Functional mapping of the interaction between TDP-43 and hnRNP A2 in vivo

Abstract

Nuclear factor TDP-43 has been reported to play multiple roles in transcription, pre-mRNA splicing, mRNA stability and mRNA transport. From a structural point of view, TDP-43 is a member of the hnRNP protein family whose structure includes two RRM domains flanked by the N-terminus and C-terminal regions. Like many members of this family, the C-terminal region can interact with cellular factors and thus serve to modulate its function. Previously, we have described that TDP-43 binds to several members of the hnRNP A/B family through this region. In this work, we set up a coupled minigene/siRNA cellular system that allows us to obtain in vivo data to address the functional significance of TDP-43-recruited hnRNP complex formation. Using this method, we have finely mapped the interaction between TDP-43 and the hnRNP A2 protein to the region comprised between amino acid residues 321 and 366. Our results provide novel details of protein–protein interactions in splicing regulation. In addition, we provide further insight on TDP-43 functional properties, particularly the lack of effects, as seen with our assays, of the disease-associated mutations that fall within the TDP-43 321-366 region: Q331K, M337V and G348C.

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

“…Although it has been demonstrated that TDP-43 itself is intrinsically aggregation-prone (57) and contains prion-like properties within its C terminus (58-60), we have found that motor neuron disease develops in the TDP-43 mutant-expressing mice without detectable TDP-43 aggregation. Furthermore, the TDP-43 C terminus has been proposed by others to play a role in association with other proteins in splicing of target genes (16)(17)(18)61), although the contribution of mutations in the C terminus to the disruption of TDP-43 splicing function was not well understood. Indeed, a study using a human cell-culture model expressing TDP-43 Q331K and TDP-43 M337V found that mutations in the TDP-43 C terminus do not alter the composition of complexes of known TDP-43-interacting hnRNPs, nor the splicing of a cystic fibrosis transmembrane conductance regulator-based splicing reporter (17).…”

“…Furthermore, the TDP-43 C terminus has been proposed by others to play a role in association with other proteins in splicing of target genes (16)(17)(18)61), although the contribution of mutations in the C terminus to the disruption of TDP-43 splicing function was not well understood. Indeed, a study using a human cell-culture model expressing TDP-43 Q331K and TDP-43 M337V found that mutations in the TDP-43 C terminus do not alter the composition of complexes of known TDP-43-interacting hnRNPs, nor the splicing of a cystic fibrosis transmembrane conductance regulator-based splicing reporter (17). In contrast, using a true in vivo context in mice that develop progressive motor neuron disease, we have found widespread splicing alterations in the adult mammalian central nervous system in the presence of nuclear, mutant TDP-43.…”

“…Structurally, the 414-aa protein consists of two RNA recognition motifs (RRM1 and RRM2) (12,13), nuclear import and export signal (14), and a glycine-rich region implicated in protein-protein interactions (15,16) that include components of the RNA splicing machinery (17,18).…”

TDP‐35 sequesters TDP‐43 into cytoplasmic inclusions through binding with RNA

“…This is distinct from the insoluble protein aggregates, formation of which is inhibited by TG-repeat nucleotide binding in vitro [18]. We have also observed that TDP-25A, a C-terminal fragment of TDP-43 containing only the RRM2 domain that binds nucleic acids much more weakly than RRM1 [37], cannot associate with TDP-43 either by RNA or synthesized ssDNA (Fig. 1).…”

RNA-binding proteins in neurological diseases

“…TDP-43 recognizes (UG) n repeat elements in target mRNAs by its RRM1 [128,129]. The glycine-rich domain mediates the interactions between TDP-43 and other proteins such as hnRNPs [130]. TDP-43 also contains a Q/N-rich prion-like element that mediates its interaction with polyQ aggregates.…”