During exon ligation, the
spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a ...conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.
U1 snRNP binds to the 5' exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, ...which together reveal at atomic resolution an almost complete network of protein-protein and RNA-protein interactions within U1 snRNP, and show how the 5' splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5'-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5'-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5'-splice sites.
The active centre of the spliceosome consists of an intricate network formed by U5, U2 and U6 small nuclear RNAs, and a pre-messenger-RNA substrate. Prp8, a component of the U5 small nuclear ...ribonucleoprotein particle, crosslinks extensively with this RNA catalytic core. Here we present the crystal structure of yeast Prp8 (residues 885-2413) in complex with Aar2, a U5 small nuclear ribonucleoprotein particle assembly factor. The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease. Suppressors of splice-site mutations, and an intron branch-point crosslink, map to a large cavity formed by the reverse transcriptase thumb, and the endonuclease-like and RNaseH-like domains. This cavity is large enough to accommodate the catalytic core of group II intron RNA. The structure provides crucial insights into the architecture of the spliceosome active site, and reinforces the notion that nuclear pre-mRNA splicing and group II intron splicing have a common origin.
Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg
site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, ...we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.
U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to ...an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.
Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal signal peptide, which is ...recognized by the signal recognition particle (SRP) when nascent polypeptide chains emerge from the ribosome. The SRP-ribosome nascent chain complex is then targeted through its GTP-dependent interaction with SRP receptor to the protein-conducting channel on endoplasmic reticulum membrane in eukaryotes or plasma membrane in bacteria. A universally conserved component of SRP (refs 1, 2), SRP54 or its bacterial homologue, fifty-four homologue (Ffh), binds the signal peptides, which have a highly divergent sequence divisible into a positively charged n-region, an h-region commonly containing 8-20 hydrophobic residues and a polar c-region. No structure has been reported that exemplifies SRP54 binding of any signal sequence. Here we have produced a fusion protein between Sulfolobus solfataricus SRP54 (Ffh) and a signal peptide connected via a flexible linker. This fusion protein oligomerizes in solution through interaction between the SRP54 and signal peptide moieties belonging to different chains, and it is functional, as demonstrated by its ability to bind SRP RNA and SRP receptor FtsY. We present the crystal structure at 3.5 A resolution of an SRP54-signal peptide complex in the dimer, which reveals how a signal sequence is recognized by SRP54.
Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5' splice site within precursor messenger RNAs and ...initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 A resolution has enabled us to build the RNA and, in conjunction with site-specific labelling of individual proteins, to place the seven Sm proteins, U1-C and U1-70K into the map. Here we present the detailed structure of a spliceosomal snRNP, revealing a hierarchical network of intricate interactions between subunits. A striking feature is the amino (N)-terminal polypeptide of U1-70K, which extends over a distance of 180 A from its RNA binding domain, wraps around the core domain consisting of the seven Sm proteins and finally contacts U1-C, which is crucial for 5'-splice-site recognition. The structure of U1 snRNP provides insights into U1 snRNP assembly and suggests a possible mechanism of 5'-splice-site recognition.
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