RNA interference in biology and disease

1
RNA interference in biology and disease

• Yang liu qing

2
Content Summarization
Introduction
Mechanisms
Application
Limitation
conclusion
3
Introduction

• RNA interference (RNAi) was first characterized
  in the nematode worm Caenorhabditis elegans by
  Fire and colleagues.

• They found that double-stranded RNA (dsRNA)
  induced a sequence-specific silencing response
  which is more efficient than customarily used
  single-stranded antisense RNA

4

• .

back
5

• ds RNA molecules can be recognized and cleaved
  into 21-23 nucleotide siRNAs by the
  RNaseIII-like enzyme termed Dicer.

Those siRNAs or synthetic siRNAs are incorporated
into the RNA-induced silencing complex (RISC)
RISC has helicase, exonuclease, endonuclease,
and homology-searching domains.
6
RNA interference in mammalian cells

• Knock-out models in experiment systems have two
  significant disadvantages
• First, it cannot be used for studying
  developmental or cell typespecific effects
• Second, this method is both labor and time
  intensive

• In theory, these problems could be resolved by
  delivering artificially synthesized siRNA
• into mammal cells.

7
Stable RNA interference

• But this effect is transient
• How to make it stably work in the
• experimental system ?

8

• To address these issues, mammalian
• expression vectors were designed to direct the
  intracellular
• synthesis of siRNAs

back
9
Research Applications of RNAi

• It can use for reverse genetic to identify a
  genes phenotype
• It can combine with genomics to perform
  large-scale genetic screens aimed at gene
  discovery

10
Therapeutic Applications of RNAi

• siRNAs can specifically target nucleic acid of
  the virus and the genes expression of cancer
  cell
• The author use leukemia therapy as a example
  leukemia can be treated through silencing of a
  fusion protein, which is most important for such
  diseases pathology

back
11
Limitations of RNAi

• Two main limitations of RNAi are

• Nonspecific effects

• off-target effects

12
Interferon induction by siRNAs
Directly or indirectly activate the transcription
of specific genes through IFN-independent pathways
dsRNA
2-5A binds and activates RNase L, resulting in
the nonspecific degradation of cellular
single-stranded RNA, and the onset of apoptosis
binds to constitutively expressed dsRNA
recognition proteins
directly mediate antiviral events
Inhibits protein synthesis
also present at basal levels to directly respond
to the initial infection
activation of NFkB and ATF2
Upregulation of IFNs
Phosphorylation of eIF2
Converts ATP to 2-5A
ISGs transcription
PKR and 2-5A synthetase
13

• Nonspecific effects can also occur in response to
  siRNA. But these effects are concentration
  dependent
• To design a highly efficient siRNA be used at
  lower concentrations, can reduce the
  concentration-dependent nonspecific side effects

14

• Nonspecific effects can occur to a much higher
  degree in response to siRNAs synthesized from Pol
  IIIdriven promoters.
• One study indicates that the 5 region of these
  enzymatically produced siRNAs contain elements
  that could be traced as responsible for ISG
  induction

15
Off-target effect
siRNA have a similar cellular machinery with
miRNA, for the loose homology requirements for
activation of miRNA function, siRNA can works as
a miRNA even in a low concentration
back
16
Conclusion

• (1) using available information and algorithms to
  design the most effective and specific siRNA
  possible so that it can be used at very low
  concentrations
• (2) using several siRNAs against the same target
  since it is unlikely that they all would have
  similar sequence-dependent off-target effects
• (3) using different control siRNAs against
  measurable irrelevant genes
• (4) rescuing the phenotype caused by an siRNA by
  ectopic expression of a version of the gene that
  cannot be silenced by the siRNA

17

• Thank You!

18
Indirectly activation effect of dsRNA
back