Biology subjects

Pulvino, M. J.

Publications and source records attributed to Pulvino, M. J..

3 recordsLinked to original sources

bioRxiv2022

A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

biochemistry↗

bioRxiv2021

Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

biophysics↗

bioRxiv2020

A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates in cells and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges an active conformation of the U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing inactive checkpoints may offer a breakthrough approach for small molecule inhibition of multi-stage macromolecular assemblies.

biochemistry↗