Non-coding RNAs ( ncRNAs )

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