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Abstract Amyloid‐beta (Aβ) peptides, primarily Aβ40 and Aβ42, are central to the formation of amyloid plaques, a pathological hallmark of Alzheimer's disease (AD). These peptides, derived from the amyloid precursor protein (APP), are aggregation prone and neurotoxic. Experimental studies aimed at understanding Aβ aggregation and interaction require pure, monomeric peptides with the native sequences, including the absence of an N‐terminal methionine. We present an optimized protocol for producing recombinant human Aβ40 and Aβ42 using a SUMO fusion system inEscherichia coli. Cleavage of the SUMO tag enables recovery of native‐sequence peptides, producing physiologically relevant monomers with high yield and purity. This method eliminates the need for chemical synthesis and offers a reliable and cost‐effective approach to producing recombinant Aβ suitable for aggregation studies, structural analyses, and interaction assays. The resulting peptides closely mimic endogenous Aβ, facilitating accurate models of Alzheimer's disease pathogenesis and supporting future therapeutics development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expression and purification of Aβ40 and Aβ42 fromEscherichia coli 

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. 

Abstract PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities.Caenorhabditis elegansPUF proteinfem-3binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5′ end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal. 

Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein’s RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1’s RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles. 

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.