Transcript Non-coding RNAs (ncRNAs).

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:
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
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
11,224 pseudogenes
15 terabites of 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.
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.
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
Mechanisms of X-inactivation
Lee JT, Science
2012
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.
– overexpressionresistant 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, Science
2012
Mechanisms of lncRNA function
Lee JT, Science
2012
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:
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)
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
19
Thomson, J. M/Hammond S
Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4
Nijiro Nohata, et al. ELSEVIER.2012
*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 primiRNAs and, when added to the C. elegans primiRNAs, confer more efficient processing in
mammalian cells.
Auyeung VC, et al. Cell 152: 844-858, 2013
Krol, Loedige &Filipowicz, Nat
Rev Genetics, 2010 Vol 11 No 9
David & McCray Jr, Nat Rev
Genetics, 2011 Vol 12 No 5
Mechanisms of gene regulation by miRNAs
Pasquinelli, Nat Rev Genetics, 2012 Vol 13 No 4
Evolutionary Conserved miRNA Cluster
26
Thomson, J. M/Hammond S
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
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:
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)
tumour suppressive and oncogenic microRNAs
T Paranjape, et al. GUT. 2009
Profiling the mature and primary microRNA transcripts
Mature
Drosha Cleavage site
Pri-miRNA
/~-OH
Thomson, J. M/Hammond S
Global Reduction in miRNAs in the Context of Cancer…
Red = Abundant
Blue= Depleted
35
Lu, J., et al Nature 435:834 2005
36
Thomson, 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 genomewide 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)
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:
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)
How do miRNAs regulate tumorigenic PCa cells?
PCa stem/progenitor cells
(AR-/lo; PSA-/lo)
Holoclones
Can (Julie) Liu
ALDHhi
CD44+
a2b1+
SP
ABCG2+
(PSA+/hi)
Prolif.
Increasingly ‘mature’ PCa cells
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
NC
miR-34a
CD44 as a DIRECT & FUNCTIONAL target of miR-34a
CD44 knockdown phenocopies miR-34a effects
CD44 knockdown phenocopies miR-34a effects
Overexpression of miR-17-19b in a Mouse Model of Human
Lymphoma
Thomson, J. M/Hammond S
65
Expression of miR17-19b Results in B-cell Lymphoma
66
He, et al Nature 435(7043):828-33 2005
miRNA as therapeutics
company
Microrna
disease
formula
Half life
Regulus
Anti-21, anti122,
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 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
Oligo+Nov34
0
Pre-clinical
Target
HDAC1
Liver cancer
(Hep3B cell
injection)