RESUMO
Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.
Assuntos
Fatores de Processamento de RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Domínio Catalítico , Sequência Conservada , Éxons , Humanos , Íntrons , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Spliceossomos/ultraestruturaRESUMO
Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5'-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.
Assuntos
Precursores de RNA/metabolismo , Spliceossomos/química , Spliceossomos/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , RNA Helicases DEAD-box/metabolismo , Éxons , Humanos , Íntrons , Modelos Moleculares , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/química , Spliceossomos/metabolismoRESUMO
Removal of an intron from a pre-mRNA by the spliceosome results in the ligation of two exons in the post-catalytic spliceosome (known as the P complex). Here, we present a cryo-EM structure of the P complex from Saccharomyces cerevisiae at an average resolution of 3.6 Å. The ligated exon is held in the active site through RNA-RNA contacts. Three bases at the 3' end of the 5' exon remain anchored to loop I of U5 small nuclear RNA, and the conserved AG nucleotides of the 3'-splice site (3'SS) are specifically recognized by the invariant adenine of the branch point sequence, the guanine base at the 5' end of the 5'SS, and an adenine base of U6 snRNA. The 3'SS is stabilized through an interaction with the 1585-loop of Prp8. The P complex structure provides a view on splice junction formation critical for understanding the complete splicing cycle.
Assuntos
Saccharomyces cerevisiae/química , Spliceossomos/química , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Splicing de RNA , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismoRESUMO
Pre-mRNA splicing involves two sequential reactions: branching and exon ligation. The C complex after branching undergoes remodeling to become the C∗ complex, which executes exon ligation. Here, we report cryo-EM structures of two intermediate human spliceosomal complexes, pre-C∗-I and pre-C∗-II, both at 3.6 Å. In both structures, the 3' splice site is already docked into the active site, the ensuing 3' exon sequences are anchored on PRP8, and the step II factor FAM192A contacts the duplex between U2 snRNA and the branch site. In the transition of pre-C∗-I to pre-C∗-II, the step II factors Cactin, FAM32A, PRKRIP1, and SLU7 are recruited. Notably, the RNA helicase PRP22 is positioned quite differently in the pre-C∗-I, pre-C∗-II, and C∗ complexes, suggesting a role in 3' exon binding and proofreading. Together with information on human C and C∗ complexes, our studies recapitulate a molecular choreography of the C-to-C∗ transition, revealing mechanistic insights into exon ligation.