RNA Methodology
: Post-transcriptional gene regulation
by RNA-binding proteins & MicroRNAs
SAIHST, SKKU
Hyeon Ho Kim
3’UTR
5’UTR
Hyeon H Kim
CR
AAAAAAAA
RNA Methodology
1
Building Blocks of DNA & RNA
Differences between DNA and RNA
1. Sugar component : in case of DNA, deoxyribose; in case of RNA, ribose
2. Thymine in DNA; Uracil in RNA
3. Stability : DNA is more stable than RNA
- presence of thymine rather than uracil in DNA is important to the stability of DNA
- hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA
Hyeon H Kim
RNA Methodology
2
Structure of RNA
Unlike DNA (long double helix), most RNAs are single-stranded and exhibits a variety of conformations
1. Structures
- Secondary structure (depending on distance of pairing nucleotides)
: Hairpin, formed by pairing of bases within 5~10 nucleotides
: Stem-loop, formed by pairing of bases that are separated by >10 to several hundred nucleotides
- Tertiary structure (Pseudoknot)
: formed by interaction of secondary loops through base pairing between complementary bases
[example: telomerase RNA core domain]
: catalytic capacities: Ribozyme
2. Ribozyme
- RNA that acts as a catalyst
- some ribozyme can catalyze Splicing : cut, remove, and ligate RNA
* Some RNAs carry out self-splicing
Hyeon H Kim
RNA Methodology
3
Structures of RNA
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RNA Methodology
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Polymerization of RNA
Transcription
- One DNA strand acts as template
- Polymerization of RNA
using rNTPs and RNA polymerase
: Nucleophilic attack by the 3’ oxygen
→ formation of phosphodiester bond
and release of pyrophosphate (PPi)
* always synthesized in the 5’ → 3’ direction
- The site on the DNA at which RNA polymerase
begins transcription is numbered +1
- Newly synthesized RNA is complementary to
the template DNA strand
* identical with the nontemplate DNA strand
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RNA Methodology
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Transcription – Step 1. Initiation
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RNA Methodology
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Transcription – Step 2/3. Elongation / Termination
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RNA Methodology
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RNA Processing : Modification & Splicing
Modification of two ends
1. Cap of 5’ end (7-methylguanylate, m7Gppp) :
- protects an mRNA from enzymatic degradation
- assists in its export to the cytoplasm
- bound by a protein factor required to begin translation in the cytoplasm
2. Polyadenylation of 3’ end
- add adenylic acid to free 3’-hydroxyl group by poly(A) polymerase
- resulting poly(A) tail contains 100~200 bases
- poly(A) polymerase is part of a complex of proteins and cleave a transcript at a specific site
RNA splicing
- internal cleavage of a transcript to excise the introns, following by ligation of the coding exons
- functional eukaryotic mRNAs retain noncoding regions on 3’ and 5’ ends (UTR, untranslated regions)
- in mammalian mRNAs: 5’ UTR (a hundred or more nucleotides long), 3’ UTR (several kb in length)
- Alternative splicing in eukaryotic genes
: multiple introns permits expression of multiple, related proteins from a single gene
: an important mechanism for production of different forms of a protein, called isoforms
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RNA Methodology
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RNA Processing : Splicing
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RNA Methodology
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3 Types of RNA : mRNA, tRNA, and rRNA
1. Messenger RNA (mRNA)
- carries the genetic information transcribed from DNA in a linear form
- mRNA is read in sets of three-nucleotide sequence, called codon, each of which specifies a
particular amino acid
2. Transfer RNA (tRNA)
- key to deciphering the codons in mRNA
- each type of amino acid has its own subset of tRNA, which bind the amino acid and carry it to the
growing end of a polypeptide chain
- correct tRNA with its attached amino acid is selected at each step because each specific tRNA
molecule contains a three-nucleotide sequence, an anticodon, that can base-pair with its
complementary codon in the mRNA
3. Ribosomal RNA (rRNA)
- associates with a set of proteins to form ribosomes
- these complex structure, which physically move along an mRNA molecule, catalyze the assembly of
amino acids into polypeptide chains
- they also bind tRNAs for protein synthesis
- ribosome are composed of a large and small subunit, each of which contains its own rRNA
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RNA Methodology
