rna interference
DESCRIPTION
rna interference mechanismTRANSCRIPT
RNA INTERFERENCE
siRNA and miRNA
Ms.ruchi yadav
lecturer
amity institute of biotechnology
amity university
lucknow(up)
RNA INTERFERENCE (RNAI)
RNA interference (RNAi) is a highly evolutionally
conserved process of post-transcriptional gene
silencing (PTGS)
by which double stranded RNA (dsRNA), when
introduced into a cell, causes sequence-specific
degradation of homogolous mRNA sequences.
It was first discovered in 1998 by Andrew Fire and
Craig Mello in the nematode worm Caenorhabditis
elegans and later found in a wide variety of organisms,
including mammals
TIMELINE
1998
1999
2000
2001
2002
1990 cosuppression of purple color in plants
dsRNA injection in worms
short RNAs
identified in
plants
RNAi shown in
vitro
RISC activity partially purified
siRNAs identified Dicer identified
RNAi used against HIV
genome-wide RNAi
screens begin
RNA INTERFERENCE
Phenomena first observed in petunia
Attempted to overexpress chalone synthase
(anthrocyanin pigment gene) in petunia. (trying to
darken flower color) Caused the loss of pigment.
Called co-suppression because suppressed
expression of both endogenous gene and transgene
RNA INTERFERENCE APPROACHES
Four types of responses induced by dsRNA
RNAI MECHANISM
The RNAi pathway can be divided into three major
steps:
First is the conversion of dsRNA input into 21-23bp small
fragments by the enzyme Dicer;
Secondly the loading of small RNAs into large
multiprotein complex RISC
Lastly the sequence specific silencing of the cognate
gene by RISC that is guided by the small RNA fragment.
SMALL INTERFERING RNA(SIRNA)
dsRNAs are cleaved into 21-23 nt segments (“small
interfering RNAs”, or siRNAs) by an enzyme called
Dicer
STRUCTURE OF DICER ENZYME
The PAZ domain, a module that binds the end of
dsRNA, is directly connected to the RNase IIIa domain
by a long alpha helix
Dicer enzyme plays two biochemically distinct roles in
the RNAi mechanics.
It functions to generate siRNA molecules
Role in loading one of the two siRNA strands onto RISC
complex
RNA-INDUCING SILENCING COMPLEX (RISC)
The siRNAs are incorporated into the (RISC) which
consists of an Argonaute (Ago) protein as one of its
main components.
The Argonaute protein is considered as the catalytic
engine or the signature component of the RISC
Ago cleaves and discards the passenger (sense)
strand of the siRNA duplex leading to activation of
the RISC.
Ago cuts mRNA targets guided by siRNA via its
endonuclease nicknamed “slicer”.
THE MECHANISM OF RNAI
MECHANISM OF RNAI
The silencing mechanism of RNAinterference involves
two steps.
Initiation step: - first the dsRNA get processed into
21-23 nucleotides small interfering RNAs
(siRNAs), which have also been called “guide
RNAs, by an RNase III like enzyme called Dicer.
Effecter step: - Then, the siRNAs assemble into
endoribonuclease-containing complexes known as
RNA-induced silencing complexes
(RISCs), unwinding in the process.
An ATP-dependent unwinding of the siRNA duplex is
required for activation of the RISC
MECHANISM OF RNAI
The active RISC then targets the homologous transcript by base
pairing interactions and cleaves the mRNA ~12 nucleotides from the
3' terminus of the siRNA and destroys the cognate RNA.
MICRO RNA (MIRNA)
first discovered in 1993 by Victor Ambros in C.
elegans:
lin-4 RNA regulates LIN-14 protein levels, but not
lin-14 mRNA levels
important for development and differentiation
second miRNA (let-7) discovered in 2000
MICRO RNA (MIRNA)
A miRNA is a ssRNA of ~22 nucleotides in length
Generated by the RNase-III-type enzymes Drosha
and Dicer from an endogenous transcript that
contains a local hairpin structure.
pri-miRNAs contain cap and poly(A) tail and are
transcribed by RNA Polymerase II
MICRO RNA (MIRNA)
Gene expression regulation
Created by similar process to siRNA
Generally prevents binding of ribosome
MICRORNA’S
MicroRNA’s (miRNA’s) are small noncoding
RNA molecules that regulate eukaryotic gene
expression at the translation level
RISC = RNA-induced Silencing Complex
MICRORNA(MIRNA)
MIRNA MECHANISM
RNase III enzyme Drosha, which cleaves the stem ~22
nt away from the terminal loop to generate an ~65-nt
pre-miRNA hairpin intermediate .
