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1、Whole genome sequences indicate 20,000 genes,3x the number in yeast.Differential RNA processing generates proteomic diversity,but why such increased complexity?But,now add 100s of distinct tissue and developmental-stage-specific non-proteinregulatory molecules,each controlling expression of 100s of
2、genes:expand coding potential complexity.RNA Interference and MicroRNAs:Tip of the Iceberg?“Most stunning development of the last decade”:discovery of multiple unexpected phenomena under RNA interference(RNAi).And,parallel development of the microRNA(miRNA)field.Disregarding the immense practical ap
3、plications of RNAi covered by Gus lectures.For example,knock down individual genes,genome loss-of-function screens,potential therapeutic use.Clearly,small RNAs are coming to be understood as profound regulators of gene expressionand function in most(not all)eukaryotic organisms,from unicellular tryp
4、anosomatids,fission yeast,and algae,to multicellular plants and animals.Subset of RNA regulatory mechanisms.Post-transcriptional regulation is as important as nuclear transcriptional regulation5-cap5-G-G AAAAAAAAApoly(A)tailpolyadenylation signal(AAUAAA)start codon(AUG)stop codon(UAA,UGA,UAG)5-UTR3-
5、UTRSynthetic DNAor RNA5-G-G AAAAAAAAAstart codon(AUG)stop codon(UAA,UGA,UAG)B)RNase H-mediatedRNA digestion5-G-G AAAAAAAAAProposed mechanisms of antisense-mediated gene modulationA)5-G-G AAAAAAAAAstart codon(AUG)stop codon(UAA,UGA,UAG)inhibition of proteintranslation(hybrid arrest)Morpholinos as kno
6、ck-down regulatorsAntisense RNA regulators.Splice-site blockers.Translational initiation codon,or ribosome scan blockers.In some ways,the expanding miRNA networks might turn out to have similar“functional logic”to transcription factor regulatory networks.First miRNAs discovered:Lee,R.C.,Feinbaum,R.L
7、.,and Ambros,V.(1993).The C.elegans heterochronicgene lin-4 encodes small RNAs with antisense complementarityto lin-14.Cell 75,843854.Wightman,B.,Ha,I.,and Ruvkun,G.(1993).Posttranscriptionalregulation of the heterochronic gene lin-14 by lin-4 mediates temporalpattern formation in C.elegans.Cell 75,
8、855862.A model of successive regulation of heterochronic gene activities by lin-4 and let-7 RNAs.(1)LIN-14 and LIN-28 expression levels are reduced by lin-4 RNA expression at end of 1st larval stage,allowing progression to late larval stages.(2)In late larval stages,expression of LIN-41 and other ge
9、nes may be similarly downregulated by let-7 RNA,relieving their repression of LIN-29 protein expression,allowing progression to adult stage.(3)Because the lin-29 mRNA does not contain sites complementary to the let-7 RNA,lin-29 is not likely to be a direct target of let-7.RNAiAny mechanism in which
10、20-30 nt single strand RNA in a complex with member of Argonaute(Ago)family of proteins.These are the RISCs:RNA-induced silencing complexesAgo is the effector moleculeSmall RNA guides the complex to appropriate targets by bp interactions.Ago provides the endonuclease activity,or somehow recruits oth
11、er activities(more later).Andrew Fire&Craig Mello(Nobel Prize 2006)Working on the C.elegans Unc-22 inactivationNull phenotype=uncoordinated twitching(1986)Injected bits of DNA into C.elegans hoping for recombination,and achieved the phenotype,but without alteration of the endogenous locus.Extra DNA“
12、turned down”unc-22 function.Among their ideas:production of antisense RNA that turned down function.Moved towards an explicit“antisense”test(pioneered earlier by other labs 1978,1984).But then,“sense”and“antisense”-directed”muscle promoter constructs showed the same phenotype.Guo and Kemphues(1985)s
13、howed that direct RNA injection could cause interference effects.And again,the“sense”RNA caused an effect.Craig Mello worked on this problem in depth:coined term RNAi(1997).(Something in the sense/antisense preparation had)Privileged status after injection,as effects were long-lived,and sometimes ge
14、nerational.Nervous system(1)Specific uptake and delivery systems for dsRNA,systemic delivery to cells(2)(2)Specificity/regulated nature of process indicated by cell-type-specificityTypical final siRNA structureSmall dsRNA,and absolute match to target RNA19 nt duplex2 nt overhang 3 endsTypical miRNA
15、structures“Bubbled”mismatch to target sequences.Overview of RNAi actionNote 2 different effects at the end step:mRNA cleavage or translationinhibitionDifferent types of small RNAs in addition to miRNAsmall interfering RNAs(siRNA)tiny non-coding RNAs(tncRNA)small modulatory RNA(smRNA)siRNAs:2128 nt R
16、NAs,produced by Dicer from long double-stranded RNAs(dsRNAs).Small RNAs in RNAi experiments are traditionally referred to as siRNAs,regardless of origin.Endogenous siRNAs discovered in various organisms.Endogenous siRNAs further categorized into at least 3 distinct sub-classes:(1)trans-acting siRNA(
17、tasiRNA)tasiRNAs in plants,21 nt RNAs encoded in intergenic regions,act on mRNAs in trans to induce mRNA cleavage.(2)repeat-associated siRNA(rasiRNA)Some endogenous siRNAs match repetitive elements in sense or antisense orientationrasiRNAs.In yeast(S.pombe)and plants(A.thaliana):rasiRNAs might funct
18、ion in transcriptional gene silencing by methylatinghistones and/or DNA.(3)small-scan RNAs(scnRNA)siRNAs of 28 nt in Tetrahymena thermophilascan for regions in the DNA for genome rearrangement.The Argonaute family protein TWI1 mediates histone methylation,which is,in turn,required for DNA eliminatio
