The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (C) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1 H NMR and circular dichroism spectroscopy. In addition, a [1,3-15 N]C phosphoramidite was used to generate H69 analogs with site-specific 15 N labels. By using this approach, different C residues can be clearly distinguished from one another in 1 H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through CN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for C such as maintaining translation fidelity.

The U6 RNA internal stem-loop (U6 ISL) is a highly conserved domain of the spliceosome that is important for pre-mRNA splicing. The U6 ISL contains an internal loop that is in equilibrium between two conformations controlled by the protonation state of an adenine (pKa = 6.5). Lower pH favors formation of a protonated C-A+ wobble pair and base flipping of the adjacent uracil. Higher pH favors stacking of the uracil, and allows an essential metal ion to bind at this position. Here, we define the minimal energy path for this conformational transition. To do this, we solved the U6 ISL structure at higher pH (8.0), in order to eliminate interference from the low pH conformer. This structure reveals disruption of the protonated C-A+ pair and formation of a new C-U pair, which explains the preference for a stacked uracil at higher pH. Next, we used Nudged Elastic Band (NEB) molecular dynamics simulations to calculate the minimum energy path between the two conformations. Our results indicate that the C-U pair is dynamic, which allows formation of the more stable C-A+ pair upon adenine protonation. After formation of the C-A+ pair, the unpaired uracil follows a minor groove base flipping pathway. Molecular dynamics simulations suggest that the extrahelical uracil is stabilized by contacts with the adjacent helix.

The essential splicing factor Prp24 contains four RNA Recognition Motif (RRM) domains, and functions to anneal U6 and U4 RNAs during spliceosome assembly. Here, we report the structure and characterization of the C-terminal RRM4. This domain adopts a novel non-canonical RRM fold with two additional flanking α-helices that occlude its β-sheet face, forming an occluded RRM (oRRM) domain. The flanking helices form a large electropositive surface. oRRM4 binds to and unwinds the U6 internal stem loop (U6 ISL), a stable helix that must be unwound during U4/U6 assembly. NMR data indicate that the process starts with the terminal base pairs of the helix and proceeds toward the loop. We propose a mechanistic and structural model of Prp24′s annealing activity in which oRRM4 functions to destabilize the U6 ISL during U4/U6 assembly.

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To facilitate the high-throughput acquisition of nuclear magnetic resonance (NMR) experimental data on large sets of samples, we have developed a simple and straightforward automated methodology that capitalizes on recent advances in Bruker BioSpin NMR spectrometer hardware and software. Given the daunting challenge for non-NMR experts to collect quality spectra, our goal was to increase user accessibility, provide customized functionality, and improve the consistency and reliability of resultant data. This methodology, NMRbot, is encoded in a set of scripts written in the Python programming language accessible within the Bruker BioSpin TopSpin™ software. NMRbot improves automated data acquisition and offers novel tools for use in optimizing experimental parameters on the fly. This automated procedure has been successfully implemented for investigations in metabolomics, small-molecule library profiling, and protein–ligand titrations on four Bruker BioSpin NMR spectrometers at the National Magnetic Resonance Facility at Madison. The investigators reported benefits from ease of setup, improved spectral quality, convenient customizations, and overall time savings.