A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43

Abstract

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein containing two consecutive RNA recognition motifs (RRM1 and RRM2) in tandem. Functional abnormality of TDP-43 has been proposed to cause neurodegeneration, but it remains obscure how the physiological functions of this protein are regulated. Here, we show distinct roles of RRM1 and RRM2 in the sequence-specific substrate recognition of TDP-43. RRM1 was found to bind a wide spectrum of ssDNA sequences, while no binding was observed between RRM2 and ssDNA. When two RRMs are fused in tandem as in native TDP-43, the fused construct almost exclusively binds ssDNA with a TG-repeat sequence. In contrast, such sequence-specificity was not observed in a simple mixture of RRM1 and RRM2. We thus propose that the spatial arrangement of multiple RRMs in DNA/RNA binding proteins provides steric effects on the substrate-binding site and thereby controls the specificity of its substrate nucleotide sequences.

An Intramolecular Salt Bridge Linking TDP43 RNA Binding, Protein Stability, and TDP43-Dependent Neurodegeneration

“…Conversely, TDP43(ΔRRM2) overexpression increased the risk of death by 42% in comparison to EGFP alone. In light of the ability of RRM1, but not RRM2, to bind RNA in vitro ( Buratti and Baralle, 2001 ; Kuo et al, 2009 ; Furukawa et al, 2016 ), these observations provide strong evidence linking RNA binding to toxicity from TDP43 overexpression.…”

“…Notably, the R151A mutation phenocopied the F147L-F149L double mutant closely, while the D247A mutation elicited an intermediate phenotype. Since the majority of high-affinity RNA binding is accomplished by RRM1 ( Buratti and Baralle, 2001 ; Ayala et al, 2005 ; Furukawa et al, 2016 ), TDP43(D247A) may still recognize many conventional TDP43 targets. Supporting this notion, the number of DEGs and alternatively spliced transcripts in cells overexpressing TDP43(D247A) were more similar to TDP43(WT) than other TDP43 variants, and TDP43(D247A) expression dis rupted mitochondrial morphology in rodent primary neurons, albeit less potently than TDP43(WT).…”

“…TDP43 contains 2 highly conserved RNA recognition motifs (RRM1 and RRM2) that exhibit distinct properties. RRM1 has a higher affinity for RNA, while RRM2 enhances the specificity of RNA recognition by RRM1 ( Buratti and Baralle, 2001 ; Ayala et al, 2005 ; Kuo et al, 2009 ; Furukawa et al, 2016 ). In Drosophila , RRM1 deletion or substitution of Trp113 to Ala eliminates RNA binding and prevents TDP43-mediated toxicity ( Ayala et al, 2005 ; Ihara et al, 2013 ).…”

Structural Insights Into TDP-43 and Effects of Post-translational Modifications

“…Studies have consistently shown preferential binding of TDP-43 to (TG)/(UG) sequences (Buratti et al, 2001;Kuo et al, 2009;Sephton et al, 2011;Colombrita et al, 2012;Bhardwaj et al, 2013;Lukavsky et al, 2013;Furukawa et al, 2016). RRM1 is known to be sufficient and essential for proper nucleic acid binding as its affinity is in the low nanomolar range for the canonical UG-rich sequence, while RRM2 is not (Kuo et al, 2009;Furukawa et al, 2016).…”

“…Kuo et al reported a large affinity increase between 4 and 6 nucleotides long, which approximately corresponds to the distance between both nucleic acid binding sites (∼22 Å) (Shodai et al, 2013; Figure 3A). Additionally, the RRMs are connected by a highly flexible loop (178)(179)(180)(181)(182)(183)(184)(185)(186)(187)(188)(189)(190)(191), thought to confer adaptability to different nucleic acid partners by allowing different orientations of the RRM domains (Auweter et al, 2006;Furukawa et al, 2016).…”