g-quadruplexes in rnas and dhx36kanai/seminar/pdf/lit_m_furuta_m2.pdf · 2020-01-15 ·...
TRANSCRIPT
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G-quadruplexes
in RNAs
and DHX36
2019/11/21
M2 Furuta
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Contents
1. Introduction
History of G-quadruplex (G4)
2. RNA G4s and DHX36
2-1. RNA G4s in translational regulation
2-2. RNA G4s in ncRNAs
3. Summary
2
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Contents
1. Introduction
History of G-quadruplex (G4)
2. RNA G4s and DHX36
2-1. RNA G4s in translational regulation
2-2. RNA G4s in ncRNAs
3. Summary
3
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Guanine in DNA
Watson-Crick base pair
DNA double helix
Watson, J. D. & Crick, F. H. Nature. 1953, 171, 737.
1. History of G-quadruplex 4
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G-quartet
Davies, R. D. et. al., Proc. Natl. Acad. Sci. USA 1962, 48, 2013.
conc.
(solution)
gelatinize
G-quartet
X-ray crystal
structure analysis
✓ Self-association in water
51. History of G-quadruplex
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G-quadruplex (oligonucleotide)
✓ Stacking of two or more quartets
✓ Stabilized by monovalent metal ions
Pradeepkumar, P. I., et. al., ACS Chem. Biol. 2019, 14, 2114.
61. History of G-quadruplex
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Synthetic G-quadruplex
Hierarchical self-assembly process
1. Hydrogen-bonded G-quartet
2. G8-M+ octamer formed by cation-dipole interactions and π stacking
3. Hexadecameric G-quadruplex with anion-nucleobase H-bonds
Davis, J. T., et. al., J. Am. Chem. Soc. 2000, 122, 4060.
Davis, J. T. Angew. Chem. Int. Ed. 2004, 43, 668.
✓ Functional… ionophore
✓ Chiral… diastereotopic faces
(Tail & Head)
71. History of G-quadruplex
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DNA G-quadruplex in cell (1)
i - iii non-telomeric regions
iv - v telomeres (G-rich regions)
Balasubramanian, S., et. al., Nat. Chem. 2013, 5, 182.
BD4… antibody for G-quadruplex structures (generated by in vitro selection with phage display)
In nuclei of human cancer cells In chromosomes
a b c d
control Pre-inc. w/ G4
DNase 1 G4 transfection
81. History of G-quadruplex
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DNA G-quadruplex in cell (2)
✓ Small molecule ligand can stabilize
G-quadruplex structure even in cells.
Balasubramanian, S., et. al., Nat. Chem. 2013, 5, 182.
Control 10 uM PDS
91. History of G-quadruplex
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G-quadruplex in many fields
Nano-
technology
Medicinal
chemistry
Molecular
biology
Supramolecular
chemistry
101. History of G-quadruplex
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G-quadruplex in many fields
Nano-
technology
Medicinal
chemistry
Molecular
biology
Supramolecular
chemistry
Today’s topics
111. History of G-quadruplex
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Contents
1. Introduction
History of G-quadruplex (G4)
2. RNA G4s and DHX36
2-1. RNA G4s in translational regulation
2-2. RNA G4s in ncRNAs
3. Summary
12
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DNA-G4 and RNA-G4
Lipps, H. J., et. al., Nucleic Acids Res. 2015, 43, 8627.
RNA-G4 (rG4)
Existence in cells?
Function?
✓ DNA-G4
transcription regulation
replication origin firing
protection of telomeres
…
2. RNA G4s and DHX36 13
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Visualization of rG4s in cells (1)
Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.
a b c d
✓ On longer exposure, staining was also distributed throughout the cytoplasm.
No BG4 Untreated
RNase 1 DNase
2. RNA G4s and DHX36 14
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Visualization of rG4s in cells (2)
Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.
Untreated
CarboxyPDS
CarboxyPDS
2. RNA G4s and DHX36 15
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Visualization of rG4s in cells (3)
Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.
