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Non-coding RNAs (ncRNAs) 1.ncRNAs: A brief intro 2.Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp 3.miRNAs: Biogenesis, measurement, functional analysis,

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Presentation on theme: "Non-coding RNAs (ncRNAs) 1.ncRNAs: A brief intro 2.Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp 3.miRNAs: Biogenesis, measurement, functional analysis,"— Presentation transcript:

1 Non-coding RNAs (ncRNAs) 1.ncRNAs: A brief intro 2.Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp 3.miRNAs: Biogenesis, measurement, functional analysis, & utility Key references: 1.Prensner JR & Chinnaiyan AM. The emergence of lincRNAs in cancer biology. Cancer Disc. 1: , Guttman M and Rinn JL. Modular regulatory principles of large non-coding RNAs. Nature 482, , Lee JT. Epigenetic regulation by long noncoding RNAs. Science 338: , 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 *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, , 2009; Khalil AM et al., PNAS 106, , 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. Science 337: , % 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 11,224 pseudogenes 15 terabites of data (32 groups; 24 exp/120 TF))

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

4 Critical features of lncRNAs

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

6 Gene expression regulation by lncRNAs Prensner JR & Chinnaiyan AM. Cancer Disc. 1: , 2011.

7 In mammals, XCI is triggered by Xist RNA to equalize gene expression between the sexes. The random form of XCI occurs ONLY once on E , when the epiblast has 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 Mechanisms of X-inactivation

8 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 histiocytic sarcoma. (Yildirim E et al., Xist RNA is a potent suppressor of hematologic cancer in mice. Cell 152, , 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: , 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 shRNA in 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 & Chinnaiyan AM. Cancer Disc. 1: , 2011.

14 Mechanisms of lncRNA function Lee JT, Science 2012

15 Mechanisms of lncRNA function Lee JT, Science 2012

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

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

19 19 Thomson, J. M/Hammond S

20 Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4

21 Nijiro Nohata, et al. ELSEVIER.2012

22 Auyeung VC, et al. Cell 152: , 2013 *For microprocessor recognition, sequences within 40 nt upstream and 40 nt downstream of the pre-miRNA hairpins are required. *Most C. elegans pri-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. elegans pri- miRNAs and, when added to the C. elegans pri- miRNAs, confer more efficient processing in mammalian cells.

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

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

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

26 26 Evolutionary Conserved miRNA Cluster Thomson, J. M/Hammond S

27 27 miR17-92 is Conserved in Vertebrates Thomson, J. M/Hammond S

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

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

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31 tumour suppressive and oncogenic microRNAs T Paranjape, et al. GUT. 2009

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

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35 35 Global Reduction in miRNAs in the Context of Cancer… Red = Abundant Blue= Depleted Lu, J., et al Nature 435:

36 36 Thomson, et al Genes Dev. 20(16):

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39 Next generation sequencing miRNA sequencing RNA sequencing HITS-CLIP sequencing

40 miRNA seq short RNAs that are about 21-25bp are first selected by column or electrophoresis. A starting quantity of ug 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

41 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.

42 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 immuneoprecipitated cross-linked Argonaute-miRNA-mRNA complexes. Starbase is the database for exploring protein-RNA and miRNA-target interactions from HITS-CLIP

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

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

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55 (PSA +/hi ) Increasingly ‘mature’ PCa cells ABCG2+  2  1+ SP CD44+ PCa stem/progenitor cells (AR -/lo ; PSA -/lo ) Holoclones Prolif. How do miRNAs regulate tumorigenic PCa cells? ALDH hi Can (Julie) Liu

56 miR-34a is Underexpressed in CD44 + PCa Cells

57 Ectopic Expression of miR-34a Inhibits Prostate Tumor Development

58 Re-expression of miR-34a in CD44 + PCa cells abolishes tumor regeneration

59 Anti-miR-34a Promotes PCa Regeneration and Metastasis

60 Systemically delivered miR-34a inhibits tumor development and metastasis PC3 Therapeutic Exp NC miR-34a

61 CD44 as a DIRECT & FUNCTIONAL target of miR-34a

62 CD44 knockdown phenocopies miR-34a effects

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65 65 Overexpression of miR-17-19b in a Mouse Model of Human Lymphoma Thomson, J. M/Hammond S

66 66 Expression of miR17-19b Results in B-cell Lymphoma He, et al Nature 435(7043):

67 miRNA as therapeutics companyMicrornadiseaseformulaHalf life RegulusAnti-21, anti- 122, Anti-10b HCC Hepatitis C GBM Single stranded modified oligos 21 days stability does not leads to destruction of miRNA. Santaris Pharma Miravirsen (anti-21) Hepatitis CLNA5 doses in 120 days, 2weeks stability Phase ii, first in human miRgenAnti-miR-208Heart failureLNAAlternative week injection Reverse heart failure (induced by high Na+ diet) miRxmiR-34aLiver cancer (Hep3B cell injection) Oligo+Nov34 0 Pre-clinicalTarget HDAC1

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