The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein.

Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System.

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites.

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes.

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity.

Unearthing SRSF1's Novel Function in Binding and Unfolding of RNA G-Quadruplexes.

SRSF1 governs splicing of over 1,500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity.

Electronegative clusters modulate folding status and RNA binding of unstructured RNA-binding proteins.

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP).

Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions.

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.

Inter-domain Flexibility of Human Ser/Arg-Rich Splicing Factor 1 Allows Variable Spacer Length in Cognate RNA's Bipartite Motifs.

Ser/Arg-rich splicing factor 1 (SRSF1 or ASF/SF2) is the prototypical member of SR proteins. SRSF1 binds to exonic splicing enhancers, which prompts inclusion of corresponding exons in the mature mRNA. The RNA-binding domain of SRSF1 consists of tandem RNA-recognition motifs (RRM1 and RRM2) separated by a 30 amino acid long linker.

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

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.

Amide additives improve RDC measurements in polyacrylamide.

Residual dipolar couplings (RDCs) provide valuable NMR parameters that can be used for structural calculation and verification. Measuring RDCs requires aligning macromolecules using one of various types of alignment media. Of different alignment media options, stretched or compressed polyacrylamide gels are advantageous due to their chemical stability.