The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) 1 , 2 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells 3 – 7 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

To understand the functional roles of RNA in the cell, it is essential to elucidate the dynamics of their production, processing and decay. A recent method for assessing mRNA dynamics is metabolic labeling with 4-thiouridine (4sU), followed by thio-selective attachment of affinity tags. Detection of labeled transcripts by affinity purification and hybridization to microarrays or by deep sequencing then reveals RNA expression levels. Here, we present a novel sequencing method (TUC-seq) that eliminates affinity purification and allows for direct assessment of 4sU-labeled RNA. It employs an OsO -mediated transformation to convert 4sU into cytosine. We exemplify the utility of the new method for verification of endogenous 4sU in tRNAs and for the detection of pulse-labeled mRNA of seven selected genes in mammalian cells to determine the relative abundance of the new transcripts. The results prove TUC-seq as a straight-forward and highly versatile method for studies of cellular RNA dynamics.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

SignificanceTranslation termination is a crucial process during protein synthesis. Class I release factors (RFs) are in charge of recognizing stop codons and consequently hydrolyzing the peptidyl-tRNA at the ribosomal P site. High-resolution crystal- and cryo-EM structures of RFs bound to the ribosome revealed a network of potential interactions that is formed between the mRNA and RFs; however, it remained enigmatic which interactions are critical for accurate stop codon recognition and peptide release. By using chemically modified stop codon nucleotides, the importance and the contribution of single hydrogen bonds to stop codon recognition was investigated. This approach revealed a detailed picture of chemical groups defining a stop codon and contributing to the discrimination against sense codons during prokaryotic and eukaryotic translation termination.

Riboswitches are metabolite-sensing, conserved domains located in non-coding regions of mRNA that are central to regulation of gene expression. Here we report the first three-dimensional structure of the recently discovered S-adenosyl-L-methionine responsive SAM-VI riboswitch. SAM-VI adopts a unique fold and ligand pocket that are distinct from all other known SAM riboswitch classes. The ligand binds to the junctional region with its adenine tightly intercalated and Hoogsteen base-paired. Furthermore, we reveal the ligand discrimination mode of SAM-VI by additional X-ray structures of this riboswitch bound to S-adenosyl-L-homocysteine and a synthetic ligand mimic, in combination with isothermal titration calorimetry and fluorescence spectroscopy to explore binding thermodynamics and kinetics. The structure is further evaluated by analysis of ligand binding to SAM-VI mutants. It thus provides a thorough basis for developing synthetic SAM cofactors for applications in chemical and synthetic RNA biology.