Welcome Everyone

Self introduction Sun, Luguo ( 孙陆果） Contact me by Professor in School of Life Sciences & National Engineering Laboratory for Druggable Gene and Protein Screening

siRNA and microRNA in RNA interference

Review RNA ( 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 RNA Transcription RNA RNA replication

Review RNA ( 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 protein transcription translation Transcriptional control Post-transcriptional control Translational control rRNAtRNA 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 active 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, 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 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. FireCraig 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-strandedSingle-stranded with hair pin Exogenous long dsRNAEndogenous transcribed RNA TargetingPerfect base pairingImperfect or perfect basepairing Specific one mRNAMultiple mRNAs effectmRNA cleavage Coding regionCoding or 3’-UTR region Translational repression or cleavage SignificanceViral 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!