G-quadruplexesin RNAs
and DHX36
2019/11/21
M2 Furuta

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

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. Summary3

Guanine in DNA

1. History of G-quadruplex

4

Watson-Crick base pairDNA double helix

Watson, J. D. & Crick, F. H. Nature. 1953, 171, 737.

G-quartet1. History of G-quadruplex

5conc.
gelatinize(solution)

X-ray crystal
structure analysis

G-quartet✓ Self-association in water

Davies, R. D. et. al., Proc. Natl. Acad. Sci. USA 1962, 48, 2013.

G-quadruplex (oligonucleotide)

1. History of G-quadruplex

6✓ Stacking of two or more quartets✓ Stabilized by monovalent metal ions

Pradeepkumar, P. I., et. al., ACS Chem. Biol. 2019, 14, 2114.

Synthetic G-quadruplex

1. History of G-quadruplex

7✓ Functional… ionophore✓ Chiral… diastereotopic faces(Tail & Head)

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.

DNA G-quadruplex in cell (1)In nuclei of human cancer cells

controlPre-inc. w/ G4

DNase 1G4 transfection

1. History of G-quadruplex

8In chromosomesi - iii non-telomeric regions
iv - v telomeres (G-rich regions)

BD4… antibody for G-quadruplex structures(generated by in vitro selection with phage display)abcd

Balasubramanian, S., et. al., Nat. Chem. 2013, 5, 182.

DNA G-quadruplex in cell (2)Control

1. History of G-quadruplex

910 uM PDS✓ Small molecule ligand can stabilize
G-quadruplex structure even in cells.

Balasubramanian, S., et. al., Nat. Chem. 2013, 5, 182.

G-quadruplex in many fields

1. History of G-quadruplex

Medicinal
chemistrySupramolecularchemistry
Nanotechnology

Molecular
biology10G-quadruplex in many fields

1. History of G-quadruplex

Medicinal
chemistrySupramolecularchemistry
Nanotechnology

Today’s topics

Molecularbiology

11

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. Summary12

DNA-G4 and RNA-G4

2. RNA G4s and DHX36

13✓ DNA-G4transcription regulation
replication origin firing
protection of telomeres…

RNA-G4 (rG4)
Existence in cells?
Function?

Lipps, H. J., et. al., Nucleic Acids Res. 2015, 43, 8627.

Visualization of rG4s in cells (1)No BG42. RNA G4s and DHX36

Untreated

aRNase 1

14bcdDNase✓ On longer exposure, staining was also distributed throughout the cytoplasm.

Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.

Visualization of rG4s in cells (2)2. RNA G4s and DHX36

15

Untreated

CarboxyPDS

CarboxyPDS

Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.

Visualization of rG4s in cells (3)Untreated

2. RNA G4s and DHX36

PDS

CarboxyPDS

PDS

CarboxyPDS16✓ Selective stabilization of endogenous RNA G4s with a RNA G4-specific ligand

Balasubramanian, S., et. al., Nat. Chem. 2014, 6, 75.

RNA G4 selectivity (v.s. DNA G4)

2. RNA G4s and DHX36

17

Other reported RNA-G4-selective ligands

CarboxyPDS✓ Hydrogen bond with 2’-OH group on the ribose✓ Multiple hydrogen-bond receptor sites✓ Accumulation in the cytoplasm of cells

Balasubramanian, S., et. al., Angew. Chem. Int. Ed. 2012, 51, 11073.
Zhou, X., et. al., Chem 2018, 4, 1314.

rG4 formation in eukaryotic cells

2. RNA G4s and DHX36

18✓ 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?

Bartel, D. P., et. al., Science 2016, 353, aaf5371.

RNA G4s in mRNA

2-1. RNA G4s intranslational regulation

19✓ Complex factors are involved in mRNA-translation regulation.

Zhou, X., et. al., Chem 2018, 4, 1314.

RNA G4s in mRNA

2-1. RNA G4s intranslational regulation

20

Today’s topics✓ Complex factors are involved in mRNA-translation regulation.

Zhou, X., et. al., Chem 2018, 4, 1314.

DHX36 (1)2-1. RNA G4s intranslational regulation

21

Structural charasteristics✓ DSM(DHX36-specific motif) andOB subdomain specificallyrecognizes G4s.
✓ DSM forms a hydrophobic core,
which surface stacks on the top of
the bound G4.

Ferré-D’Amaré, A. R., et. al., Nature 2018, 558, 465.

