In vitro reconstitution of mammalian pre-mRNA 3' processing
Final Report Abstract
Genetic information is stored in the form of DNA. In a process termed transcription, DNA sequences are selectively copied into RNA to be used for protein synthesis. The RNA molecules ferrying genetic information from the DNA in the cell nucleus to cytoplasmic ribosomes for protein synthesis are called messenger RNAs (mRNAs). The RNA is initially made as a 1:1 copy of a DNA sequence. However, in eukaryotes this so-called primary transcript is only the precursor to mRNA, being modified in a complex series of processing steps before it is used for protein synthesis. One aspect of these processing reactions is the so-called 3‘ end processing, the modification of the end of the linear RNA molecule synthesized last. Copying of the DNA proceeds past the sequence present in the mature mRNA, i. e. the precursor RNA is too long. In the first step of 3‘ end processing, the excess sequence is cleaved off: An endonuclease cuts the RNA into two fragments, of which the ‚downstream‘ one, i. e. the one closer to the 3‘ end, is degraded, whereas the ‚upstream‘ one, i. e. the one closer to the start of transcription, will be the mature mRNA, containing the protein-coding sequences. In the second step of 3‘ end processing, the upstream fragment is elongated by a poly(A) tail, a sequence consisting of about 250 A residues. The poly(A) tail is important for mRNA function in protein synthesis and the control of mRNA half-life. We have reconstituted the 3‘ end processing reaction, cleavage and polyadenylation, from proteins overproduced in insect cells or bacteria and purified. Fourteen polypeptides were essential for the reaction: A protein complex called mammalian polyadenylation specificity factor (mPSF) contains four subunits, two of which bind the central polyadenylation signal in the RNA, AAUAAA. Poly(A) polymerase (PAP) is the enzyme synthesizing the poly(A) tail, using ATP as a precursor. PAP, a single polypeptide, associates with mPSF and thus prefers AAUAAA-containing RNAs as substrates. Mammalian cleavage factor (mCF) forms a complex with mPSF and consists of three subunits, of which CPSF73 is the endonuclease that cleaves the precursor RNA. Two additional proteins play roles in the selection of the processing site in the RNA precursor: cleavage stimulation factor (CstF; three types of subunits) and cleavage factor II (CFII; two subunits). We found that another single polypeptide, RBBP6, associates with the endonuclease CPSF73 close to its active site and likely activates the nuclease. Cleavage factor I (CFI), previously thought to be essential, turned out to be merely stimulatory. This is consistent with the protein’s role in alternative polyadenylation, i. e. the selection of particular processing sites among several or many present in one precursor RNA. ATP is essential for RNA cleavage through binding to one of the CFII subunits. Our results should pave the way for further mechanistic and structural investigations of the 3‘ end processing reaction.
Publications
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Reconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6. Genes & Development, 36(3-4), 195-209.
Schmidt, Moritz; Kluge, Florian; Sandmeir, Felix; Kühn, Uwe; Schäfer, Peter; Tüting, Christian; Ihling, Christian; Conti, Elena & Wahle, Elmar