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Synthesis of Proteins on Ribosome
Key players in protein synthesis
Process of translation
1. mRNA
1. Initiation
2. Aminoacylated tRNA
2. Elongation
3. rRNA-containing ribosome
3. Termination
Methionyl-tRNAiMet
- Methionyl-tRNAiMet recognizes the AUG start codon
- AUG codon for methionine functions as the start codon in the vast majority of mRNAs
Two different methionine tRNAs * Synthesized by same aminoacyl-tRNA synthetase (MetRS)
1. tRNAiMet : initiates protein synthesis
- Only Methionyl-tRNAiMet (activated methionine attached to tRNAiMet) can bind at the appropriate
site on the small ribosomal subunit, P site
2. tRNAMet : incorporate methionine only into a growing protein chain
- The regular Met-tRNAMet and all other charged tRNA bind only to another ribosomal site, A site
*In bacteria, initiation methionine linked with a formyl group, N-fomylmethionine
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RNA Methodology
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Translation : Decoding of mRNA by tRNA
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RNA Methodology
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Synthesis of Aminoacyl-tRNA
Aminoacyl-tRNA
- Translation requires tRNAs and enzyme called aminoacyl-tRNA synthetase
- To participate protein synthesis, tRNA molecule must become chemically linked to a particular
amino acid via a high-energy bond, forming an aminoacyl-tRNA
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RNA Methodology
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Translation : Step 1. Preinitiation complex
- Large and small subunits are not engaged but are kept apart by binding of two initiation factors
- For small subunit, eIF3 and for large subunit, eIF6
Preinitiation complex
- 40S subunit containing eIF3
- eIF3 associates with eIF1A
- Ternary complex (eIF2-GTP + Met-tRNAiMet )
* Only GTP-bound eIF2 can bind to Met-tRNAiMet
* If eIF2 is phosphorylated on serine residue, eIF2 is not able to exchange GDP to GTP
→ it can not bind to Met-tRNAiMet : Anti-cancer target
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RNA Methodology
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Translation : Step 1. Initiation complex
* eIF4, cap-binding protein
1. eIF4E: bind to 5’ cap structure
2. eIF4G: bind to eIF3
3. eIF4B: architectural role positioning eIF4A
4. eIF4A: RNA helicase (unwind RNA)
Initiation complex - Preinitiation complex + eIF4 (cap-binding complex) + mRNA
Kozak sequence - (5’) ACCAUGG (3’) specific surrounding nucleotides
- helping to search for start codon
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RNA Methodology
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Translation : Step 1. Initiation
Correct binding of Met-tRNAiMet to start codon
1. the GTP bound by eIF2 is hydrolyzed to GDP: irreversible step preventing further scanning
2. eIF1, 2, 3, and 4 dissociate
3. Small subunit forms complete 80S ribosome catalyzed by eIF5 and 6
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RNA Methodology
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Translation : Step 2. Elongation
- Entry of aa-tRNA & Ribosome conformational change
-
At the completion of initiation, Met-tRNAiMet is bound to the P site,
Second aminoacyl-tRNA is brought into A site as a ternary complex in association with EF1a-GTP
When that occurs correctly, the GTP in the associated EF1a-GTP is hydrolyzed
Hydrolysis of GTP promotes a conformational change : Proofreading step
lead to tight binding of the aminoacyl-tRNA in the A site and release of EF1a-GDP complex
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RNA Methodology
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Translation : Step 2. Elongation
– Peptide bond formation & Ribosome translocation
- Formation of peptide bond : α amino group of the second amino acid reacts with the “activated”
Met on the initiator tRNA : this peptidyltransferase reaction is catalyzed by the large rRNA
- Following peptide bond synthesis, GTP in EF2-GTP complex is hydrolyzed : irreversible process
→ prevents the ribosome from moving along the RNA in the wrong direction
or from translocating an incorrect number of nucleotides : Proofreading step
- GTP hydrolysis induces conformational change of 80S ribosome
: tRNAiMet (without activated methionine) is moved to the E (exit) site on the ribosome
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RNA Methodology
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Translation : Step 3. Termination & Reuse of ribosome
Releasing factor (RF)
1. eRF1
: similar shape to tRNA
: binding to A site and recognize stop codon directly
2. eRF3
: second eukaryotic RF
: GTP-binding protein
: eRF3-GTP acts in concert with eRF1 to promote
