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RNAs in the spliceosome: Insight from cryoEM structures

Advanced Review

Lingdi Zhang, Anne Vielle, Sara Espinosa, Rui Zhao

Published Online: Feb 06 2019

DOI: 10.1002/wrna.1523

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Pre‐mRNA splicing is catalyzed by the spliceosome, a multimegadalton RNA–protein complex. The spliceosome undergoes dramatic compositional and conformational changes through the splicing cycle, forming at least 10 distinct complexes. Recent high‐resolution cryoEM structures of various spliceosomal complexes revealed unprecedented details of this large molecular machine. This review highlights insight into the structure and function of the spliceosomal RNA components obtained from these new structures, with a focus on the yeast spliceosome. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

A schematic representation of the Saccharomyces cerevisiae splicing cycle

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RNA interactions in splice site recognition. (a) The 5′ ss region is first recognized through basepairing with the 5′ end of U1 snRNA as observed in the A complex structure (PDB ID 6G90). (b) The 5′ ss region is subsequently basepaired with U6 snRNA after spliceosomal activation in the B^act complex (PDB ID 5GM6). (c) The branch point sequence is recognized through basepairing with U2 snRNA as observed in the A complex (PDB ID 6G90). (d) The 3′ ss is recognized mainly through non‐Watson–Crick basepairing between the last two nucleotides of the intron (nucleotides AG) with the 5′ ss (nucleotide G) and BP (nucleotide A) as observed in the P complex (PDB ID 6BK8). Black dashed lines indicate hydrogen bonds. (e) Base substitution at the last nucleotide G in the intron with A, C, and U result in partial or complete loss of hydrogen bonds with the 5′ ss. (f) Base substitution of the second to last nucleotide A in the intron with C, G, and U result in the loss of hydrogen bonds with the branch point A

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A stem‐like secondary structure is observed in the intronic region between the branch point sequence (BPS) and 3′ ss in the P complex structure (PDB ID 6BK8), which may bring the 3′ ss close to the 5′ ss and BPS to facilitate the recognition of the 3′ ss before the ligation reaction

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Pre‐mRNA changes its conformation dramatically through the splicing cycle. Different regions of the pre‐mRNA are shown in the color code depicted in the schematic representation of the pre‐mRNA. snRNAs are shown in gray. The same PDBs as in Figure were used to generate this figure

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The snRNA core (in the black circle) around the active site (U2/U6 helix 1 and 2, the U2 snRNA region that basepairs with the BPS, U6 ISL and ACAGAGA box, and U5 snRNP loop 1) remain largely invariant from the B^act to the ILS complex. PDB IDs for the B^act, C, C*, P, and ILS complex structures used to generate this figure are 5GM6, 5GMK, 5MQ0, 6BK8, and 5Y88, respectively

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Multiple proteins including Prp8 and Snu114 from U5 snRNP and proteins of the NTC and NTR complexes form a protein core around the active site indicated by the red circle. This protein core is fairly stable from the B^act to the ILS complex (the structure of the P complex with PDB ID 6BK8 is shown here as an example)

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CryoEM structures demonstrate that the splicing reaction is catalyzed by RNA with the help of two Mg²⁺ ions. (a) The left panel is a model of the spliceosome (presumably the B* complex) immediately before the branching reaction, generated based on the structure of the C complex. The model illustrates that the M2 ion can potentially activate the 2′ OH of branch point (BP) A, which will attack the 5′ ss, free the 5′ exon and generate a lariat intermediate. Bases at the −1 and +1 position from the BP on the 5′ intron are not shown to avoid overcrowding of the figure. The right panel shows structures of RNAs around the active site in the C complex (PDB ID 5GMK). (b) The left panel is a model of the spliceosome immediately before the ligation reaction, generated based on the structure of the P complex. The model illustrates that the M1 ion can potentially activate the 3′ OH of 5′ exon, which will attack the 3′ ss and ligate the two exons. The right panel shows structures of RNAs around the active site in the P complex (PDB ID 6BK8)

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