Deposition of the nuclear DNA/RNA-binding protein Fused in sarcoma (FUS) in cytosolic inclusions is a common hallmark of some cases of frontotemporal lobar degeneration (FTLD-FUS) and amyotrophic lateral sclerosis (ALS-FUS). Whether both diseases also share common pathological mechanisms is currently unclear. Based on our previous finding that FUS deposits are hypomethylated in FTLD-FUS but not in ALS-FUS, we have now investigated whether genetic or pharmacological inactivation of Protein arginine methyltransferase 1 (PRMT1) activity results in unmethylated FUS or in alternatively methylated forms of FUS. To do so, we generated FUS-specific monoclonal antibodies that specifically recognize unmethylated arginine (UMA), monomethylated arginine (MMA) or asymmetrically dimethylated arginine (ADMA). Loss of PRMT1 indeed not only results in an increase of UMA FUS and a decrease of ADMA FUS, but also in a significant increase of MMA FUS. Compared to ADMA FUS, UMA and MMA FUS exhibit much higher binding affinities to Transportin-1, the nuclear import receptor of FUS, as measured by pull-down assays and isothermal titration calorimetry. Moreover, we show that MMA FUS occurs exclusively in FTLD-FUS, but not in ALS-FUS. Our findings therefore provide additional evidence that FTLD-FUS and ALS-FUS are caused by distinct disease mechanisms although both share FUS deposits as a common denominator.

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments,a nd data analysis that relies on well-separated resonances.W ep resent ac ovalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs.Linkage of the DOTA-based LBT to ac ysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site.This leads to increased chemical-shift dispersion and decreased signal overlap,thereby greatly facilitating chemical-shift assignment. This approachis applicable to IDPs of varying sizes and complexity,a nd is particularly helpful for repeat-containing IDPs and lowcomplexity regions.T his results in improved efficiency for IDP analysis and binding studies.The well-established structure-function paradigm has been challenged by the discovery of intrinsically disordered functional proteins (IDPs).[1] Although lacking stable secondary or tertiary structure elements,this large class of proteins plays ac rucial role in various cellular processes.[2] It is suggested that about 40 %ofall proteins have disordered regions of 40 or more residues,w ith many proteins existing solely in the unfolded state.[3]NMR spectroscopy is aw ell-suited method for studying the residual structure,dynamics,and interactions of IDPs with atomic resolution under near-native conditions.[4] Thek ey step for these studies is the assignment of NMR resonances, which is particularly challenging for IDPs owing to poor chemical-shift dispersion, which results in severe spectral overlap.T his is due to alack of well-defined hydrogen bonds and am issing hydrophobic core with aromatic contributions.[5] To reduce spectral overlap,s everal approaches have been proposed. In the "divide and conquer" approach, chemical-shift assignments of shorter constructs obtained by standard NMR experiments are transferred to the full-length protein.[6] To assign chemical shifts of full-length unstructured proteins,e xperiments have been proposed that make use of the favorable relaxation properties of IDPs in high-dimensional experiments (up to 7D). [7] These experiments reduce resonance overlap and can potentially provide unambiguous chemical-shift assignments.A na lternative strategy is the direct detection of heteronuclei, using the beneficial chemical-shift dispersion of 13 C [8] or the increased resolution of direct detection of 15 N. [9] These approaches can be implemented easily with nonuniform sampling combined with nonFourier signal processing methods and fast-pulsing techniques in order to reduce measurement time. [10] Nevertheless,even providing that complete chemical-shift assignments can be obtained, missing chemical-shift dispersion in 2D NMR experiments complicates standard NMR approaches,including chemical-shift perturbation mapping of protein-ligand interactions and studies of protein ...