Prespliceosome structure provides insights into spliceosome assembly and regulation

Abstract

The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors. For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome, which is a focal point for regulation by alternative splicing factors. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions, however, the structural basis of the early events in spliceosome assembly remains poorly understood. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5' splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5' splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

“…During the RNA rearrangements associated with the first catalytic step of splicing, U2 snRNA is remodeled to disrupt stem IIa and form stem IIc (Hilliker et al 2007;Perriman and Ares 2007;Galej et al 2016;Rauhut et al 2016;Wan et al 2016;Yan et al 2016;Bai et al 2017;Liu et al 2017;Plaschka et al 2017;Wan et al 2017;Wilkinson et al 2017;Yan et al 2017;Plaschka et al 2018). However, before it can be reused in a subsequent round of splicing U2 snRNA must be refolded into the stem IIa form, which makes critical contacts with SF3a and SF3b proteins for pre-spliceosome assembly (Liu et al 2017;Wilkinson et al 2017;Plaschka et al 2018).…”

“…These changes lead to release of some proteins and recruitment of others to the spliceosome, ensuring the fidelity and directionality of spliceosome assembly and catalysis (Koodathingal and Staley 2013). Recent cryo-electron microscopy (cryo-EM) models of spliceosomes at distinct steps (tri-snRNP, the A, B, B act , C, and P complexes) have brilliantly illuminated the composition and conformation of the spliceosome before and after each transition Yan et al 2015;Galej et al 2016;Rauhut et al 2016;Wan et al 2016;Yan et al 2016;Bai et al 2017;Bertram et al 2017a;Bertram et al 2017b;Liu et al 2017;Plaschka et al 2017;Wan et al 2017;Wilkinson et al 2017;Yan et al 2017;Zhang et al 2017;Plaschka et al 2018;Zhan et al 2018b;Zhan et al 2018a;Zhang et al 2018). A challenge now is to understand the distinct mechanism of action of each DExD/H protein in catalyzing its specific transition.…”

“…During this step, Msl5/SF1 and Mud2/U2AF are removed from the BP as it forms an extended duplex with U2 snRNA that bulges out a conserved adenosine for attack on the 5' splice site. This duplex is packed into a crevice between Hsh155/SF3b1 and Rds3/PHF5A, where the bulged nucleotide occupies a pocket formed by both proteins in the A-complex (Plaschka et al 2018). This ATP-requiring step appears to be mediated by the Prp5 protein, a DEAD-box family member with an RNA-stimulated ATPase activity important for its function in splicing (Ruby et al 1993;O'Day et al 1996;Abu Dayyeh et al 2002;Perriman et al 2003;Xu et al 2004).…”

Transcriptional analysis supports the expression of human snRNA variants and reveals U2 snRNA homeostasis by an abundant U2 variant

“…These structures reveal massive architectural dynamics of U2 snRNP, whereas U5 and U6 snRNPs remain relatively static through spliceosomal activation, catalysis, and disassembly (Kastner et al, 2019;Plaschka et al, 2019;Yan et al, 2019). For example, when the U2 snRNA GUAGUA motif base pairs to the intron branch site early in spliceosome assembly, it forms the U2:branchsite (U2:BS) duplex, which is protected by the HEAT domains of SF3B1 (Plaschka et al, 2018). This interaction sequesters the hydroxyl group of the bulged adenosine, the first-step nucleophile, from the 5'SS and prevents formation of an active catalytic core.…”

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

“…Understanding how RNPs assemble and function, involving both one RNA-one protein interactions and multi-component interaction networks, is thus critical for characterizing many biological processes and mechanisms. To date, biochemical approaches have defined in detail protein interactions required for a number of RNP assemblies 3 , and high-resolution structural approaches [4][5][6] have revolutionized our understanding of small and large RNP architectures. Nonetheless, it remains challenging to characterize RNP assemblies and their interacting networks within living systems.…”