✓ Selective stabilization of endogenous RNA G4s with a RNA G4-specific ligand
Untreated PDS CarboxyPDS
CarboxyPDSPDS
2. RNA G4s and DHX36 16
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RNA G4 selectivity (v.s. DNA G4)
Balasubramanian, S., et. al., Angew. Chem. Int. Ed. 2012, 51, 11073.
Zhou, X., et. al., Chem 2018, 4, 1314.
✓ Hydrogen bond with 2’-OH group on the ribose
✓ Multiple hydrogen-bond receptor sites
✓ Accumulation in the cytoplasm of cells
Other reported RNA-G4-selective ligands
CarboxyPDS
2. RNA G4s and DHX36 17
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rG4 formation in eukaryotic cells
Bartel, D. P., et. al., Science 2016, 353, aaf5371.
✓ In eukaryotic cells, rG4 are globally unfolded though thousands of sites
in the transcriptome form stable rG4s in vitro.
A specialized machinery regulating rG4s formation in cells?
2. RNA G4s and DHX36 18
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RNA G4s in mRNA
Zhou, X., et. al., Chem 2018, 4, 1314.
✓ Complex factors are involved in mRNA-translation regulation.
2-1. RNA G4s in translational regulation
19
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RNA G4s in mRNA
Zhou, X., et. al., Chem 2018, 4, 1314.
✓ Complex factors are involved in mRNA-translation regulation.
Today’s topics
2-1. RNA G4s in translational regulation
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DHX36 (1)
Ferré-D’Amaré, A. R., et. al., Nature 2018, 558, 465.
Structural charasteristics
✓ DSM(DHX36-specific motif) and
OB subdomain specifically
recognizes G4s.
✓ DSM forms a hydrophobic core,
which surface stacks on the top of
the bound G4.
2-1. RNA G4s in translational regulation
21
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DHX36 (2)
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
Functional characteristics… RNA helicase which binds and unwinds rG4
(ATP-dependently)
rG4
in 5’ UTR or 3’ UTR (untranslated region)
Examples of mRNAs
Nkx2-5 (TF related to heart development)
YY1 (regulating gene expression)
PITX1 (tumor suppressor gene)
…
Translational
regulation
Translationally
active
2-1. RNA G4s in translational regulation
22
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DHX36 (3)
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
✓ DHX36-KO resulted in an increase
in rG4 formation in living cells.
2-1. RNA G4s in translational regulation
23
CarboxyPDS
(cPDS)
rG4-selective ligand
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Systems-wide analysis of DHX36 (1)
Tuschl, T., et. al., Cell 2010, 141, 129.
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
PAR-CLIP … mapping RNA-interactome of a protein
(Photoactivatable-ribonucleoside-enhanced
crosslinking and immuno-precipitation)
✓ Any preference for binding sites in exons
were observed compared to chance.
(E335A… catalytic dead)
(CDS: coding sequence)
2-1. RNA G4s in translational regulation
24
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Systems-wide analysis of DHX36 (2)
✓ DHX36 regulates gene expression in a post-transcriptional manner.
✓ DHX36’s loss results in the stabilization of target mRNAs in a helicase-
dependent manner.
RNA-seq
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
(NXPM : normalized crosslinked reads per million in PAR-CLIP)
WT vs DHX36-KO KO-Rescue w/ WT or catalytic-dead
2-1. RNA G4s in translational regulation
25
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Systems-wide analysis of DHX36 (3)
✓ DHX36 increases translational efficiency of its targets.
Ribo-seq (ribosome-footprinting) : measures a change in translational activity
Weissman, J. S., et. al., Science. 2009, 324, 218.
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
(RPF : ribosome-protected fragments)
proteasedigestion
Same as RNA-seq
Number of RBPs Translational efficiency
2-1. RNA G4s in translational regulation
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rG4-dependent SGs formation (1)
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
✓ DHX36 binding in 3’ UTR was as efficient in promoting translation as binding in 5’ UTR.
