In vitro reconstitution of mammalian pre-mRNA 3' processing

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The 3‘ ends of eukaryotic mRNAs are generated in a two-step processing reaction catalyzed by a large protein complex: Extended precursor RNAs are cleaved by an endonuclease, then the upstream cleavage product, containing the coding sequence, is extended by a poly(A) tail, whereas the downstream fragment is degraded. The two steps are separable in vitro. Having reconstituted the second step, polyadenylation, in the first funding period, we have now also succeeded in reconstituting the cleavage reaction from recombinant proteins: sixteen polypeptides, organized as five heterooligomeric complexes plus two individual proteins. We have also obtained preliminary data that the cleavage reaction involves liquid-liquid phase separation (LLPS): The reaction depends on relatively high protein concentrations and the addition of crowding reagents, which generate a high total concentration of macromolecules. Under these conditions, processing factors and substrate RNA seem to condense into a separate liquid phase in which the cleavage reaction takes place. This claim is supported by preliminary microscopic analysis and by our ability to pellet the cleavage complex, including substrate and product RNAs, by a short centrifugation at modest g force. In the proposed project, we wish to pursue the following goals: First, we will investigate certain aspects of the cleavage reaction, including the role of a poorly characterized cleavage factor, Rbbp6; the function of ATP in the cleavage reaction; and the degradation of the downstream cleavage fragment by the 5’ exonuclease XRN2. Second, we will examine our preliminary evidence for a role of LLPS in the cleavage reaction. For example, we will ask which buffer conditions (crowders), which polypeptides and which domains of the cleavage factors drive LLPS, and if cleavage is always tied to LLPS. We will try to develop a hypothesis why LLPS is necessary for the cleavage reaction. We will investigate whether cleavage products before or after polyadenylation are released from phase-separated liquid droplets, and whether polyadenylation is also favored by LLPS. Third, we will analyze aspects of the recognition of the substrate RNA. We will initially concentrate on a putative role of Rbbp6 in RNA binding and include additional polypeptides if time permits. Fourth, we aim to gain structural insight into the processing complex. In collaborations, we will analyze, by mass spectrometry, intra- and intermolecular cross-links of cleavage factors in the context of the cleavage complex, and we will contribute to a structural analysis by cryo-EM.