cleavage of the peptidyl-tRNA
: releasing complete protein chain
- Peptidyl-tRNA bond of tRNA in the P site is not cleaved
until eRF1 recognizes stop codon correctly
: Proofreading step
- A newly synthesized protein folds into its native 3-D
structure, a precess facilitated by chaperones
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RNA Methodology
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Simultaneous Translation
to increase overall rate of protein synthesis
- Two ways for increasing the overall rate of protein synthesis
1. Simultaneous translation of a single mRNA by multiple ribosome: Polysome or Polyribosome
2. Rapid recycling of ribosomal subunits
- Poly(A)-binding protein I (PARPI) can interact both an mRNA poly(A) tail and the eIF4G
- Two ends of an mRNA molecule can be bridged: Circular mRNA
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RNA Methodology
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Common Questions: the perspective of PTGR
Q1. Although promoter activity was not changed, the level of mRNA was
different
- Stabilization of mRNA
- Destabilization of mRNA
Q2. Despite the same level of mRNA, protein expression was different
- Decreased translation of mRNA
- Increased translation of mRNA
A: Post-transcriptional gene regulation
by RNA-binding proteins or MicroRNAs
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RNA Methodology
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Post-Transcriptional Gene Regulation
P-body
Stress granule
Translation
Exosome
Decay
RNA pol II
Stabilization
DNA
Transcription
Splicing
Maturation
mRNA Turnover
mRNA
mRNA
General Introduction
Global Analysis of Stress-regulated mRNA Turnover
B
A
Total RNA
Extraction
Nuclei Isolation
Total RNA Array
Labeling of Nascent RNA (33P) by
Elongation of Established Transcription Complexes
Labeling of cDNA (33P)
by Reverse Transcription
Hybridization of Reverse Transcripts
Hybridization of Transcripts
Nuclear Run-on (NRO) Array
Nuclear Run-on (NRO) Array
Total RNA Array
y = fold increase in transcription
x=
p21, Gadd45
HS
UVC
PG
HS
UVC
PG
Fold change in
steady-state levels
y=
Fold change in
transcription levels
Hyeon H Kim
11% 17%
40%
DATA ANALYSIS
x/y
Transcriptional
Downregulation
mRNA
Stabilization
control
control
mRNA
Destabilization
DP1, Cyclin D1, Cdk4,
Cdc25C, Cyclin A, Cyclin B1
RNA Methodology
32%
Transcriptional
Upregulation
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Non-Coding Region in Animal Genomes
C. elegans
H. sapiens
3 Gb
100 Mb
Coding region
Coding region
1%
15%
85%
Non-coding region
99%
Non-coding region
General Introduction
RNA-Binding Proteins vs. MicroRNAs
[A]n
[A]n
RBPs Regulate:
MicroRNAs regulate:
Pre-mRNA splicing
Nuclear maturation
Export of mRNA to cytoplasm
Integrity of the 5’ and 3’ ends
mRNA stabilization/destabilization
Subcytoplasmic mRNA localization
Translation
Small RNAs (20-22 nt)
Hyeon H Kim
Translational suppression
(incomplete complementarity)
mRNA decay
(complete complementarity)
RNA Methodology
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Conserved RNA-binding Motifs
RNA recognition motif (RRM) = RNA-binding domain (RBD)
: most common RNA-binding domain in hnRNP protein, consisting of 80 residue domain
- RRM domain
: consist of a four-stranded beta sheet flanked on one side by two alpha helices
: beta sheet forms a positively charged surface to interact with negatively charged RNA
- KH motif
: found in hnRNPK protein
: similar to RRM domain but smaller, consisting of a 3-stranded beta sheet
- RGG box
: contains five Arg-Gly-Gly (RGG) repeats with several interspersed aromatic acid (arginine-rich nature)
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RNA Methodology
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RNA-Binding Proteins (RBPs)
RBP
Function
HnRNP A1
HnRNP A2/B1
HnRNP C1/C2
HnRNP D
HnRNP F
HnRNP H
HnRNP I (PTB)
HnRNP K
HnRNP L
HnRNP Q
HnRNP U
PABP
Splicing, export
Splicing, localization
Splicing, stability
Telomeres, stability
Splicing
Splicing, polyadenylation
Splicing, polyadenylation
Transcription, translation
Export, stability
Splicing
Nuclear retention
Stability, translation
HuR
Stability, translation
αCP1,2
BRF1
TTP
Stability, translation
Stability
Stability
Hyeon H Kim
RBP
Function
Yra1
Npl3
Nab4
SF2/SRp30a
SC35/SRp30b
SRp20
9G8
Magoh
Y14
Aly/REF
RNPS1
DEK
Upf3
SRm160
TIAR, TIA-1
………
Export
Export
Polyadenylation
Splicing
Splicing
Splicing, export
Splicing, export
EJC, localization
EJC, NMD
EJC, export
Splicing, EJC, NMD
Splicing, EJC
EJC, NMD
EJC, splicing
Translation
………
RNA Methodology
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Exon Definition in Long Pre-mRNAs
In human genome, average length of an Exon ≒ 150 bases - average length of an Intron ≒ 3,500 bases
How is RNA splicing precise?
