DOCSLIB.ORG

Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle That Correlates with Splicing Complexity Across Diverse Taxa

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle That Correlates with Splicing Complexity Across Diverse Taxa Article Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa E.L. Garside 1, T.A. Whelan 2, M.R. Stark 3, S.D. Rader 3, N.M. Fast 2 and A.M. MacMillan 1 1 - Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7 2 - Biodiversity Research Center and Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 3 - Department of Chemistry, University of Northern British Columbia, Prince George, BC, Canada V2N 4Z9 Correspondence to A.M. MacMillan: Department of Biochemistry, 474 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2H7. [email protected]. https://doi.org/10.1016/j.jmb.2019.04.047 Edited by Anna Pyle Abstract Conformational rearrangements are critical to regulating the assembly and activity of the spliceosome. The spliceosomal protein Prp8 undergoes multiple conformational changes during the course of spliceosome assembly, activation, and catalytic activity. Most of these rearrangements of Prp8 involve the disposition of the C-terminal Jab-MPN and RH domains with respect to the core of Prp8. Here we use x-ray structural analysis to show that a previously characterized and highly conserved β-hairpin structure in the RH domain that acts as a toggle in the spliceosome is absent in Prp8 from the reduced spliceosome of the red alga Cyanidioschyzon merolae. Using comparative sequence analysis, we show that the presence or absence of this hairpin corresponds to the presence or absence of protein partners that interact with this hairpin as observed by x-ray and cryo-EM studies. The presence of the toggle correlates with increasing intron number suggesting a role in the regulation of splicing. © 2019 Published by Elsevier Ltd. Introduction discrete complexes and involves significant struc- tural and conformational rearrangements [2,3]. Eukaryotes contain a split-gene structure in which Recruitment of the U4/U6•U5 tri-snRNP to a coding exon sequences are interrupted by non- complex containing pre-mRNA associated with the coding intron sequences. Intron excision and exon U1 and U2 snRNPs involves the displacement of the ligation take place through two sequential transes- former at the 5′ splice site by the U6 snRNA to yield terifications catalyzed by the spliceosome (reviewed the B complex. Unwinding of a U4/U6 snRNA duplex in Ref. [1]). This large RNA–protein assembly within the B complex, mediated by the Brr2 helicase, consists of the U1, U2, U4/U6, and U5 small nuclear allows formation of a U6 internal stem loop and U2/ ribonucleoprotein particles (snRNPs), each contain- U6 snRNA structure to produce the pre-catalytic ing a unique snRNA and associated proteins, that activated spliceosome referred to as Bact. Together assemble on the pre-mRNA substrate. In the first step these U6 and U2/U6 snRNA structures comprise the of splicing, a conserved adenosine within the intron, active site of the spliceosome that catalyzes the two selected by virtue of base pairing between the U2 splicing transesterifications. Rearrangement leads to snRNA and intron branch sequence, performs a the B* complex, which catalyzes the first step and nucleophilic displacement at the 5′ splice site yielding results in the C complex. Further remodeling yields the the free 5′ exon and lariat intermediate. In the second C* complex where the second step occurs followed by step,attackofthe5′ exon at the 3′ splice site produces a transition to the post-catalytic P complex [4–6]. the ligated exons and lariat intron as products. The highly conserved U5 snRNP protein Prp8 Spliceosome assembly is a multi-step process that (61% identity between Saccharomyces cerevisiae proceeds, following snRNP biogenesis, through and humans [7]) plays key roles in the regulation of 0022-2836/© 2019 Published by Elsevier Ltd. Journal of Molecular Biology (2019) 431, 2543–2553 2544 Prp8 Lacks Toggle in Reduced Spliceosome Fig. 1. X-ray structural analysis of Prp8 RH domain. (A) Ribbon diagram of the human RH domain (PDB 4JK7) showing closed (left) and open (right) conformations. (B) Ribbon diagram of the S. cerevisiae (PDB 3E9O) RH domain. α-Helices and β-strands are colored red and yellow, respectively. spliceosome assembly and transitions through the conformations of the RH domain represent a toggle two catalytic steps while remaining intimately asso- mechanism related to proofreading, where the ciated with the spliceosomal core [8–10].Prp8 closed conformation favors accurate but inefficient contains reverse transcriptase-like, endonuclease- splicing and the open conformation favors inaccu- like, and RNase H-like (RH) domains [11] believed to rate but efficient splicing [22]. This toggle may relate be derived from an ancestral group II intron to different conformations of the U2 snRNA, involv- maturase [12,13] as well as a C-terminal Jab-MPN ing stem IIc (catalytically active) and the alternate, domain that regulates the activity of Brr2 [14–16]. mutually exclusive stem IIa (catalytically inactive; A large number of mutant prp8 alleles related to [23]). Deletion of the 17 amino acids comprising the spliceosome activation and the two transesterifica- RH domain hairpin is lethal in S. cerevisiae [20,22]. tion steps have been characterized in yeast The best-characterized spliceosomes are those (reviewed in Ref. [17]). A clustering of these alleles from humans and budding yeast. Although these led us and others to structurally and functionally spliceosomes are highly conserved, there are characterize the domain of Prp8 corresponding to significant differences between them. In order to the RH fold ([18–20]; Fig. 1A, B). Sequence analysis more fully understand the function of the RH domain was unable to predict the RH fold due to a 17-amino- of Prp8, we have examined it structurally in an acid insertion between the first and second β-strands of organism distantly related to budding yeast and the RH domain. Crystallographically, we observed two humans and also carried out a comparative bioin- conformations of the RH domain: in the closed formatic analysis across diverse eukaryotic taxa. As conformation, the insertion corresponds to an anti- a starting point, we chose Prp8 from the red alga parallel β-hairpin; while in the second, open conforma- Cyanidioschyzon merolae due to evidence of a tion, the hairpin is disrupted to yield a loop translated considerably less complex spliceosome in this ~45° back with respect to the closed structure ([21]; organism compared to either humans or yeast [24]. Fig. 1A). A hydrated magnesium ion observed in the The genome of C. merolae contains only 27 introns second conformation, bound in a site conserved from in a genome with 4803 genes [25]. A computational RNase H, possibly acts to stabilize the open structure. and biochemical analysis revealed the presence of a X-ray structural analyses of Prp8 RH domain greatly reduced spliceosome containing only 40 core mutants affecting either step of splicing, or U4/U6 proteins and completely lacking the U1 snRNA and snRNA unwinding, as characterized in yeast and its associated proteins [24]. Here we report our x-ray yeast splicing extracts, revealed a stabilization or structural analysis of the RH domain of C. merolae destabilization of one of the two RH domain Prp8 (CmPrp8) showing that while the RNase H fold conformations [21]. This transition between two is maintained, the highly conserved β-hairpin is conformations corresponded to favoring the first absent. We have extended this analysis across step of splicing (at the expense of the second) or the diverse taxa showing that the presence or absence second step of splicing (at the expense of the first), of the hairpin corresponds with intron number in as shown in both in vitro and yeast reporter splicing agreement with the observation that among red algal assays, suggesting a switch mechanism to regulate genomes spliceosome complexity is associated with splicing. Recently, it has been proposed that the two a greater number of introns [26]. We also show that Prp8 Lacks Toggle in Reduced Spliceosome 2545 Fig. 2. Curated alignment of Prp8 β-hairpin insert from 25 organisms. The 17-amino-acid insertion is highly conserved (bold, colored by similarity to the human sequence). C. merolae and 2 microsporidia (E. cuniculi, and N. bombycis) lack the insert completely, and G. intestinalis and the kinetoplastid T. cruzi lack a conserved sequence. Two conserved Asp residues are highlighted (bold, black). Taxa are grouped by evolutionary relationships: purple, Obazoa: microsporidia; orange, excavata; black, cryptista; maroon, amoebozoa; green, archaeplastida; yellow, SAR (Stramenopiles, Alveolates, and Rhizaria); blue, Obazoa: animals; red, Obazoa: fungi. Alignment begins at the start of the RH domain. the loss of this hairpin corresponds with the component of the spliceosome, Prp8 is present in predicted absence of factors that associate with the every known intron-containing taxon. Eukaryotic RH hairpin that act at multiple steps of spliceosome diversity separates phylogenetically into major assembly or progression supporting its role as a groups [27], although thorough analyses of the regulatory feature in complex spliceosomes. spliceosome have occurred in relatively few eukary- otic taxa [28]. We analyzed Prp8 sequences from 36 taxa that represent the major groups of eukaryotic diversity discussed in Burki et al. [27]; see Materials Results and Methods; curated selection of taxa in Fig. 2; all analyzed sequences in Supplemental Fig. 1). We Identification of the Prp8 RH domain of C. also increased sampling to represent taxa known to merolae and comparison across taxa have reduced genomes and low intron densities to assess whether the amino acid gap seen in CM is a Protein components of the C. merolae (CM) shared feature of lineages that have undergone splicing machinery were previously identified by spliceosomal reduction. Additional fungal taxa were searching the National Center for Biotechnology also included (see Materials and Methods). Although Information database using sequences of two or all of these exhibited the expected high degree of more species to retrieve the C.
Load more

