miRNA医学免疫学

1、miRNAs,Nan Li nanli_,“Dark matter” of the genome,Junk DNA?,98: junk DNA?,Genome,2: protein coding,80 transcribed to RNAs, which are not protein coding,DNA,RNA,Protein,nucleus,cytoplasm,Gene Expression,Regulation of,Non-coding RNA,rRNA, tRNA, snRNA, snoRNA siRNA, miRNA, piRNA, tiRNA rasiRNA, tasiRNA,

2、 tmRNA hcRNA, tncRNA, scnRNA, lncRNA,An expanding universe of non-coding RNAs,- Small RNAs, Big Biology,Slide 5,Small RNAs named Insight of the Decade by Science,What is miRNA? How are miRNAs discovered? How are miRNAs synthesized in vivo? How many miRNAs are there? Where are miRNAs located in the g

3、enome? When and where are miRNAs expressed? How miRNAs regulate gene expression? What genes do miRNAs regulate? What regulate miRNAs? What are the general functions of miRNAs? Is there any therapeutic application?,Todays topics,Discovery of miRNA,“Attempted to overexpress chalcone synthase (CHS) in

4、pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis.”,“To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually.”,Th

5、e Nobel Prize in Physiology or Medicine 2006 was awarded jointly to Andrew Z. Fire and Craig C. Mellofor their discovery of RNA interference - gene silencing by double-stranded RNA,Andrew Z. Fire,Craig C. Mello,2006,“Lin-4 acts by negatively regulating the level of LIN-14 protein. Lin-4 does not enc

6、ode a protein. Two small Lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3untranslated region (UTR) of lin-14 mRNA.”,“Let-7 encodes a temporally regulated 21-nucleotide RNA that is comple

7、mentary to elements in the 3 untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12.”,Non-coding RNA Mature form is 22-25 nt in length Usually down regulate gene expression post-transcriptionally Usually pair with complementary sequence in 3UTR Phylogenetically con

8、served,What is microRNA?,Biogenesis of miRNA,RNA-induced silencing complex,He and Hannon, 2004,What is miRNA? How are miRNAs discovered? How are miRNAs synthesized in vivo? How many miRNAs are there? Where are miRNAs located in the genome? When and where are miRNAs expressed? How miRNAs regulate gen

9、e expression? What genes do miRNAs regulate? What regulate miRNAs? What are the general functions of miRNAs? Is there any therapeutic application?,Todays topics,/,Human: 1881 precursors, 2588 mature,How many are there?,Identification of novel miRNA,Forward genetic screens: lin-4

10、, let-7, bantam, lsy-6 Direct cloning: one of the arm of a hairpin structure stable accumulation by northern Bioinformatic predictions: pre-miRNA structure conservation,Genomic organization,Thatcher and Patton, 2008,Zabrafish,Olena and Patton, 2009,cluster,Genomic organization,Expression profile of

11、miRNA,Can be temporally and spatially specific Experimental techniques for expression analysis? Northern blotting Microarray Real time PCR Next generation sequencing In situ hybridization,Tissue specific expression of miRNA,Kim and Nam 2006,Tissue specific expression of miRNA,Fang and Zhou 2012 Onco

12、logy Reports,miRNA profile in colon cancer stem cells VS non-stem cells,What is miRNA? How are miRNAs discovered? How are miRNAs synthesized in vivo? How many miRNAs are there? Where are miRNAs located in the genome? When and where are miRNAs expressed? How miRNAs regulate gene expression? What gene

13、s do miRNAs regulate? What regulate miRNAs? What are the general functions of miRNAs? Is there any therapeutic application?,Todays topics,Mechanism of function,Up-regulation VS down regulation Transcription level VS post-transcription level Cleavage VS translational repression Translational repressi

14、on at the initiation step Translational repression post-initiation mRNA destabilization,Filipowicz and Sonenberg 2008,Takacs and Giraldez 2010,Targets of miRNA,Stress response,Disease (including cancer),Regeneration,miRNA target identification,Direct experimental approaches mRNA profiling/protein qu

15、antification in miRNA overexpression/knockdown samples Crosslink and immunoprecipitation of RISC Bioinformatic approaches Experimental validation,Difficulties in target prediction for animal miRNAs: Small size, only 21-23nt in length Only display partial complementarity to targets Multiple miRNAs/mR

16、NA pairs,He 2004,Computational prediction of miRNA targets,Current target prediction rules,“Seed” pairing (residues 2-8 from 5 end of the miRNA) is the major determinant No G:U base pairs in the seed Weak sites “rule”: Weak sites that cannot silence alone cannot silence in combination,Zhao 2007,Dete

17、rminants beyond “seed” pairing Distance from stop codon Local AU-rich sequences Accessibility of the 3UTR Evolutionarily conservation of MRE Number of MREs in the 3UTR,Current prediction algorithms, False positives False negatives Inconsistency between algorithms,T

18、arget validation,Protein level changes upon alteration of miRNA expression Reporter system,What is miRNA? How are miRNAs discovered? How are miRNAs synthesized in vivo? How many miRNAs are there? Where are miRNAs located in the genome? When and where are miRNAs expressed? How miRNAs regulate gene ex

19、pression? What genes do miRNAs regulate? What regulate miRNAs? What are the general functions of miRNAs? Is there any therapeutic application?,Todays topics,Regulation of miRNA biogenesis and function,Hong-Geller and Li 2011,Transcription of pri-miRNA Editing of pri-miRNA Release of pre-miRNA Export

20、ation of pre-miRNA Creation of mature miRNA Loading of guide strands into RISCs Degradation of miRNA Silencing of target,Function of miRNA,Cancer Immune system development and response,Abnormal expression of miRNAs in cancers,Heneghan and Kerin 2010,Abnormal expression of miRNAs in cancers,miRNAs act as oncogenes and tumor suppressors,miRNA as tumor suppressor: miR-15, miR-16, let-7 m