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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: - PowerPoint PPT Presentation
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Non-coding RNAs (ncRNAs)
1. ncRNAs: A brief intro2. Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp3. miRNAs: Biogenesis, measurement, functional analysis, & utility
Key references:1. 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)
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, 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.
Science 337:1159-60, 2012
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’ regions11,224 pseudogenes15 terabites of data
(32 groups; 24 exp/120 TF))
Types of ncRNAs
Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.
Critical features of lncRNAs
Examples of lncRNAs
Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.
Gene expression regulation by lncRNAs
Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.
• 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
Mechanisms of X-inactivation
Mechanisms of X-inactivation
Lee JT, Science2012
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, 727-742, 2013).
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.– overexpressionresistant for pathogen challenge.
Gomez JA et al., Cell 152: 742-754, 2013.
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.
• 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.
Mechanisms of lncRNA function
Prensner JR & Chinnaiyan AM. Cancer Disc. 1: 391-407, 2011.
Mechanisms of lncRNA function
Lee JT, Science2012
Mechanisms of lncRNA function
Lee JT, Science2012
Non-coding RNAs (ncRNAs)
Key references:1. 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)
1. ncRNAs: A brief intro2. Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp3. miRNAs: Biogenesis, measurement, functional analysis, utility
ExonicNon-coding
MicroRNAs are transcribed by RNA Polymerase II
IntronicNon-codingand coding
Lee, et al., EMBO J. 23:4051-60 2004
Thomson, J. M/Hammond S
19Thomson, J. M/Hammond S
Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4
Nijiro Nohata, et al. ELSEVIER.2012
Auyeung VC, et al. Cell 152: 844-858, 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.
Krol, Loedige &Filipowicz, Nat Rev Genetics, 2010 Vol 11 No 9
David & McCray Jr, Nat Rev Genetics, 2011 Vol 12 No 5
Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4
Mechanisms of gene regulation by miRNAs
26
Evolutionary Conserved miRNA Cluster
Thomson, J. M/Hammond S
27
miR17-92 is Conserved in VertebratesThomson, J. M/Hammond S
mRNAs as Regulators of miRNAs
L Salmena,et al. Cell. 2011ceRNA (competing endogenous RNA)MREs (microRNA response sequences)
Non-coding RNAs (ncRNAs)
Key references:1. 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)
1. ncRNAs: A brief intro2. Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp3. miRNAs: Biogenesis, measurement, functional analysis, utility
tumour suppressive and oncogenic microRNAs
T Paranjape, et al. GUT. 2009
/~-OH
Profiling the mature and primary microRNA transcripts
Pri-miRNADrosha Cleavage site
Mature
Thomson, J. M/Hammond S
35
Global Reduction in miRNAs in the Context of Cancer…
Red = Abundant
Blue= Depleted
Lu, J., et al Nature 435:834 2005
36Thomson, et al Genes Dev. 20(16):22202-7 2006
Next generation sequencing
• miRNA sequencing• RNA sequencing• HITS-CLIP sequencing
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
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.
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
HITS-CLIP (CLIP-Seq)
Thomson D et.al., 2011
Non-coding RNAs (ncRNAs)
Key references:1. 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)
1. ncRNAs: A brief intro2. Long non-coding RNAs (lncRNAs or lincRNAs): >200 bp3. miRNAs: Biogenesis, measurement, functional analysis, utility
(PSA+/hi)
Increasingly ‘mature’ PCa cells
ABCG2+
a2b1+SP
CD44+
PCa stem/progenitor cells (AR-/lo; PSA-/lo)
Holoclones
Prol
if.
How do miRNAs regulate tumorigenic PCa cells?
ALDHhi Can (Julie) Liu
miR-34a is Underexpressed in CD44+ PCa Cells
Ectopic Expression of miR-34a Inhibits Prostate Tumor Development
Re-expression of miR-34a in CD44+ PCa cells abolishes tumor regeneration
Anti-miR-34a Promotes PCa Regeneration and Metastasis
Systemically delivered miR-34a inhibits tumor development and metastasis
PC3 Therapeutic Exp
NCmiR-34a
CD44 as a DIRECT & FUNCTIONAL target of miR-34a
CD44 knockdown phenocopies miR-34a effects
CD44 knockdown phenocopies miR-34a effects
65
Overexpression of miR-17-19b in a Mouse Model of Human Lymphoma
Thomson, J. M/Hammond S
66
Expression of miR17-19b Results in B-cell Lymphoma
He, et al Nature 435(7043):828-33 2005
miRNA as therapeuticscompany Microrna disease formula Half life
Regulus Anti-21, anti-122,Anti-10b
HCCHepatitis CGBM
Single stranded modified oligos
21 days stability
does not leads to destruction of miRNA.
Santaris Pharma
Miravirsen (anti-21)
Hepatitis C LNA 5 doses in 120 days, 2weeks stability
Phase ii, first in human
miRgen Anti-miR-208 Heart failure LNA Alternative week injection
Reverse heart failure (induced by high Na+ diet)
miRx miR-34a Liver cancer (Hep3B cell injection)
Oligo+Nov340
Pre-clinical Target HDAC1