1 / 68

Non-coding RNAs ( ncRNAs )

Non-coding RNAs ( ncRNAs ). ncRNAs : A brief intro Long non-coding RNAs ( lncRNAs or lincRNAs ): >200 bp miRNAs : Biogenesis, measurement, functional analysis, & utility. Key references: Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1:

rene
Télécharger la présentation

Non-coding RNAs ( ncRNAs )

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Non-coding RNAs (ncRNAs) ncRNAs: A brief intro Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp miRNAs: Biogenesis, measurement, functional analysis, & utility Key references: Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1: 391-407, 2011. 2. Guttman M and Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature 482, 339-346, 2012. 3. Lee JT. Epigenetic regulation by long noncoding RNAs. Science 338: 1435-9, 2012. Dean G. Tang (Molecular Biology of Cancer; 3/6/2013) Acknowledgement: Julie Liu (Tang lab); David Brown (MiRna Therapeutics); Thomson/Hammond (UNC)

  2. Encylcopedia of DNA Elements (EnCODE)/Sept 2012 Science 337:1159-60, 2012 (32 groups; 24 exp/120 TF)) 76% of the genome is transcribed; 18,400 (8,800 sRNA+9,600 lncRNA); 2.89 million DnaseI sites (1/3 in each cell type and 3,700 in all cells); >3.9 million TF binding sites >70,000 ‘promoter’ regions ~400,000 ‘enhancer’ regions 11,224pseudogenes 15 terabitesof data *Bioinformatic analysis of the chromatin marks in intergenic DNA regions and of ESTs predicts >5,000 lncRNA genes in the human genome (Guttman et al., Nature 458, 223-227, 2009; Khalil AM et al., PNAS 106, 11667-72, 2009). *Including antisense, intronic, intergenic, pseudogenes and retrotransposons. *The most ‘famous’ lincRNAs include Xist, H19, Air, Hotair, etc, which all seem to operate at the transcriptional level by binding to proteins in histone-modifying complexes and targeting them to particular genes.

  3. Types of ncRNAs Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.

  4. Critical features of lncRNAs

  5. Examples of lncRNAs Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.

  6. Gene expression regulation by lncRNAs Prensner JR & ChinnaiyanAM. Cancer Disc. 1: 391-407, 2011.

  7. Mechanisms of X-inactivation • In mammals, XCI is triggered by Xist RNA to equalize gene expression between the sexes. • The random form of XCI occurs ONLY once on E4.5-5.5, when the epiblast has 10-20 cells. • Beyond E5.5, the inactive X (Xi; the Barr body) enters into a ‘maintenance phase’ in which the same X chromosome is propagated as Xi for the remainder of female life. • The X-inactivation center (XIC), ~100kb, contains several noncoding RNA genes Xist, Tsix, Jpx/Enox, and Xite. There are also 2 protein-coding genes (Tsx and Cnbp2). The Xist promoter is the master regulator of X inactivation. • Initiation of XCI depends on Xist(the 17 kb X-inactive specific transcript) that targets and tethers PRCs to the X chromosome in cis. Xist is dispensable once the Xi is established. • Xi Xist expression • Tsix expression • XaXist expression • Tsix expression

  8. Mechanisms of X-inactivation Lee JT, Science 2012

  9. Mechanisms of X-inactivation • PRC2, H3K27me3 • RepA binding to EZH2 • Tsix acting as decoy • Conditional deletion of Xist in blood cells: • Born alive, viable, but females die around 2 months • Massive spleen in female, hyperplasia (early stage) to leukemia (late stage). • Progressive bone marrow disease, myelofibrosis, leukemia (mixed MPN/MDS), and histiocyticsarcoma. (Yildirim E et al., Xist RNA is a potent suppressor of hematologic cancer in mice. Cell 152, 727-742, 2013).

  10. Genome regulation by long noncoding RNA (H. Chang) • HOTAIR (HOX antisense intergenic RNA): on chr.12, encodes a 2.2kb lncRNA. • HOTAIR is located in the HoxC cluster, interacts with Suz12, EZH2, and LSD1 as a scaffold, and silences HoxD cluster. • HOTAIR is upregulated in many cancers (br cancer and HCC) • HOTTIP& HOTAIRM1: located at opposite ends of the HOXA locus, activating HOXA transcription. • NEST: chr.12 • Controls susceptibility of virus infection; • Interacts with WRD5, promotes H3K4me3 at INFG, and activates INFG. • overexpressionresistant for pathogen challenge. Gomez JA et al., Cell 152: 742-754, 2013.

  11. lncRNAs in prostate cancer (A. Chinnaiyan) • HOTAIR: highly expressed in breast Ca, lung Ca, but low in PCa. • PCAT-1, a novel prostate-specific regulator of cell proliferation, a transcriptional repressor, and a target of PRC2. It’s a 1.9 kb pA-containing lncRNA comprised of 2 exons and located in the Chr8q24 gene desert (Presener JR et al., Nature Biotech 29:742-9, 2011). • SChLAP1 (Second Chromosome Locus Associated with Prostate-1), a >500kb locus in a gene desert in Chr2q31, including PCAT-109, PCAT-114. • SChLAP1 over-expressed in 20% PCa patients. • Correlates with poor outcome, more aggressive samples.

