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Biology subjects

Marcia, M.

Publications and source records attributed to Marcia, M..

4 recordsLinked to original sources

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

cell biology↗

A previously-unrecognized motif of transcription factor RYBP, hotspot of cancer-related mutations, is essential for the integrity of Polycomb repressive complex 1

Polycomb repressive complex 1 (PRC1) catalyzes monoubiquitination of histone H2A on Lys119, promoting gene silencing. Cells at different developmental stages and in different tissues express different PRC1 isoforms. All isoforms share the same catalytic core (subunits RING1B and PCGF) and vary in the composition of regulatory subunits, clustering in two major classes. Canonical isoforms (cPRC1) are regulated by CBX-like subunits, while variant isoforms (vPRC1) are regulated by RYBP-like subunits. The molecular bases for how regulatory subunits affect the structural assembly of the complex and its catalytic activity are still largely unknown. To fill this knowledge gap, here we have specifically studied how RYBP regulates vPRC1 structure and function. Integrating the machine-learning algorithm AlphaFold2 and NMR, we have identified novel vPRC1 structural motifs in RING1B and RYBP. While the new RING1B motif is dispensable for vPRC1 assembly, the RYBP motif is essential for mediating inter-subunit interactions between RYBP and the catalytic RING1B-PCGF4 heterodimer. Importantly, the RYBP motif harbors cancer-related mutations systematically positioned on the same face of a putative transiently-forming -helix. Biochemical, biophysical and enzymatic characterization of purified cancer-related mutants confirm that this region is crucial for the structural stability of the complex. Overall, our data offer novel insights into the molecular architecture of vPRC1 and the effects of its regulatory subunit on the biochemical, structural, enzymatic, and physio-pathological properties of the complex.

biochemistry↗

Targeting the conserved active site of splicing machines with specific and selective small molecule modulators

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work puts solid bases for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.

biochemistry↗