TDP‐43 N‐terminal domain dimerisation or spatial separation by RNA binding decreases its propensity to aggregate

Abstract

Aggregation of the 43 kDa TAR DNA‐binding protein (TDP‐43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP‐43 N‐terminal domain dimerisation has been suggested to ameliorate TDP‐43 aggregation. However, the relationship between these factors and the solubility of TDP‐43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP‐43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP‐43‐preferable UG‐repeats, we uncovered two distinct mechanisms for modulating TDP‐43 solubility by RNA binding: One is N‐terminal domain dimerisation, and the other is the spatial separation of two TDP‐43 molecules. This study provides new molecular insights into the regulation of TDP‐43 solubility.

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes

“…As previously reported, 32 , 55 biochemical assays showed that longer GU-oligonucleotides promoted multivalent TDP-43 binding, compared to monovalent (GU)6 ( Figure 1 ). Both native EMSA assays and BS3 cross-linking-SDS-PAGE demonstrated stronger TDP-43 RRM1,2 multimerization for 34-nt Clip34nt and 36-nt (GU)16 compared to 24-nt (‘AUG12’)2.…”

“…TDP-43 LLPS regulates the native size of TDP-43 nuclear RNP complexes, which in turn dictates TDP-43 nucleocytoplasmic localization. 56 Since GU-RNA modulates TDP-43 LLPS in a length- and motif-dependent manner, 33 , 55 the observed GU-oligo-induced effects on TDP-43 localization (and function, discussed later) may be influenced by changes in TDP-43 LLPS. RNA motifs containing pure GU repeats differ in their ability to promote TDP-43 phase transition compared to GU intermixed with A and C. 55 Indeed, image analysis of oligo-induced changes in nuclear TDP-43 granules, likely composed of numerous TDP-43 RNPs, showed marked differences between oligos containing pure GU-repeats ((GU)16 and (‘AUG12’)2) versus the intermixed sequence of Clip34nt ( Figure S1 ).…”

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes

“…As previously reported, 32,55 biochemical assays showed that longer GU-oligonucleotides promoted multivalent TDP-43 binding, compared to monovalent (GU)6 (fig 1). Both native EMSA assays and BS3 crosslinking-SDS-PAGE demonstrated stronger TDP-43 RRM1,2 multimerization for 34-nt Clip34nt and 36-nt (GU)16 compared with 24-nt ('AUG12')2.…”

“…Surprisingly, oligo localization within cells did not predict the effect on TDP-43 nucleocytoplasmic localization and was inversely correlated, with ('AUG12')2 being the most nuclear and (GU)16 being equally distributed between the nucleus and cytoplasm. Future proteomic analysis could enable the identification of nuclear vs 56 Since GU-RNA modulates TDP-43 LLPS in a length-and motif-dependent manner, 33,55 the observed GU-oligo-induced effects on TDP-43 localization (and function, discussed below) may be influenced by changes in TDP-43 LLPS. RNA motifs containing pure GU repeats differ in their ability to promote TDP-43 phase transition compared to GU intermixed with A and C. 55 Indeed, image analysis of oligoinduced changes in nuclear TDP-43 granules, likely composed of numerous TDP-43 RNPs, showed marked differences between oligos containing pure GU-repeats ((GU)16 and ('AUG12')2) versus the intermixed sequence of Clip34nt (fig S1).…”

“…Future proteomic analysis could enable the identification of nuclear vs 56 Since GU-RNA modulates TDP-43 LLPS in a length-and motif-dependent manner, 33,55 the observed GU-oligo-induced effects on TDP-43 localization (and function, discussed below) may be influenced by changes in TDP-43 LLPS. RNA motifs containing pure GU repeats differ in their ability to promote TDP-43 phase transition compared to GU intermixed with A and C. 55 Indeed, image analysis of oligoinduced changes in nuclear TDP-43 granules, likely composed of numerous TDP-43 RNPs, showed marked differences between oligos containing pure GU-repeats ((GU)16 and ('AUG12')2) versus the intermixed sequence of Clip34nt (fig S1). Namely, ('AUG12')2 and (GU)16 induced an increase in TDP-43 nuclear granule number while reducing mean granule size.…”