RESUMO
Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodelling for catalysis2-6, but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.
Assuntos
Caenorhabditis elegans , Spliceossomos , Animais , Humanos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Microscopia Crioeletrônica , Íntrons/genética , Modelos Moleculares , RNA Helicases/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Nuclear Pequeno/química , Spliceossomos/metabolismo , Spliceossomos/ultraestrutura , Spliceossomos/química , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa 'heart' of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP-CD2BP2-TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome.
