RNA Interference (RNAi)

Ryan Duval Endodontics

Discovery of RNAi

• First observed in petunias• Napoli et al 1990• Observed “cosuppression”• Occurring at post-transcriptional level (plants and

fungi)• Fire et al 1998 silencing of genes in nematodes

(nice Prize ‘06)• Mammalian cells – chemically synth or expressed

from plasmid or viral vector

Goals of RNA interference

• Defending cells against parasitic genes• Defense from viruses

• Defense from transposons• Directing development and gene expression in

general

RNAi

• Gene silencing mediated by double-stranded RNA

• Silencing of gene expression– Results from cleavage and degreadation of a

target gene’s mRNA– Also results from blocking translocation of intact

mRNA

• Usually about 20 – 25 base pairs long

RNA Interference Howard Hughes Medical Institiute

Workings of RNAi

•Dicer recognizes and cuts the double-stranded RNA (dsRNA not common)

•Results: short 21 to 25 base-pair molecules called “small interfering RNAs” (siRNAs)

•siRNAs bind to several proteins (3’ overhangs)

•Forming RISC (RNA-induced silencing complex)

DICER

RNAi contd.• RISC becomes activated

when the siRNA its carying is unzipped (utilizing ATP)

• Activated RISC bind to target mRNA

• RISC subunits then cleave mRNA

• Other proteins degrade mRNA & prevent protein production

RISC• The two dsRNA pathways

– Exogenous• Coming from infection by VIRUS

w/RNA genome • By lab manipulation

– Endogenous• Pre-microRNA expressed from

RNA-coding genes in the genome

• Both pathways converge at the RISC complex

RISC• How the activated RISC

complex locates complementary mRNAs within the cell is UNKNOWN?

• Located in P-bodies (cytoplasmic bodies)

• The active components of RISC are ARGONAUTE proteins– Endonuclease– Cleaves the the target mRNA

strand complementary to bound siRNA

Overview

• 20-25nt length (siRNA)• siRNA separated into

single strands• Single strands integrated

into RISC• siRNA induce cleavage of

the mRNA• Preventing it from being

used as a translation template

Recognizing the dsRNA

• Detected and bound by effector protein

• RDE-4 in nematodes (C. elegans)

• R2D2 in Drosophila

• Both stimulate DICER

Gene knockdown

• A drastic decrease in the expression of a targeted gene

• Studying the effects of the decrease can show the physiologic role of the gene product

• RNAi may not totally abolish expression of the gene = knockdown vs knockout

RNAi applications• Silencing CD44 (hyaluronan receptor) gene in

nasopharyngeal carcinoma = malignant potential of the cells. [Jod et al 2007, Shi Oncol Rep 2007]

• Expression of p27 (common protein in oral squamous cell carcinoma), siRNA inhibited the cell proliferation in vitro and in vivo of the p27. [Kudo et al Oral Oncol 2005]

• siRNA used for the treatment of ankylosis and periodontal disease. [Yamada et al J Biol Chem 2001, 2007]

Various RNAi uses

• Topical microbicide treatments of HSV II [Jiang, Milner; Oncogene 2002]

• Knockdown host receptors for HIV [Crowe; AIDS Supp 2003]

• Silencing Hep A and Hep B genes [Kusov et al; J Virol 2006]

• Silencing Influenza gene expression [Jia, Zhang, Liu; Biotechnol Lett 2006]

• Inhibition of LPS induced osteoclast formation and cytokine stimulation [Fahid et al; JOE 2008]

Application of Small Interfering RNA for Inhibition of LPS-Induced Osteoclast Formation and Cytokine Stimulation

• Purpose: Suppression of NFATc1 (transcription factor) expression in monocytes and osteoclast cells using RNAi technique

Background

• Bone homeostasis• RANKL (TNF family)• Induces osteoclastogenesis • LPS express RANKL = osteoclast formation• Final stage of osteoclast differ. NFATc1 is

crucial part of osteoclast differentiation. • Inhibit NFATc1 pathway inhibit bone

destruction?

Materials/Methods

• Mouse hematopoetic cells osteoclasts

• siRNA transfection (silencing NFATc1

• ELISA for TNF-α/IL-6• Immunocytochemistry • Staining of nuclei

Materials/Methods

• Primers for detection of– Cathepsin K gene CSK– IL-6– TNF-α

• Compare osteoclasts transfected w/control vs NFATc1 siRNA

Results

Conclusion• Deliver siRNA into

cytoplasm w/ efficiency• Significant of TNF-α and

IL-6 in response to LPS stimulation

• Significant in # of mature osteoclasts in response to LPS

• in osteoclast-specific gene expression to LPS stimulation

RNAi Challenges

• Systemic delivery obstacle (for RNAi drugs)• How to control the amount of siRNA being

delivered (above or below therapeutic levels)• Possible stimulation of “off target” genes• Long-term effects

Any Questions?