→ rG4 resulted in their sequestration into translationally inactive subcellular
compartments? (such as stress granules (SG) or P-bodies)
Hypothesis
2-1. RNA G4s in translational regulation
27
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rG4-dependent SGs formation (2)
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
Cross-reference in dataset of PAR-
CLIP & transcripts enriched in SGs
✓ DHX36 mRNA targets are enriched in SGs.
✓ DHX36-KO induced stress response.
Western-blotting analysis
(pEIF2AK2… marker of cellular stress response)
2-1. RNA G4s in translational regulation
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rG4-dependent SGs formation (3)
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
✓ Formation of SGs dependent on rG4 was suggested.
2-1. RNA G4s in translational regulation
29
(G3BP… stress granules)
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DHX36
DHX36
DHX36
Suppression of PITX1 by DHX36 (1)
McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.
✓ DHX36 is a negative regulator
of PITX1 protein expression.
DHX36
DHX36
DHX36 knockdown Rescue experiment
2-1. RNA G4s in translational regulation
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Suppression of PITX1 by DHX36 (2)
McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.
✓ There is a link between DHX36 and miRNA-mediated regulation of PITX1.
✓ However, further study is necessary to determine the precise interplay.
Dicer knockdown Ago2 knockdown
Dicer and Ago2... proteins related to miRNA-mediated RNA silencing(see appendix for details)
2-1. RNA G4s in translational regulation
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Contents
1. Introduction
History of G-quadruplex (G4)
2. RNA G4s and DHX36
2-1. RNA G4s in translational regulation
2-2. RNA G4s in ncRNAs
3. Summary
32
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Long non-coding RNA (lncRNA)
Chang, H. Y., et. al., Mol. Cell 2011, 43, 904.
Gene activation
Gene suppression
Chromatin modification
Act on chromatin structure
✓ Non-coding RNA(ncRNA)… RNAs which is not translated into proteins
Long non-coding RNA(lncRNA)…ncRNA with lengths exceeding 200 nucleotides
Mechanisms of functions
332-2. RNA G4s in ncRNAs
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Long non-coding RNA (lncRNA)
Chang, H. Y., et. al., Mol. Cell 2011, 43, 904.
Interaction of RNA and protein
… rG4 & DHX36
34
Gene activation
Gene suppression
Chromatin modification
Act on chromatin structure
✓ Non-coding RNA(ncRNA)… RNAs which is not translated into proteins
Long non-coding RNA(lncRNA)…ncRNA with lengths exceeding 200 nucleotides
Mechanisms of functions
2-2. RNA G4s in ncRNAs
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lncRNA GSEC (1)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC was identified as a lncRNA upregulated in colorectal cancer.
✓ lncRNA GSEC showed enrichment in the cytoplasmic fraction.
lncRNA upregulated in colon tumor tissues
GSEC (FLJ39051)… G-quadruplex-forming sequence containing lncRNA
352-2. RNA G4s in ncRNAs
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lncRNA GSEC (2)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC is required for the motility of colon cancer cells.
Transwell migration chamber assay … examine cell motility (細胞遊走性)
362-2. RNA G4s in ncRNAs
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Antagonistic effect of GSEC (1)
Akiyama T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC inhibits the function of DHX36.
rG4 resolution assay
DHX36
GSEC
(GSEC 11-26… rG4 forming region)
rG4
372-2. RNA G4s in ncRNAs
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Antagonistic effect of GSEC (2)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
Luciferase PITX1 3’ UTR
(rG4 forming region)
DHX36
GSEC
✓ It is possible that GSEC is also involved in
miRNA-mediated regulation of gene expression.
Luciferase activity
382-2. RNA G4s in ncRNAs
Luciferase reporter assay
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Antagonistic effect of GSEC (3)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ DHX36 decreases cell motility in a helicase activity-dependent manner.
✓ GSEC enhances cell motility by inhibiting the function of DHX36.
