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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:

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Non-coding RNAs ( ncRNAs )

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  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)

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