分子医学3月11-12日非编码rna

1、Non-coding RNA (ncRNA)is a “functional” RNA molecule that is not translated into a protein. are highly abundant and functionally important RNAs, such as:transfer RNA (tRNA)ribosomal RNA (rRNA)snoRNAsmicroRNAssiRNAspiRNAslong ncRNAs (lnc RNAs) Noncoding RNAsFor more information, please see small cyto

2、solic RNAS (scRNAs) small nuclear RNAs (snRNAs) small nucleolar RNAs (snoRNAs) extracellular RNAs (exRNAs) microRNAs (miRNAs) small interfering RNAs (siRNAs) Piwi-interacting RNAs (piRNAs) repeat-associated small interfering RNA s (rasiRNAs) guide RNAs (gRNAs) small temporal RNAs (stRNAs) large inte

3、rgenic non-coding RNAs (linc RNAs) long non-coding RNAs (lnc RNAs) Noncoding RNAs: names & abbreviationsScience 296, 1260 (2002) How many ncRNAs are encoded by a genome? Given the absence of a diagnostic open reading frame, how can these genes be identified? How can all the functions of ncRNAs be el

4、ucidated? Noncoding RNAs: questionsScience 296, 1260 (2002)Noncoding RNAs (ncRNAs) have been found to have roles in a great variety of processes, including transcriptional regulationchromatin remodelingchromosome replicationRNA processing and modificationmessenger RNA stability and translationeven p

5、rotein degradation and translocation. An Expanding Universe of Noncoding RNAsScience 296, 1260 (2002)Current View: ncRNAs are far more abundant and important thaninitially imagined.Processes affected by ncRNAScience, 2002, 296: 1260-3Noncoding RNAsNature Reviews Genetics, 2011, 12, 861-874Different

6、mechanisms of ncRNA actionDirect base-pairing with target RNA or DNAMimic the structure of other nucleic acidsFunction as an integral partof a larger RNA-protein complexScience, 2002, 296: 1260-3Small non-coding RNAs involve in regulatory networksHuman Molecular Genetics, 2005, 14: R121-32Figure 1.

7、Regulatory networks involving small non-coding RNAs. Small non-coding RNAs regulate genome structure and gene expression at many levels. miRNAs, siRNAs, snoRNAs and other small RNAs are involved in the regulation of translation, mRNA stability and chromatin structure, as well as self-regulation (das

8、hed lines) and possibly also the control of transcription and splicing (question marks). miRNA a large class of evolutionarily conserved, noncoding, RNA originating from longer transcripts or miRNA genes mature miRNA: 1923nt (ca. 22 nt) characterized by imperfect hairpin structures function mildly o

9、n multiple targets miRNA the human genome may encode over 1800 miRNAs may target about 60% of mammalian genes miRNA complementarity is far from perfect and one miRNA can target many different sites on the same mRNA or on many different mRNAs mainly negative regulation (transcript degradation and seq

10、uestering, translational suppression) possible involvement in positive regulation (transcriptional and translational activation)Nature Reviews Genetics, 2011, 12, 861-874Biogenesis and function of microRNA miRNAmiRNA-induced silencing Complex (miRISC). A ponent gene regulatory complex that is activa

11、ted by a microRNA (miRNA) associated with an Argonaute protein and that regulates gene expression, mediated by the sequence complementarity between the miRNA and the target mRNANat Rev Mol Cell Biol. 2008, 9(3):219-30. Cap-40S initiation inhibition; 60S Ribosomal unit joining inhibition; Elongation

12、inhibition; Ribosome drop-off (premature termination); Co-translational nascent protein degradation; Sequestration in P-bodies; mRNA Decay (destabilisation); mRNA Cleavage; Transcriptional inhibition through microRNA-mediated chromatin reorganization following by gene silencingNine proposed mechanis

13、ms of miRNA action Mathematical modeling of microRNA-mediated mechanisms of translation repressionBasic techniques in microRNA research miRNA expression profilemiRNA array, miRNA cloning, miRNA RT- qPCR, MicroRNA Seq, In situ hybridization miRNA overexpressionmiRNA expression constructs miRNA target

14、 predictionTargetScan, miRanda, PicTar miRNA target validationluciferase assay, Western blot, genechip, seq miRNA functional analysisTransgenic mouse/cell: miRNA knock-in, miRNA knock-outNATURE PROTOCOLS, 2008, 3: 563-8Diagrams of detailed principle and procedureof miRNA expression profilingNATURE P

15、ROTOCOLS, 2008, 3: 563-8In situ hybridization analysis of miRNAsmicroRNA RT-qPCRTaqMan MicroRNA AssayResearch Strategy for microRNA identify the miRNA of interestscreen: miRNA array; miRNA seq discover its functionknockout; knock-down dig out the target geneprediction: bioinformaticsconfirmation: re

16、porter gene; western; expression assay establish the relationship btw miRNA and its targetsDevelopmental Cell, 2008, 15, 26171Strategy to generate miR-126 mutant mice by homologous bination.The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and AngiogenesisVascular Abnormalities in

17、 miR-126 Null MiceDevelopmental Cell, 2008, 15, 26171E15.5Luciferase assaymiR-126 target to Spred-1 3UTR, reduce both mRNA and protein level of Spred-1Developmental Cell, 2008, 15, 26171miRNA functional analysis with miRNA inhibitors and siRNADevelopmental Cell, 2008, 15, 26171Identification of mRNA

18、s and miRNAs Associated With Fragile X Mental Retardation Protein1. Isolating the FMRP-containing mRNP complex via immunoprecipitation.2. Identifying the miRNAs enriched in the FMRP-containing mRNP complex via microarray analysis.3. Determining the association between FMRP and endogenous small RNAs.