SR proteins
- RNA-binding proteins, interacting with sequences within Exons called as Exonic Splicing Enhancers
- Subset of hnRNP containing one or more RRM RNA-binding domains
- Several protein-protein interaction domains rich in serine (S) and arginine (R) residues
- Working mechanism
1. Binding of SR proteins to exonic splicing enhancer
2. Cooperative binding of U1 snRNP to a 5’ splice site and U2 snRNP to a branch point
through a network of protein-protein interactions : Cross-exon recognition complex
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RNA Methodology
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AREs Influence mRNA Stability and Translation
3’UTR
5’UTR
CR
AAAAAAAA
ARE
(U- or AU-rich element)
- UUUUAUUUAAAAGUAUUUUAAAAAGAAAAUUUAUUUAUU
UUUUAUUUUACAUUUUAUUUUUUUUUUUUUAUUGUUA -
ARE (AU-rich element)
(1) Generally present in the 3’ untranslated region (UTR) of labile mRNAs
(2) Consisted of 40 - 120 nucleotides
(3) Function as potent regulators of mRNA stability and/or translation
(4) mRNAs encoding stress-response proteins, cytokines, growth factors,
transcription factors, oncogenes, tumor suppressor genes, and cell cycle
regulatory proteins
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RNA Methodology
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Gene Regulation by HuR
STORAGE
SG
DEGRADATION
Exo
TRANSLATION
PB
DNA
HuR
TRANSCRIPTION
EXPORT
STABILIZATION
SPLICING
MATURATION
mRNA
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RNA Methodology
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Functions of HuR
HuR
Survival of
Apoptotic
Damage
ProTα
SIRT1
p21
Bcl-2
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Evasion of
Immune
Recognition
TGF-β
Galectin-1
MKP-1
Elevated
Local
Angiogenesis
VEGF
HIF-1
RNA Methodology
Invasion
and
Metastasis
Enhanced
Cell
Division
MMP-9
MTA1
uPA
EGF
GM-CSF
Fos
Myc
Cyclin A
Cyclin B1
Cyclin D1
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Enhanced Expression of HuR in Cancer
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RNA Methodology
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Screening for RBP Target mRNA:
IP mRNA:Protein Complex
Total RNA
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RT-qPCR or Microarray
IgG IP
RNA Methodology
RBP IP
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Experimental Procedure of RNP IP
Coating magnetic beads
Sepharose protein A magnetic beads
+
Antibody (5~20 mg): control IgG or RBP
Wash
5 times with NT2 buffer
Digestion of DNA in IP
Incubating with DNase I at 37’C
Digestion of proteins in IP
Incubating with Proteinase K and SDS at 56’C
Isolation of RNA
Treated with phenol, precipitated using isopropanol,
and wash with 75% EtOH
Measurement of target mRNA
RT-qPCR
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RNA Methodology
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Identification of RBP’s Motives
Identification of Motif
RBP IP (w/ control IgG)
mRNA Stabilization
Translational Enhancer
Translational Suppressor
Associated with SG/PB
HuR Motif
Lopez de Silanes et al. (2004) PNAS
TIA-1 Motif
RNA isolation
Lopez de Silanes et al. (2005) MCB
Microarray
Identification of motif
mRNA Stabilization
Translational Suppressor
mRNA Destabilization
Translational Enhancer
NF90 Motif
AUF1 Motif
Kuwano et al. (2010) NAR
Mazan-Mamczarz et al. (2009)
Hyeon H Kim
TIAR Motif
Kim et al. (2007) MCB
NAR
RNA Methodology
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General Methods
RBP Modulates Target mRNA Stabilization
mRNA Stabilization
Stimulus
mRNA Destabilization
Stimulus
RBPs
P-Body
Exosome
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RNA Methodology
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mRNA Stability Assay
A
B
mRNA level
--
B
-
A
10 -
-
Actinomycin D
100
0
1
2
3
4
5
6
Actinomycin D (h)
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RNA Methodology
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RBP Modulates Target mRNA Translation
Induction of Translation
Stimulus
Repression of Translation
Stimulus
RBPs
Ribosome
SG/PB
