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The nuts and bolts of the endogenous spliceosome

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The complex life of pre‐mRNA from transcription to the production of mRNA that can be exported from the nucleus to the cytoplasm to encode for proteins entails intricate coordination and regulation of a network of processing events. Coordination is required between transcription and splicing and between several processing events including 5′ and 3′ end processing, splicing, alternative splicing and editing that are major contributors to the diversity of the human proteome, and occur within a huge and dynamic macromolecular machine—the endogenous spliceosome. Detailed mechanistic insight of the splicing reaction was gained from studies of the in vitro spliceosome assembled on a single intron. Because most pre‐mRNAs are multiintronic that undergo alternative splicing, the in vivo splicing machine requires additional elements to those of the in vitro machine, to account for all these diverse functions. Information about the endogenous spliceosome is emerging from imaging studies in intact and live cells that support the cotranscriptional commitment to splicing model and provide information about splicing kinetics in vivo. Another source comes from studies of the in vivo assembled spliceosome, isolated from cell nuclei under native conditions—the supraspliceosome—that individually package pre‐mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Recent years have portrayed new players affecting alternative splicing and novel connections between splicing, transcription and chromatin. The challenge ahead is to elucidate the structure and function of the endogenous spliceosome and decipher the regulation and coordination of its network of processing activities. WIREs RNA 2017, 8:e1377. doi: 10.1002/wrna.1377 This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Processing > Splicing Regulation/Alternative Splicing
Nuclear pre‐mRNA processing of Pol II transcripts.
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A dual role for SNORD27 in rRNA and pre‐mRNA processing. (a) Regions of complementarity between SNORD27 and its pre‐mRNA and rRNA targets. The C (RUGAUGA), D(CUGA), and antisense boxes are indicated (AS1 and AS2). Lines indicate the sequence complementarity toward regulated pre‐mRNAs and 18S rRNA. (b) Working model for SNORD27 acting on pre‐mRNA regulation. Left: SNORD27 can associate with NOP56, NOP58, NHP2L1, and fibrillarin to form a canonical snoRNP that performs 2'‐O‐methylation on ribosomal RNA. Right: Part of the snoRNA is found in supraspliceosomes (lacking fibrillarin) and regulate pre‐mRNA splicing, where it represses exons, possibly by competing with activating splicing factors, such as U1 snRNA, or other splicing factors (SF). (Reprinted with permission from Ref . Copyright 2016)
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Competition between intronic miRNA expression and alternative splicing. A conserved novel alternative 3′SS was identified in intron 13 of MCM7 within the miR 106b‐25 cluster, between pre‐miR‐93 and 25. A diagram of MCM7 exon 13 through exon 14, with constitutive splicing at the normal 3′SS (full line, and lower left), generating MCM7 mRNA and pre‐miR106b, pre‐miR‐93, and pre‐miR‐25. Upon alternative splicing at the novel 93–25 3'SS (broken line and lower right) alternatively spliced isoform of MCM7 and pre‐miR‐106b and pre‐miR‐93 are generated.
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Alternative splicing within the supraspliceosome—a putative model. Schematic models of the supraspliceosome, in which the pre‐mRNA (introns in blue, exons in red) is connecting four native spliceosomes. Overexpression of SRSF5 causes looping out of exon 7 (Ex 7), the alternative exon of hnRNP A/B, together with its flanking introns (depicted in the lower right corner), and thus facilitates skipping of exon 7. Treatment of cells with C6‐ceramide enhances inclusion of exon 7 and looping out of introns 6 and 7 (Int 6 and Int 7, respectively). (Reprinted with permission from Ref . Coyright 2012 Elsevier)
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The supraspliceosome model. (a) A gallery of EM images of supraspliceosomes each composed of four native spliceosomes. Bar represents 20 nm. (Reprinted with permission from Ref Elsevier). (b, d) STEM dark field images of supraspliceosomes stained with a protocol that allows visualization of nucleic acids. RNA strands and loops bound with proteins are seen emanating from the supraspliceosomes. (Reprinted with permission from Ref . Copyright 1998 Elsevier). (c) A class average of supraspliceosomes in which the contacts between neighboring small subunits, which lie in the center of the particle, form a right‐angled cross that reflects a fourfold arrangement. (Reprinted with permission from Ref . Copyright 2007). (e, f) Schematic models of the supraspliceosome in which the pre‐mRNA (introns in blue, exons in red) is connecting four native spliceosomes. The supraspliceosome presents a platform onto which the exons can be aligned and splice junctions can be checked before splicing occurs. (e) The pre‐mRNA that is not being processed is folded and protected within the cavities of the native spliceosome. (f) Under the conditions presented in (b, d) the RNA kept in the cavity is proposed to unfold and loop‐out. In the looped‐out scheme, an alternative exon is depicted in the upper left corner. (Reprinted with permission from Ref . Copyright 2006 Elsevier)
