Control of the polyamine biosynthesis pathway by G2-quadruplexes
- ETH Zurich, Switzerland
Figures
Figure 1
Polyamines (PAs) and G-quadruplexes.
(a) PA biosynthesis pathway and summary of the roles/regulation of putative quadruplex sequences (PQS’s) on PA biosynthesis. Red: G2-rich motifs inhibit PA biosynthesis; Green: G2-rich motifs …
Figure 2 with 1 supplement
G2-PQS’s regulate luciferase reporter genes.
(a) Effect of PQS’s from PSP UTRs on Renilla luciferase activity in HeLa cells; PQS's ARG25Q1, AZIN15Q1, OAZ25Q1, OAZ25Q2, ODC15Q2, ODC15Q3, SMS5Q1, SAT15Q1, ARG23Q1, OAZ13Q1, OAZ33Q1 and SMS3Q1 …
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Figure 2—source data 1
Conservation of G2-PQS’s in PSP UTRs.
Assessment of PQS conservation using H-QGRS (Menendez et al., 2012), with the exception of SMS3Q1 and ARG23Q1 whose stability score is below that set by H-QGRS. SMS3Q1 and ARG23Q1 conservation was assessed through manual gene alignment. Conservation is dictated by factors such as composition, location and predicted stability. Human-mouse conservation was not observed for the reference NRAS, nor for ARG25Q1, ARG23Q1, OAZ25Q1, OAZ25Q2, SMS3Q1, OAZ13Q1 and OAZ33Q1, for which Human-primate conservation was assessed. For many PQS's, primate sequences are either not available or the UTRs are not defined (OAZ13Q1). Human-primate conservation was not observed for ARG25Q1, OAZ25Q1 and OAZ25Q2. Human-primate conservation has been reported for NRAS (Kumari et al., 2007).
- https://doi.org/10.7554/eLife.36362.005
Figure 2—figure supplement 1
PAGE migration of long-looped G2-PQS’s in PSP UTRs upon in vitro transcription.
The effect of PQS destabilizing mutations (M) in AZIN15Q1, ARG23Q1, SMS3Q1, SAT15Q1 and SMS5Q1 on in vitro transcription of Renilla luciferase mRNA on replicate gels. Smeared RNA can be observed for …
Figure 3 with 4 supplements
Biophysical properties of G2-PQSs from PSPs: Gel migration, Thioflavin T fluorescence, UV (295 nm), CD.
All biophysical studies were performed in 100 mM K+. PAGE migration and Thioflavin T staining were performed at different migration times on distinct gels. Melting temps (TM 295) are shown in the …
Figure 3—figure supplement 1
UV melt-anneal profiles for mutant G2-PQS's.
UV melt-anneal profiles for NRASM, ARG25Q1M, ODC5Q2M, OAZ25Q1M, OAZ25Q2M, SMS5Q1M and ARG23Q1M at 295 nm (performed in the presence of 100 mM K+, UV performed in 1 mM K+ for NRAS). Two replicates of …
Figure 3—figure supplement 2
Additional biophysical data.
(a) Biophysical properties of the G2-PQS from SMS3Q1 and SAT5Q1 and mutants where relevant. Gel migration, Thioflavin T fluorescence, UV (295 nm), CD. All biophysical studies were performed in 100 …
Figure 3—figure supplement 3
Full size migration gels from cropped images.
Full-length gels (shown as cropped images in Figure 3 and Figure 3—figure supplement 2).
Figure 3—figure supplement 4
Full size thioflavin gels from cropped images.
Full-length gels (shown as cropped images in Figure 3 and Figure 3—figure supplement 2).
Figure 4 with 7 supplements
G2-PQS in PSPs form quadruplexes in Hela cells and self-regulate.
(a) Effects of pyridostatin (PDS) on spermine and spermidine (n=3, *p≤0.05, **p≤0.01). (b) Effects of PDS on endogenous SMS protein at 6 and 24 h (n=3, *p≤0.05, **p≤0.01) (full length blots are in Fi…
Figure 4—figure supplement 1
Cell viabilities after PDS treatments of HeLa cells.
Effects of the quadruplex stabilizing ligand Pyridostatin (PDS) on HeLa cell viability. The error represents the standard error (SE). (n = 3,**p≤0.01).
Figure 4—figure supplement 2
Effects of PDS treatment upon spermine and spermidine levels in HeLa cells.
Example HPLC traces used for polyamine monitoring by HPLC at 229 and 198 nm, upon depletion of cellular polyamine levels following treatment with PDS for 24 hr (4, 16, 64 μM).
Figure 4—figure supplement 3
Effects of PDS treatment on PQS reporter genes in HeLa cells: un-normalized data.
Effects of the quadruplex stabilizing ligand PDS on PQS-reporters in HeLa cells. Un-normalized Renilla data for the three reporters of Figure 4b and c after PDS treatment of cells.
Figure 4—figure supplement 4
Effects of PDS treatment on endogenous polyamine synthesis proteins in HeLa cells.
Full-length blots.
Figure 4—figure supplement 5
Polyamine addition and depletion reporter assays.
The error bars represent the standard error (SE) of at least two biological replicates. Normalized data.
Figure 4—figure supplement 6
Polyamine addition and depletion reporter assays.
The error bars represent the standard error (SE) of at least two biological replicates. Un-normalized data.
Figure 4—figure supplement 7
Polyamine depletion and spermine synthase inhibition.
Example HPLC traces used for polyamine monitoring by HPLC at 198, 229 and 254 nm, upon depletion of cellular polyamine levels following treatment with DFMO and APCHA. Confirmation of APCHA …
Figure 5 with 3 supplements
Structure of AZIN1wt.
(a) Derived model for the equilibrium between two possible hairpin conformers and the G2-quadruplex of AZIN1wt; K+ is expected to favor the G2-quadruplex; Na+ or Mg2+ are expected to favor the …
Figure 5—figure supplement 1
1H NMR spectra of AZIN1wt, NRAS, ODC15Q2, ARG25Q1, OAZ25Q1 and OAZ25Q2.
(a) Overlay of 1D 1H NMR spectra corresponding to the imino region of AZINwt in 100 mM KCl (blue) and 100 mM NaCl (red). (b) Overlay of 1D 1H NMR spectra corresponding to the imino region of AZINwt …
Figure 5—figure supplement 2
1H 15N-HSQC and 1H 1H 2D NOESY of AZIN1wt.
(a) 1H-15N-HSQC spectrum corresponding to the imino region of AZIN1wt (in the presence of 10% 15N-labelled RNA) in 200 mM KCl. (b) 1H-15N HSQC spectrum corresponding to the imino region of AZIN1pos …
Figure 5—figure supplement 3
Polyamine-related biophysical analysis of AZIN15Q1.
AZIN1 G2 quadruplex is induced by polyamines in vitro (a) Effect of spermine on UV melt profiles of (i) AZIN15Q1 (295 nm), (ii) control AZIN15Q1M3, (iii) control AZIN15Q1M2, (iv) control AZIN15Q1M …
Additional files
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Supplementary file 1
- https://doi.org/10.7554/eLife.36362.023
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Supplementary file 2
- https://doi.org/10.7554/eLife.36362.024
