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1. Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

   Zaharias, S.; Zhang, Z.; Davis, K.; Fargason, T.; Cashman, D.; Yu, T.; Zhang, J. (July 2021, Journal of biological chemistry)

   Musier-Forsyth, Karin (Ed.)

   RNA-binding proteins play crucial roles in various cellular functions, and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15) as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3 (SRSF3). Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, while underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts. 

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2. Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid

   https://doi.org/10.1093/nar/gkab797  

   Chen, Shi; Xing, Li; Zhang, Douglas; Monferrer, Alba; Hermann, Thomas (September 2021, Nucleic Acids Research)

   Abstract Design and preparation of layered composite materials alternating between nucleic acids and proteins has been elusive due to limitations in occurrence and geometry of interaction sites in natural biomolecules. We report the design and kinetically controlled stepwise synthesis of a nano-sandwich composite by programmed noncovalent association of protein, DNA and RNA modules. A homo-tetramer protein core was introduced to control the self-assembly and precise positioning of two RNA–DNA hybrid nanotriangles in a co-parallel sandwich arrangement. Kinetically favored self-assembly of the circularly closed nanostructures at the protein was driven by the intrinsic fast folding ability of RNA corner modules which were added to precursor complex of DNA bound to the protein. The 3D architecture of this first synthetic protein–RNA–DNA complex was confirmed by fluorescence labeling and cryo-electron microscopy studies. The synthesis strategy for the nano-sandwich composite provides a general blueprint for controlled noncovalent assembly of complex supramolecular architectures from protein, DNA and RNA components, which expand the design repertoire for bottom-up preparation of layered biomaterials. 

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3. Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade–TniQ complex

   https://doi.org/10.1073/pnas.2422895122  

   Patel, Amun C; Sinha, Souvik; Arantes, Pablo R; Palermo, Giulia (April 2025, Proceedings of the National Academy of Sciences)

   TheVibrio choleraeCascade–TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of “knocking-in” genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open–closed conformational change that is key for the complex’s function. We show that when Cascade–TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction promotes the bundle’s transition toward the open state, priming the complex for DNA binding. As the target DNA binds the guide RNA, the opening of the Cas8 bundle becomes more favorable, exposing positively charged residues and facilitating their interaction with DNA, which ultimately leads the DNA-binding process to completion. These outcomes provide a dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade–TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms. 

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4. RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

   https://doi.org/10.1073/pnas.2116222119  

   Gavrilov, Alexey A.; Sultanov, Rinat I.; Magnitov, Mikhail D.; Galitsyna, Aleksandra A.; Dashinimaev, Erdem B.; Lieberman Aiden, Erez; Razin, Sergey V. (January 2022, Proceedings of the National Academy of Sciences)

   Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis - and trans -acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs. 

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5. EquiPNAS: improved protein–nucleic acid binding site prediction using protein-language-model-informed equivariant deep graph neural networks

   https://doi.org/10.1093/nar/gkae039  

   Roche, Rahmatullah; Moussad, Bernard; Shuvo, Md Hossain; Tarafder, Sumit; Bhattacharya, Debswapna (January 2024, Nucleic Acids Research)

   Abstract Protein language models (pLMs) trained on a large corpus of protein sequences have shown unprecedented scalability and broad generalizability in a wide range of predictive modeling tasks, but their power has not yet been harnessed for predicting protein–nucleic acid binding sites, critical for characterizing the interactions between proteins and nucleic acids. Here, we present EquiPNAS, a new pLM-informed E(3) equivariant deep graph neural network framework for improved protein–nucleic acid binding site prediction. By combining the strengths of pLM and symmetry-aware deep graph learning, EquiPNAS consistently outperforms the state-of-the-art methods for both protein–DNA and protein–RNA binding site prediction on multiple datasets across a diverse set of predictive modeling scenarios ranging from using experimental input to AlphaFold2 predictions. Our ablation study reveals that the pLM embeddings used in EquiPNAS are sufficiently powerful to dramatically reduce the dependence on the availability of evolutionary information without compromising on accuracy, and that the symmetry-aware nature of the E(3) equivariant graph-based neural architecture offers remarkable robustness and performance resilience. EquiPNAS is freely available at https://github.com/Bhattacharya-Lab/EquiPNAS. 

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