Interconnections between m6A RNA modification, RNA structure, and protein-RNA complex assembly.

Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein-RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N6-methyladenosine (m6A) is the most common base modification on mRNAs and regulatory noncoding RNAs and has been shown to impact RNA structure and directly modulate protein-RNA interactions. In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 m6A writer complex is specifically targeted to RNA assisted by protein-RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the m6A modification affects RNA folding and protein-RNA interactions.

Eukaryotic Box C/D methylation machinery has two non-symmetric protein assembly sites.

Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2'-OH of RNA. The central 'guide RNA' has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C'/D' motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C'/D' motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2'-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C'/D' motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2'-O-methylation enzymes.

High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.

Structural and functional roles of 2'-O-ribose methylations and their enzymatic machinery across multiple classes of RNAs.

RNA complexity is augmented by numerous post-transcriptional modifications, which influence RNA function by modulating its structure and interactome. One prominent modification is methylation at the ribose 2'-hydroxyl group. 2'-O-methylation has been found in all RNA classes, with rRNA and tRNA being extensively modified. The exact function of 2'-O-methylation at specific RNA sites is still not understood, with a few notable exceptions. The relevance of 2'-O-methylation for cell survival and well-being is proven by the large effort that the cell spends in maintaining a diverse and highly regulated methylation machinery. Here, we review the current knowledge on the impact of 2'-O-methylation on structure and function of different RNAs as well as on the factors determining substrate specificity in the enzymatic machinery.

Compensatory adaptations of structural dynamics in an intrinsically disordered protein complex.

Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This "unfolding-upon-binding" is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin-OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate-binding and -release events in IDP interaction networks.