In vitro reconstitution of mammalian pre-mRNA 3' processing

真核生物mRNA的3'末端是由一个大的蛋白质复合物催化的两步加工反应产生的：延伸的前体RNA被核酸内切酶切割，然后含有编码序列的上游切割产物被poly（A）尾延伸，而下游片段被降解。这两个步骤在体外是可分离的。在第一个资助期重建了第二步，聚腺苷酸化，我们现在也成功地重建了重组蛋白的切割反应：16个多肽，组成5个异源寡聚复合物加上2个单独的蛋白质。我们还获得了裂解反应涉及液-液相分离（LLPS）的初步数据：该反应取决于相对高的蛋白质浓度和拥挤试剂的添加，这产生了高总浓度的大分子。在这些条件下，加工因子和底物RNA似乎凝结成一个单独的液相，在其中发生切割反应。这一声明得到了初步显微镜分析和我们通过在适度g力下短暂离心沉淀切割复合物（包括底物和产物RNA）的能力的支持。在拟议的项目中，我们希望追求以下目标：首先，我们将研究切割反应的某些方面，包括一个不好表征的切割因子Rbbp 6的作用; ATP在切割反应中的功能;和5'外切核酸酶XRN 2对下游切割片段的降解。其次，我们将研究我们的初步证据的作用，LLPS的裂解反应。例如，我们将询问哪些缓冲条件（拥挤）、哪些多肽和切割因子的哪些结构域驱动LLPS，以及切割是否总是与LLPS相关。我们将尝试提出一个假设，为什么LLPS是必要的裂解反应。我们将研究多聚腺苷酸化之前或之后的裂解产物是否从相分离的液滴中释放出来，以及多聚腺苷酸化是否也受到LLPS的青睐。第三，我们将分析底物RNA的识别方面。我们将首先集中在一个假定的作用Rbbp 6在RNA结合，包括额外的多肽，如果时间允许。第四，我们的目标是从结构上深入了解加工复合体。在合作中，我们将分析，通过质谱，内和分子间交联的裂解因子的背景下的裂解复合物，我们将有助于通过冷冻EM的结构分析。

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.