Open AccessReconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6
Author(s) -
Moritz Schmidt,
Florian Kluge,
Felix Sandmeir,
U. Kühn,
Peter Schäfer,
Christian Tüting,
Christian Ihling,
Elena Conti,
Elmar Wahle
Publication year - 2022
Publication title -
genes and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.136
H-Index - 438
eISSN - 1549-5477
pISSN - 0890-9369
DOI - 10.1101/gad.349217.121
Subject(s) - polyadenylation , cleavage and polyadenylation specificity factor , cleavage stimulation factor , biology , cleavage (geology) , cleavage factor , rna , messenger rna , protein subunit , microbiology and biotechnology , post transcriptional modification , rna binding protein , biochemistry , gene , paleontology , fracture (geology)
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.