Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6. 2022

Moritz Schmidt, and Florian Kluge, and Felix Sandmeir, and Uwe Kühn, and Peter Schäfer, and Christian Tüting, and Christian Ihling, and Elena Conti, and Elmar Wahle
Institute of Biochemistry and Biotechnology, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany.

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.

UI MeSH Term Description Entries
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D012322 RNA Precursors RNA transcripts of the DNA that are in some unfinished stage of post-transcriptional processing (RNA PROCESSING, POST-TRANSCRIPTIONAL) required for function. RNA precursors may undergo several steps of RNA SPLICING during which the phosphodiester bonds at exon-intron boundaries are cleaved and the introns are excised. Consequently a new bond is formed between the ends of the exons. Resulting mature RNAs can then be used; for example, mature mRNA (RNA, MESSENGER) is used as a template for protein production. Precursor RNA,Primary RNA Transcript,RNA, Messenger, Precursors,RNA, Ribosomal, Precursors,RNA, Small Nuclear, Precursors,RNA, Transfer, Precursors,Pre-mRNA,Pre-rRNA,Pre-snRNA,Pre-tRNA,Primary Transcript, RNA,RNA Precursor,mRNA Precursor,rRNA Precursor,snRNA Precursor,tRNA Precursor,Pre mRNA,Pre rRNA,Pre snRNA,Pre tRNA,Precursor, RNA,Precursor, mRNA,Precursor, rRNA,Precursor, snRNA,Precursor, tRNA,Precursors, RNA,RNA Primary Transcript,RNA Transcript, Primary,RNA, Precursor,Transcript, Primary RNA,Transcript, RNA Primary
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D026723 Polyadenylation The addition of a tail of polyadenylic acid (POLY A) to the 3' end of mRNA (RNA, MESSENGER). Polyadenylation involves recognizing the processing site signal, (AAUAAA), and cleaving of the mRNA to create a 3' OH terminal end to which poly A polymerase (POLYNUCLEOTIDE ADENYLYLTRANSFERASE) adds 60-200 adenylate residues. The 3' end processing of some messenger RNAs, such as histone mRNA, is carried out by a different process that does not include the addition of poly A as described here. Polyadenylations
D039221 mRNA Cleavage and Polyadenylation Factors Factors that are involved in directing the cleavage and POLYADENYLATION of the of MESSENGER RNA near the site of the RNA 3' POLYADENYLATION SIGNALS. Cleavage and Polyadenylation Factors,Polyadenylation Factors,Cleavage and Polyadenylation Factor,Polyadenylation Factor,Pre-mRNA Polyadenylation Factor,Polyadenylation Factor, Pre-mRNA,Pre mRNA Polyadenylation Factor
D039223 Cleavage And Polyadenylation Specificity Factor An RNA-binding protein that recognizes the AAUAAA RNA SEQUENCE at the 3' end of MRNA. It contains four subunits of 30, 73, 100 and 160 kDa molecular size and combines with CLEAVAGE STIMULATION FACTOR to form a stable complex with mRNA that directs the 3' cleavage and polyadenylation reaction. CPSF 30K,CPSF Protein,CPSF-100-kDa Subunit,CPSF-160-kDa Subunit,CPSF-30-kDa Subunit,CPSF-73-kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 100-kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 160-kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 30-kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 73-kDa Subunit,Cleavage-Polyadenylation Specificity Factor,CPSF 100 kDa Subunit,CPSF 160 kDa Subunit,CPSF 30 kDa Subunit,CPSF 73 kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 100 kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 160 kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 30 kDa Subunit,Cleavage And Polyadenylation Specificity Factor, 73 kDa Subunit,Cleavage Polyadenylation Specificity Factor,Subunit, CPSF-100-kDa,Subunit, CPSF-160-kDa,Subunit, CPSF-30-kDa,Subunit, CPSF-73-kDa

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