Biology subjects

Stuani, C.

Publications and source records attributed to Stuani, C..

3 recordsLinked to original sources

bioRxiv2023

Axon guidance genes are regulated by TDP-43 and RGNEF through the rate of long-intron processing.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. Previous work has shown that RGNEF inclusions in the spinal motor neurons of ALS patients co-localise with TDP-43, the major RNA binding protein aggregating in the brain and spinal cord of ALS patients. To further characterise their relationship, we have compared the transcriptomic profiles of neuronal-like cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these factors on the processivity of long introns can explain their mode of action. Our findings highlight that neurodegenerative processes at the RNA level can often represent the result of combinatorial interactions between different RNA binding factors, leading to a better understanding of pathogenic mechanisms occurring in patients where more than one specific protein may be aggregating in their neurons.

neuroscience↗

bioRxiv2023

hnRNP A1 induces aberrant CFTR exon 9 splicing via a newly discovered Exonic Splicing Silencer element.

RNA-protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a non-functional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends primarily on variations in the polymorphic (TG)mTn locus upstream of exon 9. At the pre-mRNA level, it generates an extended UG-rich binding site for TDP-43, associated with hnRNP A1 recruitment, and prevention of exon 9 3 splicing site (3ss) recognition. While TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs (RRM1 and RRM2) and a glycine-rich domain, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Thermal Calorimetry (ITC). The affinities are submicromolar and ITC data suggest that the separate RRMs as well as tandem RRMs form 1:1 complexes. NMR titrations reveal that hnRNP A1 interacts with model CTFR 3ss sequences in a fast exchange regime at the NMR timescale. Splicing assays finally show that this hnRNP A1 binding site represents a previously unknown exonic splicing silencer element. Together, our results shed light on the mechanism of aberrant CFTR exon 9 splicing.

biochemistry↗

bioRxiv2020

Phe-Gly Motifs Drive Fibrillization of TDP-43's Prion-Like Domain Droplets

TDP-43 assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g. muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct endpoint species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs, and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions, and illustrate structural features that distinguishes between aggregate forms.

biophysics↗