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RNA Methodology
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Analysis of Translational Efficiency
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RNA Methodology
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OD254
Polysome-associated mRNA Analysis
Cytoplasmic Lysates
FreeMonosomes
10%
Polysomes
28S –
18S –
50%
Sucrose Gradients
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RNA Methodology
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Nascent Translation Assay:
A
** ** **
less translated
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B
** **
** **
** **
** **
** **
35S
labeling
IgG
A
B
** **
** **
** **
** **
** **
more translated
RNA Methodology
35S-Protein
41
General Introduction
RNA-Binding Proteins vs. MicroRNAs
[A]n
[A]n
RBPs Regulate:
MicroRNAs regulate:
Pre-mRNA splicing
Nuclear maturation
Export of mRNA to cytoplasm
Integrity of the 5’ and 3’ ends
mRNA stabilization/destabilization
Subcytoplasmic mRNA localization
Translation
Small RNAs (20-22 nt)
Hyeon H Kim
Translational suppression
(incomplete complementarity)
mRNA decay
(complete complementarity)
RNA Methodology
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Differences between siRNA and miRNA
Imperfect match
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Perfect match
RNA Methodology
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MicroRNA Biogenesis
Ago
RNA pol II
miRNA-coding gene
Transcription
Ago
Primary miRNA
(pri-miRNA)
NUCLEUS
CYTOSOL
Assembly
Mature miRNA
(miRNA)
Drosha complex
m7G
AAAAAAA
2nt Cleavage : Dicing
1st Cleavage : Cropping
Importin 5
Precursor miRNA
(pre-miRNA)
Dicer complex
Breakthrough Discoveries in miRNA Biology
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RNA Methodology
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History of microRNAs
Year
Hallmarks
1993
Lin-4 recognition as a small ncRNA
2000
Let-7 discovery
2000
RNAi “Unit”: 21~23 nts
2001
Dicer in miRNA biogenesis pathway
2002
miRNAs deregulation in cancer
2004
miRNA as a diagnostic biomarker
2005
miRNA-target interaction relevant to cancer
2005
Altered expression of miRNAs affects tumor formation/growth in vivo
2005
Connection between miRNAs and the Myc oncogene
2005
Inhibition of miRNA by antagomirs in mammalians
2006
Regulation of miRNAs by epigenetics in mammalian cells
2006
Deregulation of miRNAs in cardiovascular diseases
2007
Nuclear import of miRNAs
2007
Deregulation of miRNAs in neurodegenerative diseases
History of microRNAs
Year
Hallmarks
2007
miRNA target sequences in 5’UTR
2007
Deregulation of miRNAs in autoimmune diseases
2007
Deregulation of miRNAs in cancer metastasis
2007
miRNAs as transcription upregulators
2008
miRNAs target gene promoter and upregulate gene expression
2008
Detection of miRNAs in serum/plasma
2008
LNA-anti-miRNAs in primates
2008
miRNAs target coding sequence
2009
Proof of concept: miRNA delivery as cancer therapy
2010
Clinical application of LNA-anti-miRNAs
2010
miRNA as molecular decoys
2010
mRNA destabilization by miRNAs
2010
Overexpression of single miRNA is sufficient to cause cancer
Almeida et al. 2011
MicroRNA Target Prediction
miRanda
miRNA target prediction for human, drosophila and zebrafish
genomes
miRBase
a comprehensive repository for miRNAs and their predicted
targets
miRDB
an online database for miRNA target prediction and
functional annotations in animals
miRNAMap
a genomic maps of microRNA genes and their target genes
in mammalian genomes
miR2Disease
a database providing comprehensive resource of miRNA
deregulation in various human diseases
TarBase
a comprehensive database of experimentally supported
animal microRNA targets
PicTar
microRNA targets for vertebrates, fly and nematodes
TargetScan
a search for the presence of conserved sites that match the
seed of each miRNA
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RNA Methodology
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Databases for MicroRNA Expression
microRNA.org
predicted microRNA targets & target downregulation scores.
