Biology subjects

Casanal, A.

Publications and source records attributed to Casanal, A..

3 recordsLinked to original sources

bioRxiv2026

Molecular basis of tRNA modification by the human m5C methyltransferase NSUN2

RNA 5-methylcytidine (m5C) is a prevalent modification that drives RNA stability and function. In humans, m5C is deposited on distinct RNA substrates by DNMT2/TRDMT1 and the NSUN family, to regulate diverse cellular processes, but how m5C writers recognise their substrates remains unclear. NSUN2 is a major m5C methyltransferase with broad roles in cell physiology and strong links to cancer and neurodevelopmental disorders 1. Here, we reconstitute an active human NSUN2-tRNA complex and capture its post-catalytic, tRNA-bound structure at 3.1 [A] resolution. Using an integrated approach combining biochemistry, cryo-electron microscopy, crosslinking mass spectrometry and molecular dynamics simulations, we show that NSUN2 remodels the tRNA to access the variable-loop target cytidine. Recognition is driven by RNA architecture, with NSUN2 exploiting the L-shaped tRNA scaffold to position the target base in the catalytic centre. We further show that Gly679 at the NSUN2-tRNA interface is important for the stability of the complex, providing a mechanistic basis for how the disease-associated Gly679Arg substitution can impair tRNA binding. Together, these findings establish an RNA-structure-guided mechanism for NSUN2 substrate recognition and methylation and provide general principles for m5C deposition on cellular RNAs and their fundamental role in disease.

molecular biology↗

bioRxiv2023

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2019

Breaking a Strong Amide Bond: Structure and Properties of Dimethylformamidase.

Dimethylformamidase (DMFase) breaks down the human-made synthetic solvent N,N-dimethyl formamide(DMF) used extensively in industry(1). DMF is not known to exist in nature and was first synthesized in 1893. In spite of the recent origin of DMF, certain bacterial species such as Paracoccus, Pseudomonas, and Alcaligenes have evolved pathways to breakdown DMF and use them as carbon and nitrogen source for growth(2, 3). The work presented here provides a molecular basis for the ability of DMFase from Paracoccus to function in exacting conditions of high solvent concentrations, temperature and ionic strength to catalyze the hydrolysis of a stable amide bond. The structure reveals a multimeric complex of the 2{beta}2 type or (2{beta}2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed folds and as yet of unknown evolutionary origin. The active site is made of a distinctive mononuclear iron that is coordinated by two tyrosine residues and a glutamic acid residue. The hydrolytic cleavage of the amide bond is catalyzed at the Fe3+ site with a proximal glutamate probably acting as the base. The change in the quaternary structure is salt dependent with high salt resulting in the larger oligomeric state. Kinetic characterization reveals an enzyme that shows cooperativity between subunits and the structure provides clues on the interconnection between the active sites. Significance StatementN,N-dimethyl formamide(DMF) is a commonly used industrial solvent that was first synthesized in 1893. The properties that make DMF a highly desired solvent also makes it a difficult compound to breakdown. Yet, certain bacteria have evolved to survive in environments polluted by DMF and have enzymes that breakdown DMF and use it as their carbon and nitrogen source. The molecular structure of the enzyme that breaks down the stable amide bond in these bacteria, reveals two new protein folds and a unique mononuclear iron active site. The work reported here provides the structural and biochemical framework to query the evolutionary origins of the protein, as well as in engineering this enzyme for use in bioremediation of a human made toxic solvent.

biochemistry↗