welcome everyone. self introduction sun, luguo ( 孙陆果) contact me by email:...
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Welcome EveryoneWelcome Everyone
Self introduction
Sun, Luguo ( 孙陆果)
Contact me by email: [email protected]
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!