s to C. elegans

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piwi- P element induced wimpy testis

Piwi is a core component of nuclear RNAi machinery

Piwi is associated with endogenous piRNA in the nucleus

Piwi is required for both transcriptional and post transcriptional gene silencing

Biosynthesis of microRNAs

AAAAAA

AAAAAA

mirTranscription

Drosha Processing

Pri-miR

DroshaDGCR8

Pre-miRExportin 5

Dicer TRBP

Ago2Mature miR

miRISC Assembly

Nuclear Export

mRNA Targeting

AAAAA

AAAAA

Localization into P-body

Deadenylation

ARE-mediated decay

mRNA cleavage

Pol II

Cytoplasm

Nucleus

Dicer Processing

microRNA based gene regulation

Scheme for the proposed Interdisciplinary pipeline leading to development of Nutraceuticals

A Green Light for Biofuel: RNAi engineered microalgae for efficient capture of Solar Energy

Fig: Commercially important metabolic pathways in microalgae,2008

Drug and vaccine development.

Gene therapy for Infectious diseases

Viral resistance.

RNAi in Stem Cell Research

Functional genomics.

Transgenic plant and animal production

High-throughput RNAi screening for novel Drugs in Cancer

Turning siRNA in potential drugs

Diseases targeted by siRNA therapy

All virus and infectious diseases, Cancer, Diabetes, Cardiovascular disease,Innate immunity, Arthritis, All genetics disorders and many more

Sirna develops RNAi based therapy for Macular Degeneration

Inhibition of respiratory viruses by nasally administered siRNA

Therapeutic silencing of Cholesterol gene by systemic administration of modified siRNA

SiRNA bank in CCMB

HIV All open reading frames JEV 7 GenesSARs-Cov 2 GenesCancer genes 13 GenesCardiovascular 3 Genes

HCVHPV

3 Genes2 Genes

Baculovirus 158 open reading frames

BaculoVirus resistant Silkworm (initial resistant variety)

SiRNA vector based resistance

Critics says

Our efforts in siRNA based therapy(in collaboration with Dr. M Pal-Bhadra (IICT)

Pro

ble

ms

Direct delivery Vector based delivery

Embryo soaking

NanotubeFeeding

New generation AAV2 and retroviral vectors

Delivery in stem cells

Systemic spreading of silencing signalin Drosophila

Viral resistance and Gene therapy

Our Gang

Collaborators

Utpal Bhadra (CCMB), India Steve Cohen, Singapore Giacomo Cavalli (CNRS) France Andrew chess Harv. Univ, USAAxel Imhof (Univ.of Munich) GermanyJim Birchler (Univ.of Missouri) USATom Tsuchl (Rockefeller Univ.) USASarah Elgin (Washington Univ.) USA

Funding .Wellcome Trust Senior Fellowship- UK

Human Frontier Young Investigator grant, G8 country Howard Huge Medical Grant (Applied) USA

Wellcome Trust Equipment Grant- UK