19、n.Biogenesis and function unclear,and classification uncertain:tncRNA(tiny non-coding RNA):nematodes,similar to tasiRNAs,although origin from long dsRNA precursor needs to be verified.Neural Stem Cells smRNA(small modulatory RNA):20 nt modulates transcription possibly by interacting with a transcrip
20、tion factor.Biogenesis pathways of smRNAs are as yet unknown.miRNAsSmall 19-25 nt RNAs(hence the“22 nt”designation),derived from endogenous hairpin-shaped larger precursors by orchestrated processing reactions(Kim,2005).DefinitionCurrent convention,defined as a ssRNA of 22 nt,by RNase-III-type enzym
21、e Dicer from endogenous hairpin-containing larger transcripts.When a small RNA is discovered(e.g.,cDNA cloning)must meet criteria:Expression size confirmed on a size-fractionated RNA sample(northern blot,RT-PCR,primer extension,RNase protection assay,microarray).Northern blot good as shows mature 22
22、 nt form and 70 nt hairpin precursor.The small RNA sequence is present in one arm of the hairpin precursor,lacking large internal loops/bulges.Precursors 6080 nt in animals,but more variable in plants.(3)The RNA sequence(s)should be phylogenetically conserved.200 species per genome1%and therefore on
23、e of the largest gene families.Target recognition 6 bp:each miRNA can/could recognize 100s of distinct target mRNAs.Other specificity steps?Computational analysis(Bartel and others):perhaps up to 1/3 of the genome could be regulated by miRNAs.Unique combinations of miRNAs expressed might affect or d
24、ampen the utilization of 1000sof mRNAs.How they all functionally interact and crossregulate,or why different labs get different results are currently open questions.Recently found,under certain conditions,that miRNPs do not repress but activate translation(Vasudevan et al.,2007).Furthermore,the fact
25、 that both piRNAs(see later and next class)and miRNAs can function oppositely(i.e.,either as repressors or activators).Leads to the question:How is the mode of action determined?Certain transcription factors have both functions,with overall composition and 3-D organizationof complexes determining ac
26、tivator or repressor activity.Is this going to be similar for activator and repressor RISCs?Other basic questions:How are some miRNAs expressed tissue-specifically(e.g.,Wienholds et al.,2005)?Promoters almost completely unstudied.Do different miRNAs have different stabilities?Do any have nuclear fun
27、ctions?So far,miRNP studies have been“separated”from other aspects of post-transcriptional control:But we must now consider:are miRNP pathway coordinated with other cellular regulatory mechanisms?In this respect,miRNA-mediated translational activation has been linked to interactions with theRNA-bind
28、ing protein,fragile X mental retardation-related protein(FXR1)(Vasudevan et al.,2007).Magnitude of regulationMany times,miRNAs provide modest(2-fold)levels of regulation(e.g.,Bartel and Chen,2004).Extremely relevant in some cases(consider dosage compensation),but more dramatic regulation of specific
29、 targets or in certain cellular contexts likely to be seen(see Giraldez talk later this week).P bodies(Processing bodies)How is everything sorted,shuttled,and compartmentalized?Cytoplasm may have functional compartments;suggested by discovery of processing bodies(P bodies),stress granules,and other
30、cytoplasmic foci,including some found exclusively in specialized cells(e.g.,germ cells)review:Eulalio et al.,2007;Parker and Sheth,2007P bodies and stress granules:sites of mRNA degradation and/or storage,and potential roles in miRNA function.Wide range of activities,in many species bantam RNA(D.mel
31、anogaster)suppresses apoptosis,stimulates cell proliferation.Temporal&tissue-specific expression:bantam RNA regulates tissue formation in development.Another nematode miRNA,lsy-6RNA:genetic screen for leftright asymmetry of chemoreceptor neuron formation.Lsy-6 miRNA targets cog-1,which encodes a tra
32、nscription factor.MammalsmiR-181 controls haematopoiesis,as-yet-unknown target(s).miR-196 repress function of HoxB8,encoding a transcription factor important in developmental regulation.miR-196 RNAs first example of animal miRNAs causing mRNA cleavage,rather thantranslational repression.Plant miRNAs
33、Generally higher complementarity to target mRNAs,results in target cleavage.Interesting point:most known targets of plant miRNAs are transcription factors,particularly those involved in developmental regulation and cell differentiation.miRNA regulation:various processes in many speciesmiRNAs:“standa
34、rd”Polymerase II-transcribed genesPol III initially believed to mediate miRNA transcription,because it transcribes most small RNAs,(e.g.,tRNAs).But,pri-miRNAs are sometimes several kb,with stretches of 4 U residues,which would terminate RNA pol III.Several poly(A)+transcripts comprising miRNA sequen
35、ces and pieces of adjacent mRNAs found in EST(expressed sequence tag)and gene structure analyses.Expression profiles of miRNAs indicate miRNAs under elaborate spatiotemporal control,more like RNA pol II genes(idea:not ubiquitous).Fully functional mature miRNA can be generated from a plasmid that contains a pri-miRNAsegment driven by a heterologous pol II promoter.