RNA INTERFERENCE:
OVERVIEW
RNA INTERFERENCE:
OVERVIEW
• SENSE OR ANTISENSE RNAs LED TO
NEGLIGIBLE DECREASE OF TARGET RNAs
(Caenorhabditis elegans) - Andrew Fire and Craig
Mello (1998)
• dsRNA EFFECTIVELY DEGRADED
CYTOPLASMIC mRNA
• SYSTEMIC AND HERITABLE
What is RNA interference?
•RNAi is a way to silence gene expression
•To perform RNAi, dsRNA homologous
to the targeted gene is made and
then introduced into cells
dsRNA
nucleus
•Any mRNA with high sequence homology
to the dsRNA may be silenced
• Gold standard technique in gene silencing
in the nematode, C elegans
• Easy introduction of dsRNA into this
organism
• Systemic and heritable (RNA-dependent
RNA polymerases)
microRNAs (miRNAs)
• Regulation of gene expression during
development, differentiation and apoptosis
• RNA polymerase II
• Similar to mRNA with respect to regulation
MOLECULAR
MECHANISM(S)
• Drosha is an RNase III-like enzyme
• Releases the characteristic stem-loop structure of the 70 bp
precursor-miRNA from the primary transcript (premiRNA)
• Pre-miRNA is transported from the nucleus to the
cytoplasm by exportin-5
• Dicer processes pre-miRNA transcripts to generate the
mature miRNA of 21-23 nt in lenght
• siRNA duplexes of 21-25 bp are also generated from long
dsRNAs in the cytoplasm by Dicer
• Antisense strand of the mature si/miRNAs is retained in
the active ribonucleoprotein effector complexes(RISC or
miRNP) and acts as a guide to target homologous mRNA
• siRNAs are fully complementary and induce cleavage and
degradation of target mRNA
• miRNAs preferentially bind only with partial homology to
the 3´ untranslated region of their cognate mRNAs and
inhibit translation
• Both siRNA and miRNA can induce PTGS through
degradation or translation inhibition
siRNAs have a defined structure
19 nt duplex
’
2 nt 3 overhangs
DESIGNING siRNA
• The base pair at the 5´ end of the antisense (guide) strand should have
a lower thermodynamic stability compared with 3´ end (3-5 A/U
nucleotides at the 3´- terminus of the sense strand)
• 27-29 mers may be more active than 21-mers (Dicer processing of
these longer dsRNAs leads to direct and more efficient incorporation
into RISC)
• Low GC content (30% - 52%)
• Avoid internal inverted repeats which can form secondary
structures
• To prevent cross-reactive silencing, a BLAST search of
potential target sequences should be performed
Practical aspects of RNAi
•
Therapeutic treatment
– Cancer
– Viral diseases
– Infectious diseaees