19、4. Determining the association between FMRP and specific miRNAs via Northern blot.Northern blot is used to detect the association between Fragile X mental retardation protein and miR-199a in mouse brain. The RNAs of INPUT (Lane 2), immunoprecipitate (IP) from wild type (Lane 3), and IP from knockout

20、 (Lane 4) were probed for miR-199a. The 32P labeled RNA size marker (Ambion) for 10 to 100 nucleotides was run in parallel (Lane 1).Prediction of human microRNA (miRNA) targetsRetroviral Constructs for miRNA Expression(A) A double-copy retroviral vector for miRNA expression. A polymerase III express

21、ion cassette containing the human H1 promoter (H1) and the T5 termination signal is placed in the U3 region of the viral 3 long terminal repeat (LTR). Viral infection can be monitored by the expression of a green fluorescent protein (GFP) marker driven by the constitutive murine 3-phosphoglycerate k

22、inase promoter (PGK). (B) A diagram depicting a primary (pri)-miRNA gene fragment that contains the mature miRNA and 125-nucleotide flanking sequences.Nature Reviews Genetics, 2011, 12, 861-874Therapies targeting miRNAs in human disease siRNASmall interfering RNAs (siRNAs). A class of doublestranded

23、 RNAs (dsRNAs) of 21 nucleotides in length. siRNAs are produced from longer double-stranded (bimolecular) RNAs or long hairpins, often of exogenous origin, and usually target homologous sequences at the same locus or elsewhere in the genome for destruction (gene silencing), the phenomenon termed RNA

24、i. However, the distinction between miRNAs and siRNAs is ing blurred as both are produced by similar pathways and have similar mechanisms of action . Methods for Producing siRNAs and shRNAs Chemical synthesis In vitro transcription (IVT) Dicer/RNase III digestion Expression vector Procedure1. Order

25、pre-designed siRNAs onlineOR 1. Design siRNA and order custom siRNAs online1. Design siRNAs2. Order DNA oligonucleotides3. Perform IVT reaction4. Anneal5. Treat with DNase and ssRNA- specific RNase6. Perform column purification1. Create transcription template2. Perform IVT reaction3. Anneal4. Clean

26、up dsRNA5. Digest with6. Dicer/RNase III7. Purify 1. Design siRNAs2. Order DNA oligonucleotides3. Clone into linearized vector4. Isolate clones5. Verify sequence6. Perform large scale prepRequirementsTwo 21-mer RNA oligonucleotidesTwo 29-mer DNA oligonucleotidesTranscription template (200800 bp regi

27、on flanked by promoters)Two 5560-mer DNA oligonucleotidesAbility to fluorescently label siRNAYesYes YesNo (however some vectors code for Green Fluorescent Protein)Useful for long-term studies?NoNo NoYesSmall nucleolar RNAs, 60-300 bp, represent an abundant, evolutionarily ancient group of noncoding

28、RNAs.Function: sequence-specific 2-O-methylation and pseudouridylation of RNA (rRNA) nucleolytic processing of rRNAs (splicing) synthesis of telomeric DNA.Cell, Vol. 109, 145148, April 19, 2002, snoRNAStructure and Function of Box C/D and Box H/ACA snoRNAsCell, Vol. 109, 145148, April 19, 2002,Mecha

29、nism of snoRNA in rRNA modificationc | Small nucleolar RNAs (snoRNAs) are predominantly located in introns. Following splicing, debranching and trimming, mature snoRNAs are either exported, in which casethey function in ribosomal RNA (rRNA) processing, or remain in the nucleus, where theyare involve

30、d in alternative splicing and additional yet unknown functions. Nature Reviews Genetics, 2011, 12, 861-874Cell, Vol. 109, 145148, April 19, 2002, lncRNA more than 200 nt involved in many biological processes dual functions: ligand for protein & base-pairing with nucleic acid (both DNA and RNA) mecha

31、nism: not clear yet one mechanism: mediate epigenetic modifications of DNA by recruiting chromatin remodelling complexes to specific loci (Nature, 2010, 464: 10716) Cell, Vol. 109, 145148, April 19, 2002, lncRNATrends Mol Med.2011,17(6):337-46 nucleate chromatinrecruit chromatin modifying factors se

32、rve as a scaffold higher-order chromatin loops dynamic assembly of nuclear structures Modes of lncRNA ActivityCell.2011;145(2):178-81 Cell.2011;145(2):178-81 Methods for studying lncRNAs (A) Protein interactions: RNA-immunoprecipitation (RIP)chemical-cross-linked RIPnative RIP (nRIP) UV-crosslinked immunoprecipitation (CLIP) (e.g., RIP-Seq, HITS-CLIP/CLIP-Seq)(B) DNA interactions: chromatin RNA immunoprecipitation (ChRIP)chromatin oligo-affinity precipitation (ChOP) chromatin isolation by RNA purification (ChIRP) capture hybridization of RNA target