Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Splicing machine shifts into gear Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure called Bact. Previously, little was known about the pathway leading to the U2/U6 active site and how proteins aid in folding the U2/U6 RNA. Using cryo–electron microscopy to determine structures of two human pre-Bact complexes, Townsend et al. uncovered an intricate cascade of coordinated structural changes involving mutually exclusive interactions that facilitate the directionality of the activation process. These structures reveal the assembly pathway of the U2/U6 catalytic RNA and the mechanism whereby proteins facilitate its folding. Science, this issue p. eabc3753 A cryo–electron microscopy study reveals the structure of human pre-Bact spliceosomes. INTRODUCTION The spliceosome, including its catalytic center, is formed anew on each pre-mRNA intron, through a pathway involving multiple, successive assembly intermediates. Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure. As of now, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. RATIONALE To elucidate the complex rearrangements that occur during transformation of a spliceosomal B complex into an activated spliceosome (i.e., Bact complex), we blocked spliceosome assembly at previously uncharacterized, intermediate stages of activation and determined the structure of purified human pre-Bact complexes. RESULTS The cryo–electron microscopy (cryo-EM) structures of two distinct, human pre-Bact complexes (denoted pre-Bact-1 and pre-Bact-2) that lack a mature catalytic U2/U6 RNA structure were obtained at core resolutions of 3.9 and 4.2 Å, and a pseudo-atomic model of each complex was generated using an integrative structural biology approach. Their composition and molecular architecture indicate that pre-Bact-1 is a precursor to pre-Bact-2, and chase experiments demonstrate that they are functional spliceosome intermediates. The pre-Bact-1 and pre-Bact-2 structures, aided by biochemical analyses, provide new insight into the order of protein exchanges during Bact formation. They also elucidate a number of mutually exclusive protein-protein and protein-RNA interactions that ensure a productive pathway of ribonucleoprotein (RNP) rearrangements needed to form the U2/U6 catalytic RNA and reveal new roles for the so-called B-specific proteins. They show that there is a stepwise repositioning of BRR2 and U2 small nuclear RNP during activation, the latter being a prerequisite for bringing U2 and U6 small nuclear RNA (snRNA) into sufficiently close proximity to form U2/U6 helix I. Furthermore, they indicate that the proteins TCERG1, WBP11, CTNNBL1, and KIN17, which interact transiently with the spliceosome, stabilize intermediate RNP conformational states of the pre-Bact complexes. The pre-Bact-1 and pre-Bact-2 cryo-EM structures reveal that a U6 internal stem-loop (ISL) with a distinctive conformation, which is stabilized by WBP11 in pre-Bact-1, and U2/U6 helix Ib are initially formed, followed by U2/U6 helix Ia and the U6 catalytic triplex. Facilitated by several B-specific proteins, the scaffold protein PRP8 retains its open conformation in both pre-Bact complexes, thereby providing sufficient three-dimensional (3D) space for the formation of the U6 ISL and helix Ib, which are docked already to their cognate PRP8 binding sites in the pre-Bact complexes. Other spliceosomal proteins accommodating the U2/U6 network are largely in place in both pre-Bact complexes and are thus poised to aid PRP8 in folding the U2 and U6 snRNAs. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby formation of a catalytically active U2/U6 RNA network is facilitated by spliceosomal proteins, with a conformational change in the scaffold protein PRP8 playing a key role in facilitating its final 3D folding. CONCLUSION The cryo-EM structures of two human pre-Bact complexes presented here reveal an intricate cascade of highly coordinated structural changes during the activation phase of the human spliceosome, involving mutually exclusive interactions that facilitate the directionality of the activation process. In addition, they provide new insights into the strategy used by the spliceosome to assemble its catalytic RNA network and how a conformational rearrangement in PRP8 facilitates the 3D folding of the catalytically active U2/U6 RNA. Protein-guided 3D folding of U2/U6 RNA during spliceosome activation. Structural organization of U2 and U6 snRNA and the proteins cradling them in pre-Bact-2 and Bact spliceosomal complexes. A PRP8 conformational change moves U2/U6 RNA elements docked on PRP8 toward the U6 ISL and also repositions PRP8-docked proteins, restricting space within the now closed PRP8 cavity and thereby aiding the final tertiary folding of the U2/U6 catalytic RNA. Single letters followed by numbers indicate nucleotides. Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo–electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.