The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA–protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as subcomplexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.

•Crystal structures of dimeric Smu1 and a hetero-tetrameric Smu1-RED complex•Smu1 dimerizes via a hydrophobic interface involving its LisH motif•An N-terminal region of Smu1 interacts with short, central helices of RED•Smu1-RED complex is structurally similar to transcriptional co-repressor complexesThe proteins Smu1 and RED have been jointly implicated in the regulation of alternative splicing, mitosis, and influenza virus infection, but how they interact and whether their diverse cellular functions are coupled is unknown. We identified an N-terminal region of Smu1 and a central region of RED that stably interact. Structural analyses revealed that the RED-binding region of Smu1 contains an N-terminal LisH motif linked to a core domain and a C-terminal α helix that folds back onto the LisH motif. Smu1 dimerizes via its LisH motif and C-terminal α helix and undergoes global conformational changes upon RED binding. In the ensuing hetero-tetrameric Smu1-RED complex, two molecules of RED use short α helices to bind hydrophobic grooves of two Smu1 core domains. Our results show how Smu1 and RED form a functional module that exhibits intriguing similarities to transcriptional co-repressor complexes, arranging multiple additional protein-protein interaction sites for contacting splicing and/or chromatin factors.Download high-res image (234KB)Download full-size image

•Crystal structures of Prp38-MFAP1 and Snu23-Prp38-MFAP1 complexes are elucidated•MFAP1 and Snu23 bind Prp38 via ER/K motif-stabilized single α helices•The helicity of the unbound state tunes the strength of single α helix interactions•Proteins with multiple single α helix epitopes are abundant in spliceosomesThe spliceosomal B complex-specific protein Prp38 forms a complex with the intrinsically unstructured proteins MFAP1 and Snu23. Our binding and crystal structure analyses show that MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α helices, which have previously been recognized only as rigid connectors or force springs between protein domains. A variant of the Prp38-binding single α helix of MFAP1, in which ER/K motifs not involved in Prp38 binding were mutated, was less α-helical in isolation and showed a reduced Prp38 affinity, with opposing tendencies in interaction enthalpy and entropy. Our results indicate that the strengths of single α helix-based interactions can be tuned by the degree of helix stabilization in the unbound state. MFAP1, Snu23, and several other spliceosomal proteins contain multiple regions that likely form single α helices via which they might tether several binding partners and act as intermittent scaffolds that facilitate remodeling steps during assembly of an active spliceosome.Download high-res image (120KB)Download full-size image