ATP regulates RNA-driven cold inducible RNA binding protein phase separation

Abstract

Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate-forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate-forming protein. Here, using the arginine-glycine/arginine-glycine-glycine (RG/RGG)-rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD⁺ , NADH, NADP⁺ , and NADPH directly interact with CIRBP, involving both the folded RNA-recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA-driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid-liquid phase separation studies of RG/RGG and other condensate-forming proteins in order to better mimic the cellular environment in the future.

Keywords: ATP; CIRBP; RG/RGG region; RNA-binding protein; disordered protein; liquid-liquid phase separation.

FIGURE 1

HeLa Cell metabolites contain binding partners of CIRBP. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or with addition of 1 or 4 units of HeLa metabolites extract (pink and magenta, respectively). Overlay of 1D ¹H‐CPMG spectra of HeLa metabolites extract in absence (cyan) or presence of increasing concentrations of CIRBP ranging from 8.5 to 25.5 μM (orange, magenta and green) and zoomed in a region corresponding to ADP and NAD+ in (b) and lactate, alanine and glutamate in (c)

FIGURE 2

CIRBP directly interacts with (di)nucleotides. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or with addition of 10 mM of either AMP, ADP, or ATP (pink, orange, and cyan, respectively). Chemical shift perturbations (CSPs) of the ¹H‐¹⁵N HSQC CIRBP cross‐peaks between the CIRBP free and either AMP, ADP or ATP bound forms are shown in a bar‐plot in (b). Unassigned residues are indicated in grey. The CIRBP region between amino‐acids 91–172 is not shown as most of the corresponding ¹H‐¹⁵N HSQC CIRBP cross‐peaks are unassigned. (c) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or with addition of 10 mM of either NAD⁺ or NADH (green and violet, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP cross‐peaks between free CIRBP and either NAD⁺‐ or NADH‐bound forms are shown in bar‐plots in (d). Unassigned residues are indicated in grey. The CIRBP region between amino‐acids 91–172 is not shown as most of the corresponding ¹H‐¹⁵N HSQC CIRBP cross‐peaks are unassigned. (e) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or upon addition of 10 mM of either NADP⁺ or NADPH (blue and magenta, respectively). CSPs of the ¹H‐¹⁵N HSQC CIRBP cross‐peaks between free and either NADP⁺ or NADPH bound forms are shown in a bar‐plot in (f). Unassigned residues are indicated in grey. The CIRBP region between amino‐acids 91–172 is not shown as most of the corresponding ¹H‐¹⁵N HSQC CIRBP cross‐peaks are unassigned. CIRBP, cold inducible RNA binding protein

FIGURE 3

ATP binding to CIRBP overlaps with the putative RNA‐binding interface. (a) ¹H, ¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or with increasing ATP concentration, ranging from 1 to 20 mM (blue gradient). (b) CSP plot of CIRBP ¹H‐¹⁵N cross‐peak corresponding to valine 10 as function of ATP concentration. Using GraphPad Prism 8.4 we estimated the corresponding affinity with an associated K_D of 2.9 ± 1.4 mM using a nonlinear one binding site fitting function. (c) Mapping of the CSPs of CIRBP¹H‐¹⁵N cross‐peaks (residue 1–90) upon binding to 10 mM ATP on the published structure of the RRM of CIRBP (PDB code: 5TBX). The electrostatic potential surface representation of the CIRBP^RRM is shown. (d) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP at 17 μM without (black) or with addition of 3.3 mM of aspartate or 10 mM of either alanine, glutamine, lactate, glucose or glutamate (blue, magenta, yellow, light grey, grey, and orange, respectively). CIRBP, cold inducible RNA binding protein

