A combined NMR and EPR investigation on the effect of the disordered RGG regions in the structure and the activity of the RRM domain of FUS

Abstract

Structural disorder represents a key feature in the mechanism of action of RNA-binding proteins (RBPs). Recent insights revealed that intrinsically disordered regions (IDRs) linking globular domains modulate their capability to interact with various sequences of RNA, but also regulate aggregation processes, stress-granules formation, and binding to other proteins. The FET protein family, which includes FUS (Fused in Sarcoma), EWG (Ewing Sarcoma) and TAF15 (TATA binding association factor 15) proteins, is a group of RBPs containing three different long IDRs characterized by the presence of RGG motifs. In this study, we present the characterization of a fragment of FUS comprising two RGG regions flanking the RNA Recognition Motif (RRM) alone and in the presence of a stem-loop RNA. From a combination of EPR and NMR spectroscopies, we established that the two RGG regions transiently interact with the RRM itself. These interactions may play a role in the recognition of stem-loop RNA, without a disorder-to-order transition but retaining high dynamics.

Figure 1

(A) Organization of full-length FUS and FUS^165–422; (B) primary sequence of FUS^165–422. The residues selected for replacement with cysteine and MTSL-labelling are marked with asterisk.

Figure 2

(A) Far-UV CD spectrum (200–250 nm) of FUS^165–422; (B) 2D ¹H-¹⁵N HSQC spectra of FUS^165–422. The NMR spectrum was acquired at 298 K with a 22.3 T Bruker Avance III spectrometer equipped with a TCI probehead.

Figure 3

(A) Labelling positions selected for G-rich, RGG_1-2 and RRM domains of FUS^165–422. (B,C) Experimental (black line) and simulated (red line) X-band (9.8 GHz) CW-EPR spectra of D180R1, D258R1, S412R1, A349R1 and S360R1 spin labelled mutants of FUS^165–422.

Figure 4

Left panels—¹H-¹⁵ N HSQC NMR spectra of MTSL-labelled (red) and unlabelled (black) FUS^165–422 mutants (A) D180R1, (B) D258R1, (C) A349R1, (D) S360R1 and (E) S412R1 with the assignment of the relevant cross peaks; Central panels—mapping on the protein surface of the RRM domain (PDB ID 2LA6) of the relevant changes in signal intensity. Residues are coloured from red to orange according to PRE magnitude (F–J); unaffected residues are coloured in grey. The labelled position in the RRM is coloured in yellow. Right panels: Paramagnetic relaxation enhancement values reported as normalized intensity ratio (I/I°) for FUS^165–422 mutants (F) D180R1, (G) D258R1, (H) A349R1, (I) S360R1 and (J) S412R1.

Figure 5

(A) Far-UV CD spectra (200–250 nm) of FUS^165–422 in the presence of hnRNPA2/B1 stem-loop RNA; (B) Far-UV CD spectra (200–250 nm) of hnRNPA2/B1 stem-loop RNA; (C) 2D ¹H-¹⁵N HSQC spectrum of FUS^165–422 in the presence of hnRNPA2/B1 stem-loop RNA (red) superimposed with the spectrum of the free protein in solution (black) with the assignment of selected cross peaks.

Figure 6

X-band (9.8 GHz) CW-EPR spectra without (black line) and in the presence of 1:1 hnRNPA2/B1 stem-loop RNA (red line) recorded spin labelled FUS^165–422 mutants (A) D258R1 and (B) A349R1 and S360R1.

Figure 7

(A) Background corrected Q-band (9.8 GHz) DEER traces (black line) and corresponding fitted curve (red line) for D258R1/A349R1 and A349R1/S412R1 mutants of FUS^165–422 in without and in the presence of hnRNPA2/B1 stem-loop RNA; (B) corresponding distance distribution without (black line) and in the presence of stem-loop RNA (red line) recorded for D258R1/A349R1 (top panel) and A349R1/S412R1 (bottom panel) mutants of FUS^165–422.