  12. SChLAP1 expression: • Nuclear staining in VCaP, 22Rv1, LNCaP. • In situ hybridization of FFPE PCa revealed high expression levels. • Biological functions: • Promotes invasion, (not strong phenotype in proliferation) • KD with shRNAin vitro (in cells) and in vivo (cardiac injection) results decrease in invasion and met. spread. • Molecular mechanisms: • Microarray of gene expression of a serial of PCa KD of SchLAP1. • Reversed relationship with SWN/SNF complex. • Pull down SNF5, also pull down SChLAP1. • ChIP –seq of SNF5 in RWPE-SchLAP1 OE shows reduced binding.

  13. Mechanisms of lncRNA function Prensner JR & ChinnaiyanAM. Cancer Disc. 1: 391-407, 2011.

  14. Mechanisms of lncRNA function Lee JT, Science 2012

  15. Mechanisms of lncRNA function Lee JT, Science 2012

  16. Non-coding RNAs (ncRNAs) ncRNAs: A brief intro Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp miRNAs: Biogenesis, measurement, functional analysis, utility Key references: Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1: 391-407, 2011. 2. Guttman M and Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature 482, 339-346, 2012. 3. Lee JT. Epigenetic regulation by long noncoding RNAs. Science 338: 1435-9, 2012. Dean G. Tang (Molecular Biology of Cancer; 3/6/2013)

  17. MicroRNAs are transcribed by RNA Polymerase II Thomson, J. M/Hammond S Exonic Non-coding Intronic Non-coding and coding Lee, et al., EMBO J. 23:4051-60 2004

  18. Thomson, J. M/Hammond S

  19. Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4

  20. NijiroNohata, et al. ELSEVIER.2012

  21. *For microprocessor recognition, sequences within 40 nt upstream and 40 nt downstream of the pre-miRNA hairpins are required. *Most C. eleganspri-miRNAs lack determinants required for processing in human cells. *Pairing in the basal stem is important. *Primary sequence features, including the basal UG, the CNNC, and the apical GUG motifs, contribute to efficient processing in human cells. *79% of the conserved human miRNAs contain at least one of the three motifs. *These motifs are not enriched in C. eleganspri- miRNAs and, when added to the C. eleganspri- miRNAs, confer more efficient processing in mammalian cells. Auyeung VC, et al. Cell 152: 844-858, 2013

  22. Krol, Loedige &Filipowicz, Nat Rev Genetics, 2010 Vol 11 No 9

  23. David & McCray Jr, Nat Rev Genetics, 2011 Vol 12 No 5

  24. Mechanisms of gene regulation by miRNAs Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4

  25. Evolutionary Conserved miRNA Cluster Thomson, J. M/Hammond S

  26. Thomson, J. M/Hammond S miR17-92 is Conserved in Vertebrates

  27. mRNAs as Regulators of miRNAs ceRNA (competing endogenous RNA) MREs (microRNA response sequences) L Salmena,et al. Cell. 2011

  28. Non-coding RNAs (ncRNAs) ncRNAs: A brief intro Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp miRNAs: Biogenesis, measurement, functional analysis, utility Key references: Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1: 391-407, 2011. 2. Guttman M and Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature 482, 339-346, 2012. 3. Lee JT. Epigenetic regulation by long noncoding RNAs. Science 338: 1435-9, 2012. Dean G. Tang (Molecular Biology of Cancer; 3/6/2013)

  29. tumoursuppressive and oncogenic microRNAs T Paranjape, et al. GUT. 2009

  30. Profiling the mature and primary microRNA transcripts Mature Drosha Cleavage site Pri-miRNA /~-OH Thomson, J. M/Hammond S

  31. Global Reduction in miRNAs in the Context ofCancer… Red = Abundant Blue= Depleted Lu, J., et al Nature 435:834 2005

  32. Thomson, et al Genes Dev. 20(16):22202-7 2006

  33. Next generation sequencing • miRNA sequencing • RNA sequencing • HITS-CLIP sequencing

  34. miRNA seq • short RNAs that are about 21-25bp are first selected by column or electrophoresis. A starting quantity of 50-100ug total RNA is required for gel purification and size selection. • Adaptor ligation adds DNA adapters to both ends of the small RNAs, which act as primer binding sites during RT and PCR amplification. • Then these small adaptor-ligated RNAs will be RT and PCR and then sequencing

  35. RNA seq • provides information on the level of RNA transcribed from a particular genome, can be use to identify miRNA targetome. • Total RNA is first isolated and then Poly A library is constructed by using poly T primer for all the coding RNAs, followed by NGS and transcriptome alignment. • Disadvantage: will miss the mRNA targets that are regulated by miRNA at translational repression.

  36. HITS-CLIP seq • High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP), is a genome-wide means of mapping protein–RNA binding sites in vivo. • UV irradiation to crosslink RNA to associated RNA-binding proteins, then IP using antibody against argonaute protein (AGO2 orAGO1), followed by deep-sequencing. • It identifies direct target sequences through the sequencing of RNAs from immuneoprecipitatedcross-linked Argonaute-miRNA-mRNA complexes. • Starbase is the database for exploring protein-RNA and miRNA-target interactions from HITS-CLIP

  37. HITS-CLIP (CLIP-Seq) Thomson D et.al., 2011

  38. Non-coding RNAs (ncRNAs) ncRNAs: A brief intro Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp miRNAs: Biogenesis, measurement, functional analysis, utility Key references: Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1: 391-407, 2011. 2. Guttman M and Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature 482, 339-346, 2012. 3. Lee JT. Epigenetic regulation by long noncoding RNAs. Science 338: 1435-9, 2012. Dean G. Tang (Molecular Biology of Cancer; 3/6/2013)

More Related