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Mechanism of RNA splicing and step‐wise assembly of spliceosome in vitro. (a) Mechanism of pre‐mRNA splicing. First step: The 2′ hydroxyl of a specific adenosine base at the branch site of a pre‐mRNA becomes nucleophilic and makes a nucleophilic substitution type 2 (SN2) attack on the phosphodiester moiety at the 5′SS (marked in red). Two intermediates are formed: the 5′ exon in a free form (red) and the 3′ exon‐intron lariat. Second step: In a second SN2 type reaction, the free 3′ hydroxyl anion of the 5′ exon attacks the phosphodiester moiety at the 3′SS (marked in blue) to yield the spliced RNA and the spliced‐out intron in a lariat form. (b) Schematic presentation of the stepwise assembly of the spliceosome in vitro. When pre‐mRNA is added to a nuclear extract of cells (color code as in panel (a)) complex E is formed by the binding of U1 snRNP (green) to the 5′SS of the pre‐mRNA. Complex A is then formed by an ATP‐dependent interaction of U2 snRNP with the branch site. Addition of the tri‐snRNP gives rise to complex B. This step is followed by major rearrangements in RNA:RNA and RNA:protein interactions, destabilization of U1 and U4 snRNPs and formation of activated complex Bact. This is followed by generation of complex B*, which catalyszes the first step of splicing, generating complex C, which catalyzes the second step of splicing. For simplification, the scheme focuses on the U snRNPs.
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Structure of the native spliceosome. (a, b) Two different views of the structure of the native spliceosome reconstructed at 20‐Å resolution from cryo‐images. (c) The high‐density mass region of the native spliceosome (blue) represents the stable RNAs within the structure of the native spliceosome. The large subunit of the native spliceosome is thus a suitable candidate to harbor the five‐spliceosomal U snRNPs. (Reprinted with permission from Ref . Copyright 2004 Cell Press; Published by Elsevier) (d) A unique spatial arrangement of the U snRNPs within the native spliceosome emerges from in silico studies. The native spliceosome is transparent; U4/U6.U5 tri‐snRNP is colored by functional regions, with U5 snRNP in pink and the region attributed to loop I in black; U4/U6 in beige‐orange; U2 snRNP subcomponent SF3b is in green; and U1 snRNP is blue. (Reprinted with permission from Ref . Copyright 2012 Elsevier)
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The catalytic center and the active site of the spliceosome. (a) Base–pairing interactions among U2 snRNA, U6 snRNA, and the intron lariat‐3′‐exon. Base‐pairing interactions in the RNA triplex, between A41‐G40, U68 and A47‐G48‐C49, are identified by solid lines. (b) An overall cartoon representation of the catalytic center of the spliceosome, three perpendicular views. The intron lariat, Helix II of the U2/U6 duplex, and Stem I of U5 snRNA are included to indicate the relative orientation. (c) A close‐up view on the catalytic center. Helix I of the U2/U6 duplex is placed to the left of the two catalytic Mg2+ ions, and the ISL of U6 snRNA is close to Loop I of U5 snRNA. (d) A close‐up view on the active site. Among the two catalytic Mg2+ ions, the first (M1) is coordinated by phosphate groups from U68, G66, and G48 of U6 snRNA, and the second (M2) is bound by phosphates from U68, G48, and A47. Notably, A47 and G48 are part of the RNA triplex. (e) Both the catalytic center and U5 snRNA are anchored on Spp42 (Prp8 in Saccharomyces cerevisiae). Two perpendicular views are shown. The catalytic center is placed into a positively charged, catalytic cavity on Spp42. Interactions between the active site and Loop I of U5 snRNA are mediated in a channel below the surface (left panel). This may allow retention of the 5′‐exon after the first‐step reaction. (Reprinted with permission from Ref . Copyright 2015 American Association for the Advancement of Science)
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Structure of splicing intermediates assembled in vitro and splicing complexes isolated from cells’ nuclei. (a) Structure of in vitro assembled complex B∆U1 reconstructed at 40‐Å resolution from cryo‐negatively stained images. (b) Structure of S. pombe U5.U2/U6 splicing complex, reconstructed at 29‐Å resolution from cryo‐images. (c) Structure of in vitro assembled complex C, reconstructed at 30‐Å resolution from cryo‐negatively stained images. (d) Structure of the native spliceosome, reconstructed at 20‐Å resolution from cryo‐images. (e) Structure of S. pombe splicing complex U5.U2/U6, reconstructed at 3.6‐Å resolution from cryo‐images. Bar represents 10 nm. 3D images of the above splicing complexes are presented in Figure S1.
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RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
RNA Processing > Splicing Regulation/Alternative Splicing

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