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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. Controlled by disorder: Phosphorylation modulates SRSF1 domain availability for spliceosome assembly

   https://doi.org/10.1002/pro.70070  

   Fargason, Talia; Powell, Erin; De_Silva, Naiduwadura_Ivon_Upekala; Paul, Trenton; Zhang, Zihan; Prevelige, Peter; Zhang, Jun (February 2025, Protein Science)

   Abstract Serine/arginine‐rich splicing factor 1 (SRSF1) is key in the mRNA lifecycle including transcription, splicing, nonsense‐mediated decay, and nuclear export. Consequently, its dysfunction is linked to cancers, viral evasion, and developmental disorders. The functionality of SRSF1 relies on its interactions with other proteins and RNA molecules. These processes are regulated by phosphorylation of its unstructured arginine/serine‐rich tail (RS). Here, we characterize how phosphorylation affects SRSF1's protein and RNA interaction and phase separation. Using NMR paramagnetic relaxation enhancement and chemical shift perturbation, we find that when unphosphorylated, SRSF1's RS interacts with its first RNA‐recognition motif (RRM1). Phosphorylation of RS decreases its interactions with the protein‐binding site of RRM1 and increases its interactions with the RNA‐binding site of RRM1. This change in SRSF1's intramolecular interactions increases the availability of protein‐interacting sites on RRM1 and weakens RNA binding of SRSF1. Phosphorylation alters the phase separation of SRSF1 by diminishing the role of arginine in intermolecular interactions. These findings provide an unprecedented view of how SRSF1 influences the early‐stage spliceosome assembly. 

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3. The juxtamembrane linker of synaptotagmin 1 regulates Ca2+ binding via liquid-liquid phase separation

   https://doi.org/10.1038/s41467-023-44414-5  

   Mehta, Nikunj; Mondal, Sayantan; Watson, Emma T.; Cui, Qiang; Chapman, Edwin R. (January 2024, Nature Communications)

   Abstract Synaptotagmin (syt) 1, a Ca²⁺sensor for synaptic vesicle exocytosis, functions in vivo as a multimer. Syt1 senses Ca²⁺via tandem C2-domains that are connected to a single transmembrane domain via a juxtamembrane linker. Here, we show that this linker segment harbors a lysine-rich, intrinsically disordered region that is necessary and sufficient to mediate liquid-liquid phase separation (LLPS). Interestingly, condensate formation negatively regulates the Ca²⁺-sensitivity of syt1. Moreover, Ca²⁺and anionic phospholipids facilitate the observed phase separation, and increases in [Ca²⁺]_ipromote the fusion of syt1 droplets in living cells. Together, these observations suggest a condensate-mediated feedback loop that serves to fine-tune the ability of syt1 to trigger release, via alterations in Ca²⁺binding activity and potentially through the impact of LLPS on membrane curvature during fusion reactions. In summary, the juxtamembrane linker of syt1 emerges as a regulator of syt1 function by driving self-association via LLPS. 

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4. Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition

   https://doi.org/10.1111/febs.16301  

   Roy, Sayantani; Boral, Soumendu; Maiti, Snigdha; Kushwaha, Tushar; Basak, Aditya J.; Lee, Woonghee; Basak, Amit; Gholap, Shivajirao L.; Inampudi, Krishna K.; De, Soumya (December 2021, The FEBS Journal)

   Human RNA‐binding motif 3 protein (RBM3) is a cold‐shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N‐terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a βαββαβ topology, which is found in many RNA‐binding proteins. NMR‐monitored titration experiments and molecular dynamic simulations show that the beta‐sheet and two loops form the RNA‐binding interface. Hydrogen bond, pi–pi and pi–cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross‐linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold‐shock protein. Temperature‐dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature‐dependent oligomerization. 

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5. Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates

   https://doi.org/10.1093/nar/gkac1179  

   Morse, Michael; Sefcikova, Jana; Rouzina, Ioulia; Beuning, Penny J.; Williams, Mark C. (December 2022, Nucleic Acids Research)

   Abstract The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion. 

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