Nucleic Acids Research最新文献

Integrative biomolecular modeling of RNA relies on refined structural collections and accurate experimental data that reflect binding and folding behavior. However, predicting such collections remains challenging due to the rugged energy landscape and extensive conformational heterogeneity of large RNAs. To overcome these limitations, we applied a Förster resonance energy transfer (FRET)-guided strategy to identify RNA conformational states consistent with single-molecule FRET (smFRET) experiments. We predicted 3D structures of a ribosomal RNA tertiary contact comprising a GAAA tetraloop and a kissing loop using three popular RNA 3D modeling tools, namely RNAComposer, FARFAR2, and AlphaFold3, yielding a collection of candidate conformations. These models were structurally validated based on Watson-Crick base-pairing patterns and filtered using an eRMSD threshold. For each retained structure, we computed the accessible contact volume of the Cy3/Cy5 dye pair using FRETraj to predict FRET distributions. These distributions were then compared and weighted against experimental smFRET data to identify conformational states compatible with the observed FRET states. Our results demonstrate that experimental transfer efficiencies can be reproduced using in silico predicted RNA 3D structures. This FRET-guided workflow, combined with structural validation, lays the foundation for capturing the highly diverse conformational states characteristic of flexible RNA motifs.

Production of the eukaryotic ribosomal subunits (40S and 60S) is a highly dynamic process in which numerous assembly factors (AFs) coordinate structural rearrangements of pre-ribosomal complexes to achieve their mature, functional architectures. Across the domains of life, GTPases leverage their functions as molecular switches to induce conformational changes that drive key steps in subunit maturation. Three GTPases, GTPBP4, GNL2, and GNL3, have been detected in nucleolar/nucleoplasmic human pre-60S complexes. Here, we compositionally analyze the pre-ribosomal particles associated with each of these GTPases and demonstrate the requirement of these enzymes, and their abilities to bind and hydrolyze GTP, for distinct steps in pre-ribosomal RNA processing. We further reveal that the GNL3 paralog, GNL3L, also associates with pre-ribosomes, and we map GNL3L binding sites on pre-rRNAs as well as identifying RNA contact sites on GNL3L. Lack of GNL3L impairs synthesis of the 60S rRNAs and expression of GTPase-inactive GNL3L causes defects in early steps of pre-rRNA processing. Impaired GTP hydrolysis by GNL3L leads to its accumulation on pre-60S particles, together with other AFs with proximal binding sites. Our data further demonstrate that the GTPase activity of GNL3L is required for maintaining 60S subunit levels, protein synthesis, and cellular proliferation.

The nucleotide excision repair (NER) system removes UV photoproducts from the genome in the form of small, excised, damage-containing DNA oligonucleotides (sedDNAs). However, the methods that are traditionally used to detect these repair products in human cells have several limitations, including the presence of other contaminating large DNAs and labeling efficiency. We therefore developed a novel microplate-based competitive immunoassay platform to precisely measure sedDNAs containing pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] and cyclobutane pyrimidine dimer (CPD) following UV exposure. The immunoassay employs polyethylene glycol to separate sedDNAs from larger contaminating DNA molecules along with immobilized UV-irradiated oligonucleotides and damage-specific antibodies. Our results demonstrate that this method is highly sensitive and specific, enabling the detection of NER-dependent sedDNAs generated by minimal UV doses and within a short time frame after UV exposure. Moreover, quantification of (6-4)PP- and CPD-containing sedDNAs in this assay allowed the detection of up to 22-fold more (6-4)PP-sedDNAs and up to 7-fold more CPD-sedDNAs than by conventional 3'-end labeling. Thus, this novel immunoassay shows remarkable sensitivity and accuracy for quantifying sedDNAs in UV-irradiated cells and provides a convenient platform for advancing our understanding of the molecular mechanisms of the NER system.

DNA is highly negatively charged, making its structure strongly dependent on the ionic environment. DNA twist-a central DNA property-varies with ion concentration and identity. Prior studies have focused on salt concentrations below 1 M, and it is unclear whether twist trends persist at higher concentrations. It has been proposed that at high salt, DNA transitions from its canonical B-form to C-form, originally observed by fiber diffraction. Here, we use single-molecule magnetic tweezers to measure DNA twist in high concentrations of LiCl, NaCl, KCl, and CsCl. For all salts, twist initially increases approximately as ∼[salt]1/2, but plateaus and even decreases above 3 M. LiCl causes the largest twist increase, by ≤ 0.9° bp-1, compared with physiological salt, still far below the suggested C-form values of 2-3° bp-1. We perform extensive all-atom molecular dynamics simulations for DNA in LiCl solutions with different force fields. For parmbsc1, we observe good agreement with experiments when ion activities are taken into account. We find that simulations initiated in the C-form rapidly convert to the B-form, while the B-form remains stable. Our results demonstrate ion-specific, systematic changes in DNA twist beyond 1 M salt, but do not support a transition to the C-form for DNA.