welcome everyone. self introduction sun, luguo ( 孙陆果） contact me by email:...

Welcome EveryoneWelcome Everyone

Self introduction

Sun, Luguo ( 孙陆果）

Contact me by email: sunlg388@nenu.edu.cn

Professor in School of Life Sciences & National Engineering Laboratory for Druggable Gene and Protein Screening

siRNA and microRNAin RNA interference

ReviewRNA ( RiboNucleic Acid )

RNA:•the direct product of genes

•one of the three major macromolecules

•essential for all known forms of life

Structure ： *consists of a nucleobase (A,U,C,G), a ribose and a phosphate group

*single-stranded with complex three-dimensional structures

DNA RNATranscription

RNA RNARNA replication

ReviewRNA ( RiboNuclic Acid )

RNA:•the direct product of genes

•one of the three major macromolecules

•essential for all known forms of life

Structure ： *consists of a nucleobase (A,U,C,G), a ribose and a phosphate group

*single-stranded with complex three-dimensional structures

Various Types of RNA

For protein expression

DNA mRNA proteintranscription translation

Transcriptionalcontrol

Post-transcriptionalcontrol

Translational control

rRNA tRNA

Review

Involved in protein expression Involved in controlling

protein expression

Review

Various Types of RNA

Overview

RNA interference (RNAi)

RNAi --a system within living cells that takes

part in controlling which genes are ac

tive and how active they are

Central to RNAi: short (small) interfering RNA (siRNA)

microRNA (miRNA)

•Functions

•Biogenesis & Working Mechanism•History

•Comparison

Summary: RNA interference

Si RNA and miRNA

CONTENTs

siRNA (Short interfering RNA)

-- a class of double-stranded

RNA molecules, 20-25

nucleotides in length, which

bind to the complementary

portion of the target mRNA

and tag it for degradation

-- gene targeting

--Post-transcriptional silencing

Schematic representation of a siRNA molecule: a 19-21 bp RNA core duplex, followed by a 2 nucleotides 3’ overhang on each strand

• Instead of enhancing purple color, addition of pigment-producing transgenes eliminated purple color

• Called co-suppression or postranscriptional gene silencing

• Similar phenomenon observed in fungus , called quelling

Transgene in Plants, 1990

History of siRNA

Wild-type Transgene

• In 1995, Su G et al found sense RNA to work just as well as antisense RNA for suppressing gene expression in C. elegans

*Actually the sense RNA has been contaminated with little antisense RNA so dsRNA formed

History of siRNA

Antisense RNA Study, 1995

Antisense RNA

C. elegans

• In 1998, Andrew F et al found that dsRNA was at least tenfold more potent as a silencing trigger than sense or antisense RNA

History of siRNA

--dsRNA had to include exons; introns and promoter didn’t work

--ssRNA doesn’t work as well as dsRNA

--small amounts of dsRNA can wipe out an excess of mRNA

Fire, A.S, Xu. M.K. Montgomery. S. A. Kostas. S. E. Driver. and C.C.Mello. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(1998).P809

Andrew Z. Fire Craig C. Mello

Mechanism of siRNA

Dicer: RNase III like, cleave dsRNA into 21~22nt siRNA,

RISC: RNA Induced Silencing Complex

RISC uses siRNA as guide to degrade the target mRNA

•Originated from exogenous dsRNA

Perfect complement, always targeting the coding region

•Induces a naturally occurring gene silencing mechanism

Exogenous dsRNA

Viral dsRNA

Transposon dsRNA

RNA synthesis by RdRP

‘aberrant’ ssRNA dsRNA

•works in many organisms

Functions of siRNA

• Pathogen resistance• Genome stabilization• Normal regulation of

gene expression• ……

siRNA technique is developed based on siRNA mechanism, becoming a powerful tool.

miRNAs (Micro RNA)

--short RNA molecule, about 21 to 23 bases in length, encoded in the genomes, bind to complementary sequences on target mRNAs, usually resulting in gene silencing

-- gene targeting

--Post-transcriptional silencing

History of miRNA

•Victor A. et al. study of the gene lin-14 in C. elegans development.

lin-4, the first identified miRNA in 1993

•A short RNA product encoded by lin-4 gene regulated lin-14 protein level.

Translational inhibition

•Mature 22 nt Lin4 RNA is partially complementary to 3’-UTR of lin-14 mRNA and inhibit the translation of lin-14

History of miRNA

let-7, the second miRNA in 2000

•Let-7 repressed lin-41, lin-14, lin-28, lin-42, and daf-12 expression during developmental stage transitions in C. elegans

•let-7 was soon found to be conserved in many species

•Except translational inhibition, mRNA stability is also compromised

Lin4 and Let7 control C. Elegans development

•Lin4 and Let7 miRNAs control differentiation

•As usual, they act by silencing targets

Biogenesis of miRNA

full length pri-miRNA

Hairpin precursor ~70 nt (pre-miRNA)

Mature miRNA ~22 nt (miRNA)

Gene

Export to cytoplasm

transcription

Exportin 5

Drosha

Dicer

RISC

targeting mRNA

miRNA action mode

Mechanism of miRNA

•Binding to 3’-UTR to inhibit translation(Partially base-pairing)

•Binding to coding region to mediate mRNA degradation(perfect or near perfect base-pairing)

Functions of miRNA

• Found almost in all eukaryotes• Natural occurring• Highly conserved• Multiple targets• A common mechanism for gene

expression regulation

• Important in development

• Related with diseases happening

• Potential therapy tools

•Cancer•Heart diseases•Nerve diseases

Summary and Comparison of siRNA and miRNA

Similar:

--Natural occurring

--small RNAs (around 22 nt )

--target mRNA

--processed by Dicer

--RISC as heart of their working mechanism

--overlapped mechanism in mediating mRNA degradation

--Posttranscriptional regulation mechanism

Different:

siRNA miRNA

structure

precursor

Double-stranded Single-stranded with hair pin

Exogenous long dsRNA Endogenous transcribed RNA

Targeting Perfect base pairing Imperfect or perfect basepairing

Specific one mRNA Multiple mRNAs

effect mRNA cleavage

Coding region Coding or 3’-UTR region

Translational repression or cleavage

Significance Viral defense and genome stability

Endogenous gene expression regulation

Summary and Comparison of siRNA and miRNA

Summary of RNAi

RNAi: a powerful tool

•Research applications

•Potent: Highly efficient•Convenient•Time-saving•Highly specific

•Therapeutic applications

--A new strategy of reverse genetics & a novel way of gene knock-out

-- Counter viral infection by specifically destroying the mRNA of the pathogenic viruses,such as HIV and HBV

-- Counter cancers by specifically down-regulate the expression of genes related to oncogenesis

That is all for today!