Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive neurodegenerative disorder that affects the nerve cells responsible for controlling voluntary muscles. There is currently no cure for ALS, and its causes remain largely unknown. However, recent studies have shown that mutations in a protein called TDP-43 are a common hallmark of the disease. In a new study published in Scientific Reports, researchers have discovered that these mutations interfere with the recruitment of RNA recognition motifs to G-quadruplex RNA, providing new insights into the mechanisms underlying ALS.
The binding properties of TDP-43, a protein that has been linked to ALS, have been analyzed in vitro using segments containing two RNA-recognition motifs (RRMs). However, the RRM is not the only module that participates in RNA recognition. To obtain a more accurate analysis, the researchers purified a full-length untagged TDP-43 natural dimer and analyzed its RNA binding properties using two dendritic G4-mRNAs that bind TDP-43 in vivo. The effects of 10 ALS-linked amino acid substitution mutations were also determined. All the mutant TDP-43 proteins showed reduced interaction with G4-RNA using surface plasmon resonance. The researchers used the G4-RNAs and TDP-43 proteins that had the G287S or M337V mutations, which had the lowest G4-RNA binding properties.
Far-UV circular dichroism spectroscopy was used to confirm the effects of ALS-linked mutations on the overall structure. The secondary structure compositions of TDP-43 wild-type and mutant proteins were estimated using the Beta Structure Selection algorithm. TDP-43G287S had a higher helical content, whereas TDP-43M337V showed a decrease and an increase in beta-turn. This led the researchers to hypothesize that the mutations induced changes in both the secondary and tertiary structures.
The researchers also compared the interactions with G4-RNA under unified experimental conditions using an electrophoretic mobility shift assay. The binding of G4-RNA to specific interacting proteins with multiple binding modules often does not migrate normally when using a non-denaturing acrylamide gel. By adding 2.5, 5, and 10 times the amount of protein to G4-RNA, they confirmed a dose-dependent over-shift in the G4-RNA mobility. The two mutant proteins showed a reduction in shifting bands, consistent with the SPR and secondary structure prediction data.
The researchers also examined the possible influence of GST-fused GR mutant proteins on the G4-RNA immobilizing sensor chip using SPR. However, no significant difference was observed in the interaction with G4-RNA between the wild-type GST-GR and mutant GST-GRs. The researchers then analyzed the possible impact of GR mutations on the conformations of G4-RNA using CD spectroscopy. The presence of the mutant proteins increased the positive peak due to conformational alternation of G4-RNAs, which is generally characterized as an altered stacking arrangement of guanine tetrads. In addition, binding of the mutant proteins affected the negative peak of PSD-95 G4-RNA.
These unexpected results suggest that mutations in the GR region do not affect GR-G4-RNA binding but influence other molecular characteristics of GR, such as the modulation of G4-RNA structures and/or intramolecular signal transmission to the RRM for modulation of its RNA-binding activity. It is possible that the GR region alone cannot withstand even slight structural changes in gels forced to operate under low-salt conditions. Therefore, the researchers reinforced the association by UV cross-linking and confirmed the complex by SDS-PAGE. The cross-linking and gel shift assay supported the SPR results, and two mutations did not affect the interaction of GST-GR with G4-RNAs.
In conclusion, the study provides new insights into the complex interactions between RNA and proteins in ALS. By investigating the effects of TDP-43 mutations on the recruitment of RNA recognition motifs to G-quadruplex RNA, the researchers have shed light on a key mechanism that may contribute to the development of ALS. This knowledge could pave the way for the development of new therapeutic strategies that target these mechanisms and ultimately help to treat this devastating disease. Further studies are needed to fully understand the complex interactions between RNA and proteins, but this research represents an important step forward in the fight against ALS.