FIGURE 4

The RG/RGG region of CIRBP are involved in nucleotide binding. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RSY at 30 μM without (black) or with addition of 10 mM of ATP or NAD⁺ (grey, left and right panel, respectively). (b) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RGG at 30 μM without (black) or with addition of 10 mM of either AMP, ADP, or ATP (magenta, orange, and blue, respectively). CSPs of the ¹H‐¹⁵N HSQC CIRBP^RGG cross‐peaks between the CIRBP free and either AMP, ADP or ATP bound forms are shown in a bar‐plot in (c). Unassigned residues are indicated in grey. (d) ¹⁵N{¹H} heteronuclear NOE values are plotted versus CIRBP^RGG residue numbers in presence (cyan) or absence (black) of ATP. Error bars for heteronuclear NOE values were derived from error propagation calculation using standard deviation of 10 arbitrarily chosen noise peaks in saturated and unsaturated spectra. CIRBP, cold inducible RNA binding protein

FIGURE 5

The RG/RGG region of CIRBP is involved in dinucleotide binding. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RGG at 30 μM without (black) or upon addition of 10 mM of either NAD⁺ or NADH (green and violet, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP^RGG cross‐peaks between free and either NAD⁺ or NADH bound forms are shown in a bar‐plot in (b). The unassigned residues are indicated in grey. (c) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RGG at 30 μM without (black) or upon addition of 10 mM of either NADP⁺ or NADPH (blue and magenta, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP^RGG cross‐peaks between free and either NADP⁺ or NADPH bound forms are shown in a bar‐plot in (d). unassigned residues are indicated in grey

FIGURE 6

The RRM of CIRBP is involved in (di)nucleotide binding. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RRM at 30 μM without (black) or upon addition of 10 mM of either AMP, ADP or ATP (magenta, orange, and blue, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP^RRM cross‐peaks between free and either AMP, ADP or ATP bound forms are shown in a bar‐plot in (b). Unassigned residues are indicated in grey. (c) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RRM at 30 μM without (black) or upon addition of 10 mM of either NAD⁺ or NADH (green and violet, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP^RRM cross‐peaks between free and either NAD⁺ or NADH bound forms are shown in a bar‐plot in (d). Unassigned residues are indicated in grey. (e) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RRM at 30 μM without (black) or upon addition of 10 mM of either NADP⁺ or NADPH (blue and magenta, respectively). CSPs of ¹H‐¹⁵N HSQC CIRBP^RRM cross‐peaks between free and either NADP⁺ or NADPH bound forms are shown in a bar‐plot in (f). Unassigned residues are indicated in grey. CIRBP, cold inducible RNA binding protein

FIGURE 7

ATP binding to the CIRBP RG/RGG region attenuates RNA binding and is regulated by arginine methylation. (a) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled CIRBP^RGG at 30 μM in presence or absence of 10 mM ATP (right and left panel, respectively) and without (black) or with addition of 15 μM (UG)₁₂ RNA (orange, violet). The intensity ratio of the ¹H‐¹⁵N HSQC CIRBP^RGG cross‐peaks between the ATP (orange) or ATP/RNA (violet) free and bound forms are shown in a bar‐plot in (b). The unassigned residues are indicated in grey. (c) ¹H‐¹⁵N HSQC spectrum of ¹⁵N‐labeled _metCIRBP^RGG at 30 μM in presence (orange) or absence of 10 mM ATP (black). CSPs of the ¹H‐¹⁵N HSQC methylated _metCIRBP^RGG (orange) or unmethylated CIRBP^RGG (blue) cross‐peaks between the ATP free and bound forms are shown in a bar‐plot in (d). The unassigned residues are indicated in grey. CIRBP, cold inducible RNA binding protein

FIGURE 8

ATP modulates LLPS of CIRBP. Turbidity assay to quantify phase separation of full‐length CIRBP with fixed CIRBP concentration (17 μM) and increasing RNA concentration in (a), with fixed CIRBP and RNA concentration (17 and 7.5 μM, respectively) and increasing ATP concentration in (b). Values represent means ±SEM (n = 3). (c) DIC microscopy images of CIRBP at 17 μM in presence of 7.5 μM RNA and/or 5 mM ATP. Scale bar, 10 μm. (d) Model of ATP/RNA‐mediated regulation of CIRBP LLPS. CIRBP, cold inducible RNA binding protein; DIC, differential interference contrast; LLPS, liquid–liquid phase separation