The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2′-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5′-terminal 2′-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein–protein interactions causative of cellular toxicity.

Gene regulation ensures that the appropriate genes are expressed at the proper times. Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of post-transcriptional gene regulation. Studies on this phenomenon indicated that it exerted significant impact on the regulation of gene expression by regulating export and translation delay, which allows synthesis of specific proteins in response to a stimulus, e.g. under stress conditions or at strictly controlled time points, e.g. during cell differentiation or development. Here, we found that transcription in microsporocytes, during prophase of the first meiotic division, occurs in pulsatile manner. After each pulse, the transcriptional activity is silenced, but the transcripts synthesized at this time are not exported immediately to the cytoplasm, but are retained in the nucleoplasm and Cajal bodies (CBs). In contrast to nucleoplasm, mature transcripts were not found in CBs. Only non-fully-spliced transcripts with retained introns were stored in the CBs. Retained introns are spliced at precisely defined times, and fully mature mRNAs are released into the cytoplasm, where the proteins are produced. These proteins are necessary for further cell development during meiotic prophase. Our findings provide new insight into the regulatory mechanisms of gene expression based on mRNA retention in the nucleus during the development of generative cells in plants. Similar processes were observed during spermatogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

Gene regulation ensures that the appropriate genes are expressed at the proper time. Nuclear retention of incompletely spliced or mature mRNAs is emerging as a novel, previously underappreciated layer of post-transcriptional regulation. Studies on this phenomenon indicated that it exerts a significant influence on the regulation of gene expression by regulating export and translation delay, which allows the synthesis of specific proteins in response to a stimulus or at strictly controlled time points, e.g. during cell differentiation or development. Here, we show that transcription in microsporocytes of European larch (Larix decidua) occurs in a pulsatile manner during prophase of the first meiotic division. Transcriptional activity was then silenced after each pulse. However, the transcripts synthesized were not exported immediately to the cytoplasm but were retained in the nucleoplasm and Cajal bodies (CBs). In contrast to the nucleoplasm, we did not detect mature transcripts in CBs, which only stored non-fully-spliced transcripts with retained introns. Notably, the retained introns were spliced at precisely defined times, and fully mature mRNAs were released into the cytoplasm for translation. As similar processes have been observed during spermatogenesis in animals, our results illustrate an evolutionarily conserved mechanism of gene expression regulation during generative cells development in Eukaryota.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

SUMMARY In recent years, research has increasingly focused on the key role of post‐transcriptional regulation of messenger ribonucleoprotein (mRNP) function and turnover. As a result of the complexity and dynamic nature of mRNPs, the full composition of a single mRNP complex remains unrevealed and mRNPs are poorly described in plants. Here we identify canonical Sm proteins as part of the cytoplasmic mRNP complex, indicating their function in the post‐transcriptional regulation of gene expression in plants. Sm proteins comprise an evolutionarily ancient family of small RNA‐binding proteins involved in pre‐mRNA splicing. The latest research indicates that Sm could also impact on mRNA at subsequent stages of its life cycle. In this work we show that in the microsporocyte cytoplasm of Larix decidua , the European larch, Sm proteins accumulate within distinct cytoplasmic bodies, also containing polyadenylated RNA. To date, several types of cytoplasmic bodies involved in the post‐transcriptional regulation of gene expression have been described, mainly in animal cells. Their role and molecular composition in plants remain less well established, however. A total of 222 mRNA transcripts have been identified as cytoplasmic partners for Sm proteins. The specific colocalization of these mRNAs with Sm proteins within cytoplasmic bodies has been confirmed via microscopic analysis. The results from this work support the hypothesis, that evolutionarily conserved Sm proteins have been adapted to perform a whole repertoire of functions related to the post‐transcriptional regulation of gene expression in Eukaryota. This adaptation presumably enabled them to coordinate the interdependent processes of splicing element assembly, mRNA maturation and processing, and mRNA translation regulation, and its degradation.