2-1. RNA G4s intranslational regulation

DHX36 (2)22Functional characteristics… RNA helicase which binds and unwinds rG4(ATP-dependently)
rG4
in 5’ UTR or 3’ UTR (untranslated region)

Translational
regulation

Examples of mRNAs
Nkx2-5 (TF related to heart development)YY1 (regulating gene expression)
PITX1 (tumor suppressor gene)…

TranslationallyactivePaeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

DHX36 (3)2-1. RNA G4s intranslational regulation

23

CarboxyPDS(cPDS)
rG4-selective ligand✓ DHX36-KO resulted in an increasein rG4 formation in living cells.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

Systems-wide analysis of DHX36 (1)2-1. RNA G4s intranslational regulation

24

PAR-CLIP … mapping RNA-interactome of a protein(Photoactivatable-ribonucleoside-enhanced
crosslinking and immuno-precipitation)(CDS: coding sequence)(E335A… catalytic dead)✓ Any preference for binding sites in exons
were observed compared to chance.

Tuschl, T., et. al., Cell 2010, 141, 129.
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

Systems-wide analysis of DHX36 (2)2-1. RNA G4s intranslational regulation25

RNA-seq
WT vs DHX36-KO

KO-Rescue w/ WT or catalytic-dead(NXPM : normalized crosslinked reads per million in PAR-CLIP)✓ DHX36 regulates gene expression in a post-transcriptional manner.
✓ DHX36’s loss results in the stabilization of target mRNAs in a helicasedependent manner.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

Systems-wide analysis of DHX36 (3)2-1. RNA G4s intranslational regulation

26

Ribo-seq (ribosome-footprinting) : measures a change in translational activity
Number of RBPs

Translational efficiency(RPF : ribosome-protected fragments)

proteasedigestion✓ DHX36 increases translational efficiency of its targets.

Same as RNA-seq

Weissman, J. S., et. al., Science. 2009, 324, 218.
Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

rG4-dependent SGs formation (1)2-1. RNA G4s intranslational regulation

27✓ DHX36 binding in 3’ UTR was as efficient in promoting translation as binding in 5’ UTR.
→ rG4 resulted in their sequestration into translationally inactive subcellularcompartments? (such as stress granules (SG) or P-bodies)

Hypothesis

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

rG4-dependent SGs formation (2)Cross-reference in dataset of PARCLIP & transcripts enriched in SGs

2-1. RNA G4s intranslational regulation

28

Western-blotting analysis(pEIF2AK2… marker of cellular stress response)✓ DHX36 mRNA targets are enriched in SGs.
✓ DHX36-KO induced stress response.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

rG4-dependent SGs formation (3)2-1. RNA G4s intranslational regulation

29(G3BP… stress granules)✓ Formation of SGs dependent on rG4 was suggested.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

Suppression of PITX1 by DHX36 (1)DHX36 knockdown

DHX36

2-1. RNA G4s intranslational regulation

30

Rescue experiment

DHX36

DHX36

DHX36

DHX36

✓ DHX36 is a negative regulatorof PITX1 protein expression.

McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.

Suppression of PITX1 by DHX36 (2)Dicer knockdown

2-1. RNA G4s intranslational regulation31

Ago2 knockdown

Dicer and Ago2... proteins related to miRNA-mediated RNA silencing(see appendix for details)✓ There is a link between DHX36 and miRNA-mediated regulation of PITX1.

✓ However, further study is necessary to determine the precise interplay.

McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.

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. Summary32

Long non-coding RNA (lncRNA)

2-2. RNA G4s in ncRNAs

33✓ Non-coding RNA(ncRNA)… RNAs which is not translated into proteinsLong non-coding RNA(lncRNA)…ncRNA with lengths exceeding 200 nucleotides
Mechanisms of functions
Gene activation
Chromatin modification

Act on chromatin structure

Gene suppression

Chang, H. Y., et. al., Mol. Cell 2011, 43, 904.

Long non-coding RNA (lncRNA)

2-2. RNA G4s in ncRNAs

34✓ Non-coding RNA(ncRNA)… RNAs which is not translated into proteinsLong non-coding RNA(lncRNA)…ncRNA with lengths exceeding 200 nucleotides
Mechanisms of functions
Gene activation
Chromatin modification

Act on chromatin structure

Gene suppression

Interaction of RNA and protein… rG4 & DHX36

Chang, H. Y., et. al., Mol. Cell 2011, 43, 904.

lncRNA GSEC (1)2-2. RNA G4s in ncRNAs35

GSEC (FLJ39051)… G-quadruplex-forming sequence containing lncRNA

lncRNA upregulated in colon tumor tissues✓ GSEC was identified as a lncRNA upregulated in colorectal cancer.
✓ lncRNA GSEC showed enrichment in the cytoplasmic fraction.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

lncRNA GSEC (2)2-2. RNA G4s in ncRNAs

36

Transwell migration chamber assay … examine cell motility (細胞遊走性)✓ GSEC is required for the motility of colon cancer cells.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

Antagonistic effect of GSEC (1)2-2. RNA G4s in ncRNAs37

rG4 resolution assay
DHX36
GSEC

rG4(GSEC 11-26… rG4 forming region)✓ GSEC inhibits the function of DHX36.

