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 ( ) Ten years later, came in animals (1997) (Pal-Bhadra M., Bhadra U and JA Birchler (1997). Cell 90:

4 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 RNA Non- coding RNA UnRNA Uracil riched RNAs siRNAmiRNARasiRNA 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

7 RNAi-mediated pathways TGSPTGS

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

9 High packaging of chromosomes required for normal cell division Disruption of high packaging by the disruption of RNAi Active RNAi Intact RNAi Defective RNAi 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

10 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: (2006)

11 Establishment and Maintenance of long- distance chromosomal interactions Grimaud et al. Cell 124: (2006) Copies NucleusGene-1Gene-2Merge Active RNAi Restoration RNAi Defective RNAi (Loss of Interaction)

12 is RNAi so simple ?

13 Screening for RNAi machinery components RNAi sensor worms Science. 308, 1164 (2007) In vitro transcription In-vitro dsRNA 90 new components in C. elegans

14 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.

15 Screening for RNAi machinery components w W w W W W promoter RNAi sensor

16 X Gene 50 KB 20 MB 500 KB First Screening Second Screening Mutation detection

17 Genome wide genetical screening (from our lab) identified 73 components of RNAi in Drosophila 23 highly homologou s to C. elegans

18 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 mir Transcription Drosha Processing Pri-miR Drosha DGCR8 Pre-miR Exportin 5 Dicer TRBP Ago2 Mature miR miRISC Assembly Nuclear Export mRNA Targeting 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 JEV7 Genes SARs-Cov2 Genes Cancer genes 13 Genes Cardiovascular3 Genes HCV HPV 3 Genes 2 Genes Baculovirus158 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) Problems Direct delivery Vector based delivery Embryo soaking Nanotube Feeding New generation AAV2 and retroviral vectors Delivery in stem cells Systemic spreading of silencing signal in Drosophila Viral resistance and Gene therapy

Our Gang

Collaborators Utpal Bhadra (CCMB), India Steve Cohen, Singapore Giacomo Cavalli (CNRS) France Andrew chess Harv. Univ, USA Axel Imhof (Univ.of Munich) Germany Jim Birchler (Univ.of Missouri) USA Tom Tsuchl (Rockefeller Univ.) USA Sarah 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