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