Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43

Abstract

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

“…These results suggest that RRM1 and RRM2 interact in the absence of RNA, with interdomain interactions stabilizing the native state of each RRM domain by 0.9 kcal mol Ϫ1 (Table 1). Indeed, a recent NMR study on the tethered RRM domain revealed that in the absence of RNA, the two RRMs do not tumble independently of one another (43), further supporting mutual stabilizing interactions between the domains. The Trp FL also suggests that RRM1 is completely unfolded at the first intermediate, I1, of the CD equilibrium unfolding profile.…”

“…Note that the solution structure of RRMc is unknown and predicted to be much different from that mapped on RRM2 (Protein Data Bank entry 1wf0) due to removal of secondary structural elements in the protein core. E, NMR structure of the tethered RRMs with RNA (Protein Data Bank entry 4bs2) with RNA shown in gray (43). The secondary structure elements are colored as shown in C and D. The RNA-binding residues and intrinsic fluorophores are shown as sticks.…”

“…In these scenarios, the formation and accumulation of TDP-43 aggregates generate toxicity or impair normal TDP-43 cellular function, resulting in cell death. Mounting evidence supports a loss-of-function phenotype (11), where sequestration of functional protein into cytoplasmic aggregates would limit the pool of available functional nuclear TDP- 43.…”

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

“…1A). The NLS and NES regulate the shuttling of TDP-43 between the nucleus and the cytoplasm (5), whereas the RRM1 and RRM2 are responsible for binding to nucleic acids including single-or double-stranded DNA/RNA (5)(6)(7)(8). The prion-like domain mediates protein-protein interactions between TDP-43 and other hnRNP members (9), which also hosts most known ALS-associated TDP-43 mutations.…”

Backbone-independent nucleic acid binding by splicing factor SUP-12 reveals key aspects of molecular recognition

“…Limited similarity to the preceding guanine lies only in a common syn-conformation with 3 0 endo sugar pucker. Finally, in the complex of tandem RRM domains of TDP-43 in complex with GUGUGAAUGAAU (PDB 4BS2) 18 , the nucleotide conformation of the bound G3-U4-G5 is again similar to that of G4, G5 and G6 in complex with SUP-12, but with dissimilar types of protein contact. Interestingly, a guanine is recognized by both RRM domains reminiscent of the interface between ASD-1 and SUP-12 in the ternary complex.…”