Journal: Genes Dev / Year: 2022 Title: Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6. Authors: Moritz Schmidt / Florian Kluge / Felix Sandmeir / Uwe Kühn / Peter Schäfer / Christian Tüting / Christian Ihling / Elena Conti / Elmar Wahle / Abstract: The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By ...The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.
History
Deposition
Jan 25, 2022
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Header (metadata) release
Mar 2, 2022
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Map release
Mar 2, 2022
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Update
Mar 2, 2022
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Current status
Mar 2, 2022
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7462 / Average exposure time: 9.5 sec. / Average electron dose: 63.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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