Biology subjects

Bandeiras, T. M.

Publications and source records attributed to Bandeiras, T. M..

3 recordsLinked to original sources

bioRxiv2026

The R2T(P) complex orchestrates the SHQ1 driven early steps of box H/ACA snoRNP maturation

The chaperones RuvBL1 and RuvBL2 are members of the AAA+ ATPase family and participate in diverse cellular processes, including DNA repair, transcriptional regulation, and assembly of macromolecular complexes such as snoRNPs. The biogenesis of box H/ACA snoRNPs additionally requires the assembly factor SHQ1. These protein-RNA complexes are essential for ribosome biogenesis and telomerase stability and are linked to diseases such as dyskeratosis congenita and cancer. Despite detailed knowledge of mature complexes, their assembly mechanisms and how they can be modulated remain unclear. We characterize a trimeric interaction between SHQ1 and RuvBL1:RuvBL2, providing insight into early maturation of the protein-only precursor of box H/ACA snoRNPs. SHQ1 binds the flexible domain II of RuvBL1:RuvBL2, corresponding to the dodecamerization interface, suggesting disruption of this interface and promotion of hexamer formation. We further purified a complex containing RuvBL1:RuvBL2, SHQ1, and DKC1, the catalytic component of H/ACA snoRNPs and a client of SHQ1. This demonstrates that SHQ1 and DKC1 can simultaneously associate with RuvBL1:RuvBL2, potentially facilitating DKC1 release and subsequent snoRNA binding. Additionally, we identified a direct interaction between SHQ1 and RPAP3, a co-chaperone of RuvBL1:RuvBL2 (within the R2TP). Hence, RPAP3 may be responsible for recruiting SHQ1:DKC1 to hexameric RuvBL1:RuvBL2 and/or assist in the AAA+ mediated dissociation of the dimer. Since SHQ1 shares a domain with PIH1D1, an integral member of the R2TP complex, our findings suggest that early H/ACA snoRNP maturation may involve the R2T instead of the previously proposed R2TP complex.

biophysics↗

bioRxiv2025

R2TP-like Quaternary Chaperones: a comprehensive overview to understand the dynamic R2SP complex

The human R2SP complex belongs to the R2TP-like quaternary chaperone family and consists of RUVBL1, RUVBL2, SPAG1 and PIH1D2. R2SP is crucial for the correct assembly of motile cilia (SPAG1 null mutations cause Primary Ciliary Dyskinesia) and the organization of the synaptic zone. RUVBL1/2 ATPases are the powerhouse of this molecular machinery, while SPAG1 and PIH1D2 would be adaptors that interact with specific clients to promote their quaternary assembly. Despite these functional data, little is known about the structure of R2SP and the precise mode of action of these R2TP-like complexes. We have combined biochemical and structural approaches (NMR, structural mass spectrometry and cryo-EM) to investigate the 3D organization of the human R2SP complex, its mode of assembly and ATPase activity. Our study reveals a three-dimensional structure similar to that of the canonical R2TP complex, but also highlights differences in the mode of action of its RUVBL1/2 ATPase core as well as the binding of its adaptors SPAG1 and PIH1D2.

biophysics↗

bioRxiv2023

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets.

biophysics↗