Overexpression of DHX36 Knockdown of DHX36 and GSEC
392-2. RNA G4s in ncRNAs
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Schematic model of GSEC functions
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC binds to DHX36 via rG4 and inhibits rG4-resolution activity.
402-2. RNA G4s in ncRNAs
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Contents
1. Introduction
History of G-quadruplex (G4)
2. RNA G4s and DHX36
2-1. RNA G4s in translational regulation
2-2. RNA G4s in ncRNAs
3. Summary
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Summary
✓RNA G-quadruplexes (rG4s) are thought to be regulated by
complex factors, which includes DHX36, RNA helicase.
✓DHX36 mainly gives a positive regulation to the translation of
mRNAs via its helicase activity.
✓Long non-coding RNA (lncRNA) GSEC acts as a decoy for
DHX36 binding to prevent the action of DHX36 on other targets.
423. Summary
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Appendix
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Diastereotopic faces of G-quartet 1. History of G-quadruplex 44
✓ The attached sugars result in the chiral G-quartet having diastereotopic faces,
a “head” and a “tail”.
“Head” face
=
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Global unfolding of rG4s
Bartel, D. P., et. al., Science 2016, 353, aaf5371.
DimethylSulfate
2. RNA G4s and DHX36 45
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Localization of DHX36
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
✓ DHX36 is a cytoplasmic helicase.
✓ Isoforms 1 and 2 shows no
differences in localization.
✓ DHX36 interacts with mRNA.
FlagHA-DHX36(iso1 or iso2) expressed HEK293 cells
✓ DHX36’s main targets are cytoplasmic mRNAs.
Purification of polyadenylated RNA after crosslinking
2-1. RNA G4s in translational regulation
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RNA-seq analysis of DHX36 binding
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.
✓ DHX36 binds G-rich target mRNAs in cells that form rG4s in vitro.
2-1. RNA G4s in translational regulation
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rG4s in the PITX1 mRNA 3’-UTR
McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.
✓ PITX1 3’-UTR contains three rG4s
capable of interacting with DHX36.
2-1. RNA G4s in translational regulation
DHX36
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DHX36 and miRNA machinery (1)
Steiz, J. A., et. al., Cell. 2007, 128, 1105.
✓ Ago2 acts also as a translational upregulator dependent on the context of
3’UTR and bound cofactors.
✓ This literature could explain in part the result that Ago2 knockdown abrogated
DHX36 siRNA-mediated PITX1 upregulation.
2-1. RNA G4s in translational regulation
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DHX36 and miRNA machinery (2)
Schratt, G., et. al., Genes Dev. 2013, 27, 991.
✓ DHX36 acts as a specific binding partner of the miR-134 microRNA in neuronal cells.
✓ It demonstrates a role in microRNA trafficking.
Hypothesis of miRNA-mediated translational regulation mechanism
1. The interaction between DHX36 and PITX1 mRNA is mediate by a microRNA.
2. In certain context, DHX36 may be involved in the sub-cellular localization of
PITX1 mRNA.
2-1. RNA G4s in translational regulation
Dendritic puncta
(樹状突起)
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Regulation mechanism by miRNA
http://leading.lifesciencedb.jp/1-e001
✓ Dicer… Rnase which produces
RNA duplex (21-24 bp)
✓ Ago protein… cleave RNA duplex
into single strand RNA
✓ Ago + ssRNA → RISC (RNA-
induced silencing complex)
2-1. RNA G4s in translational regulation
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Transwell migration chamber assay
https://www.funakoshi.co.jp/contents/64388
2-2. RNA G4s in ncRNAs 52
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GSEC binding to DHX36 via rG4 (1)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC binds to DHX36 even in living cells.
RIP(RNA immunoprecipitation) & qRT-PCR
2-2. RNA G4s in ncRNAs 53
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GSEC binding to DHX36 via rG4 (2)
Akiyama, T., et. al., Oncogene 2017, 36, 1191.
✓ GSEC directly interacts with DHX36
through a G-quadruplex structure.
2-2. RNA G4s in ncRNAs 54