Experimentally observed expression patterns
HMDD
Human MicroRNA Disease Database (HMDD) is a database
that contains the experimentally supported miRNA-disease
association data, which are manually curated from
publications. The dysfunction evidence or miRNAs
and literature PubMed ID are also given
TransmiR
a web query-driven database integrating the experimentally
supported transcription factor and miRNA regulatory
relations
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RNA Methodology
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Detection of miRNA
The detection methods of miRNA
1. Based on molecular hybridization (such as Northern Blot)
- Insensitive, time-consuming and large amount of RNA.
2. miRNA Q-PCR Detection Kit
- Nucleic acid detection standard technology
- Rapid, specific and sensitive to detect miRNA
3. Two ways are available
- Using stem loop-specific primer (TaqMan)
- Using polyA polymerase and Universal primer (SYBR Green)
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RNA Methodology
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RT-qPCR for miRNA
Hyeon H Kim
RNA Methodology
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TaqMan miRNA Assay
TaqMan-based RT-qPCR of miRNAs
Step 1. Stem–loop RT
Stem–loop RT primers bind to at the 3′
portion of miRNA molecules and are reverse
transcribed with reverse transcriptase.
Step 2. Real-time PCR
Then, the RT product is quantified using
conventional TaqMan PCR that includes
miRNA-specific forward primer, reverse
primer and a dye-labeled TaqMan probes.
NAR vol. 33 no. 20 e179
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RNA Methodology
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QuantiMir Assay: System Bioscience (SBI)
1. PolyA polymerase
2. Universal reverse primer
3. Forward primer: miRNA itself
4. SYBR Green system
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RNA Methodology
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IP-RNP Experiments: IP of RISC (Ago2)
RISC IP
To check specific binding of miRNA
-
Target mRNA
miRNA
Transfection of pre-miRNA
: increased enrichment of target mRNA in Ago2 IP
RISC
Ago2
Ago2 Antibody
Hyeon H Kim
Transfection of anti-miRNA
: decreased enrichment of target mRNA in Ago2 IP
RNA Methodology
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Validation of miRNA Binding to Target mRNA: MS2 System
MS2 system
MS2-binding protein
MS2
MS2-binding protein
Target 3’UTR
MS2
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RNA Methodology
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HuR negatively regulates c-Myc expression
1
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2
RNA Methodology
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HuR binds to 3’UTR of c-Myc mRNA
1
2
3
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RNA Methodology
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c-Myc 3’UTR is required for HuR-mediated suppression
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RNA Methodology
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Several possibilities for HuR-mediated c-Myc suppression
1. Does HuR silencing affect c-Myc mRNA stability or protein stability?
2. Does HuR silencing affect the levels of let-7b or let-7c
3. Does let-7b or let-7c affect the enrichment of c-Myc mRNA in HUR IP?
1
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2
RNA Methodology
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Let-7 b/c controls c-Myc expression
Hyeon H Kim
let-7b
UGAGGUAGUAGGUUGUGUGGUU
let-7c
UGAGGUAGUAGGUUGUAUGGUU
RNA Methodology
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Several possibilities for HuR-mediated c-Myc suppression
1. Does HuR silencing affect c-Myc mRNA stability or protein stability?
2. Does HuR silencing affect the levels of let-7b or let-7c
3. Does let-7b or let-7c affect the enrichment of c-Myc mRNA in HUR IP?
2
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3
RNA Methodology
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HuR requires let-7 binding sites for c-Myc suppression
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RNA Methodology
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Another possible mechanism
Cooperative binding of HuR and let-7 into c-Myc 3’UTR ?
5’UTR
CR
3’UTR
AAAAAAAA
?
let7
RISC
Hyeon H Kim
RNA Methodology
HuR
63
Ago2 is associated with let-7-mediated c-Myc suppression
RISC IP
c-Myc mRNA
let-7
RISC
Ago2
Ago2 Antibody
Hyeon H Kim
RNA Methodology
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HuR is required for let-7 binding to c-Myc mRNA
1
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2
RNA Methodology
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HuR is required for let-7 binding to c-Myc mRNA
MS2 system
pMS2
pAB-MS2
AB
2
let-7b
let-7 levels in YFP IP
MS2-binding protein
MS2
MS2-binding protein
c-Myc 3’UTR-AB
MS2
Hyeon H Kim
MS2
RNA Methodology
MS2
let-7c
MS2
AB-MS2
1
0
Ctrl
siRNA
HuR
siRNA
Ctrl
siRNA
HuR
siRNA
66
Proposed Model for Cooperative Regulation of c-Myc
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RNA Methodology
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