Journal: Science / Year: 2022 Title: Structural basis of branch site recognition by the human spliceosome. Authors: Jonas Tholen / Michal Razew / Felix Weis / Wojciech P Galej / Abstract: Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and ...Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17 U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.
Protein or peptide: PHD finger-like domain-containing protein 5A
Complex: BPS oligo
RNA: BPS oligo
Ligand: ZINC ION
Ligand: water
+
Supramolecule #1: A-like U2 snRNP
Supramolecule
Name: A-like U2 snRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10 Details: Spliceosomal complex A-like U2 snRNP generated by binding BPS oligo to 17S U2 snRNP that was isolated from nuclear extract of HEK293F cells by affinity chromatography.
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
TFS KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 15681 / Average exposure time: 1.0 sec. / Average electron dose: 52.03 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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