RNA Interference RNAi

1
RNA Interference (RNAi)

• Ryan Duval
• Endodontics

2
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

3
Goals of RNA interference

• Defending cells against parasitic genes
• Defense from viruses
• Defense from transposons
• Directing development and gene expression in
  general

4
RNAi

• Gene silencing mediated by double-stranded RNA
• Silencing of gene expression
• Results from cleavage and degreadation of a
  target genes mRNA
• Also results from blocking translocation of
  intact mRNA
• Usually about 20 25 base pairs long

RNA Interference Howard Hughes Medical Institiute
5
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
6
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

7
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

8
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

9
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

10
Recognizing the dsRNA

• Detected and bound by effector protein
• RDE-4 in nematodes (C. elegans)
• R2D2 in Drosophila
• Both stimulate DICER

11
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

12
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

13
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

14
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

15
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?

16
Materials/Methods

• Mouse hematopoetic cells ? osteoclasts
• siRNA transfection (silencing NFATc1
• ELISA for TNF-a/IL-6
• Immunocytochemistry
• Staining of nuclei

17
Materials/Methods

• Primers for detection of
• Cathepsin K gene CSK
• IL-6
• TNF-a
• Compare osteoclasts transfected w/control vs
  NFATc1 siRNA

18
Results
19
Conclusion

• Deliver siRNA into cytoplasm w/? efficiency
• Significant ? of TNF-a 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

20
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

21
Any Questions?