Akiyama T., et. al., Oncogene 2017, 36, 1191.

Antagonistic effect of GSEC (2)2-2. RNA G4s in ncRNAs38

Luciferase reporter assay
DHX36
GSEC
Luciferase

PITX1 3’ UTR(rG4 forming region)

Luciferase activity✓ It is possible that GSEC is also involved inmiRNA-mediated regulation of gene expression.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

Antagonistic effect of GSEC (3)Overexpression of DHX36

2-2. RNA G4s in ncRNAs39

Knockdown of DHX36 and GSEC

✓ DHX36 decreases cell motility in a helicase activity-dependent manner.
✓ GSEC enhances cell motility by inhibiting the function of DHX36.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

Schematic model of GSEC functions

2-2. RNA G4s in ncRNAs

40✓ GSEC binds to DHX36 via rG4 and inhibits rG4-resolution activity.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

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

41

Summary

3. Summary

42✓ RNA G-quadruplexes (rG4s) are thought to be regulated bycomplex factors, which includes DHX36, RNA helicase.

✓ DHX36 mainly gives a positive regulation to the translation ofmRNAs 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.

Appendix

Diastereotopic faces of G-quartet

1. History of G-quadruplex=“Head” face

✓ The attached sugars result in the chiral G-quartet having diastereotopic faces,a “head” and a “tail”.

44Global unfolding of rG4s

2. RNA G4s and DHX36

45

DimethylSulfateBartel, D. P., et. al., Science 2016, 353, aaf5371.

2-1. RNA G4s intranslational regulation

Localization of DHX36

46

FlagHA-DHX36(iso1 or iso2) expressed HEK293 cells✓ DHX36 is a cytoplasmic helicase.
✓ Isoforms 1 and 2 shows nodifferences in localization.

Purification of polyadenylated RNA after crosslinking✓ DHX36 interacts with mRNA.

✓ DHX36’s main targets are cytoplasmic mRNAs.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

RNA-seq analysis of DHX36 binding

2-1. RNA G4s intranslational regulation

47✓ DHX36 binds G-rich target mRNAs in cells that form rG4s in vitro.

Paeschke, K., et. al., Nat. Commun. 2019, 10, 2421.

rG4s in the PITX1 mRNA 3’-UTR

DHX36

2-1. RNA G4s intranslational regulation

48✓ PITX1 3’-UTR contains three rG4s
capable of interacting with DHX36.

McKenna, S. A., et. al., Nucleic Acids Res. 2014, 42, 3346.

DHX36 and miRNA machinery (1)2-1. RNA G4s intranslational regulation49✓ Ago2 acts also as a translational upregulator dependent on the context of3’UTR and bound cofactors.

Steiz, J. A., et. al., Cell. 2007, 128, 1105.

✓ This literature could explain in part the result that Ago2 knockdown abrogated
DHX36 siRNA-mediated PITX1 upregulation.

DHX36 and miRNA machinery (2)2-1. RNA G4s intranslational regulation50

Dendritic puncta(樹状突起)✓ DHX36 acts as a specific binding partner of the miR-134 microRNA in neuronal cells.
✓ It demonstrates a role in microRNA trafficking.

Schratt, G., et. al., Genes Dev. 2013, 27, 991.

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 ofPITX1 mRNA.

Regulation mechanism by miRNA

2-1. RNA G4s intranslational regulation

51✓ Dicer… Rnase which produces
RNA duplex (21-24 bp)✓ Ago protein… cleave RNA duplex
into single strand RNA✓ Ago + ssRNA → RISC (RNAinduced silencing complex)

http://leading.lifesciencedb.jp/1-e001

Transwell migration chamber assay

2-2. RNA G4s in ncRNAs

52

https://www.funakoshi.co.jp/contents/64388

GSEC binding to DHX36 via rG4 (1)2-2. RNA G4s in ncRNAs53

RIP(RNA immunoprecipitation) & qRT-PCR✓ GSEC binds to DHX36 even in living cells.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.

GSEC binding to DHX36 via rG4 (2)2-2. RNA G4s in ncRNAs

54✓ GSEC directly interacts with DHX36
through a G-quadruplex structure.

Akiyama, T., et. al., Oncogene 2017, 36, 1191.