Drosha leaves a characteristics 2-nt 3` overhang
The pre-miRNA is transported to the cytoplasm by
Exportin-5,where it interacts with a second RNase III
enzyme called Dicer.
Dicer binds the 2-nt 3` overhang found at the base of
the pre-miRNA hairpin and cleaves ~22nt away from the
base, removing loop & leaving another 2-nt 3` overhang
.
The resultant duplex intermediate interacts with RISC
components,
PRI-MIRNA PROCESSING
MIRNA MECHANISM
Ago 2 binding of siRNA and passenger strand cleavage.
MIRNA VS. SIRNA PATHWAYS
MIRNAS AS CANCER GENES
MIRNAS AS CANCER GENES
Overexpression of miRNAs—for instance, by
amplification of the miRNA-encoding locus—could
decrease expression of the target, such as a tumor
suppressor gene.
Underexpression of miRNAs—for instance, by
deletion or methylation of the miRNA locus—could
result in increased expression of a target such as
an oncogene.
MIRNAS PLAY IMPORTANT ROLES IN ALL ASPECTS
OF LIFE
Brain development (miR-430)
Patterning of nervous system (miR-273)
Pancreatic islet-cell development (miR-375)
Adipocyte differentiation (miR-143)
Limb patterning (miR-196)
Heart development (miR-1)
Programmed cell death (miR-14)
GENOMIC ORGANIZATION OF MIRNAS
INTEGRATION OF RNAI IN DRUG DISCOVERY
RNAI, A NEW THERAPEUTIC STRATEGY
AGAINST VIRAL INFECTION
Prevents viral infection
Inhibits the expression of viral antigens
Suppresses the transcription of viral genome
Blocks viral replication
Silences viral accessory genes
Hinders the assembly of viral particles & Displays
roles in virus-host interactions
RNA-INTERFERENCE-BASED THERAPIES
COMPUTATIONAL METHODS FOR IDENTIFICATION OF SIRNA
siRNA targeted sequence is usually 21 nt in length.
Avoid regions within 50-100 bp of the start codon and
the termination codon
Avoid stretches of 4 or more bases such as
AAAA, CCCC
Avoid regions with GC content <30% or > 60%.
Avoid repeats and low complex sequence
Avoid single nucleotide polymorphism (SNP) sites
Perform BLAST homology search to avoid off-target
effects on other genes or sequences .
Avoid sequences that share a certain degree of
homology with other related or unrelated genes
TOM TUSCHL'S RULES (HTTP://WWW.ROCKEFELLER.EDU)
The first set of empirical rules for siRNA design was
compiled by Tuschl’s group
Select targeted region from a given cDNA sequence
beginning 50-100 nt downstream of start condon
First search for 23-nt sequence motif AA (N19). If no
suitable sequence is found, then,
Search for 23-nt sequence motif NA(N21) and convert
the 3' end of the sense siRNA to TT
Or search for NAR(N17)YNN
Target sequence should have a GC content of around
50% less than 60%
R=Adenine or Guanine (Purines); Y=Thymine or
Cytosine (Pyrimidines); N = Any.
RNAI TARGET SELECTION
SIRNA VALIDATION TOOLS
DEQOR, a web-based tool for the Design and Quality
Control of siRNAs
GenScript - siRNA Design
IDT SciTools RNAi Design
Ambion scientists find that ~50% of siRNAs
designed using this tool will reduce target gene
expression by >50%.
TARGET PREDICTION ALGORITHMS
HTTP://WWW.RNAIWEB.COM/RNAI/RNAI_WEB/
MIRNAS
HTTP://WWW.RNAINTERFERENCE.ORG/SEQUE
NCES.HTML
CLONING OF MIRNAS
RULES USED IN SVM RNAI
GC content 30-52% preferred.
At least 3 "A/U" at sense position 15-19 preferred.
Internal hairpin is penalized.
"A" at position 19 is preferred.
"A" at position 3 is preferred.
"U" at position 10 is preferred.
"G/C" at position 19 is penalized.
"G" at position 13 is penalized.
Consecutive repeats (e.g., AAAA, GGGG) of more
than 3 bases are penalized.
SVM RNAI 3.6
SVM RNAI 3.6