Recommended publications
  • Mechanism of Regulation of Spliceosome Activation by Brr2 And
    I Mechanism of regulation of spliceosome activation by Brr2 and Prp8 and links to retinal disease Dissertation for the award of the degree “Doctor of Philosophy” (Ph.D.) in the Molecular Biology Program Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Sina Mozaffari-Jovin born in Sabzevar, Iran Göttingen 2012 II Members of the thesis committee: Prof. Dr. Reinhard Lührmann (Reviewer) Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Reinhard Jahn Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Ralf Ficner Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Göttingen Date of submission of Thesis: December, 14th, 2012 III Affidavit I declare that my Ph.D. thesis entitled “Mechanism of regulation of spliceosome activation by Brr2 and Prp8 and links to retinal disease” has been written independently and with no other sources and aids than quoted. Sina Mozaffari-Jovin Göttingen, 2012 IV “The knowledge of anything, since all things have causes, is not acquired or complete unless it is known by its causes” Avicenna (Father of Modern Medicine; c. 980- June 1037) V Table of Contents 1 Abstract ......................................................................................................................... 1 2 Introduction .................................................................................................................. 4 2.1 The chemistry
    [Show full text]
  • Distinct Domains of Splicing Factor Prp8 Mediate Different Aspects of Spliceosome Activation
    Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation Andreas N. Kuhn*, Elizabeth M. Reichl†, and David A. Brow‡ Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706-1532 Communicated by James E. Dahlberg, University of Wisconsin Medical School, Madison, WI, May 21, 2002 (received for review March 25, 2002) Prp8 is the largest and most highly conserved protein in the splicing. Although Prp8 is two-thirds identical between yeast and spliceosome yet its mechanism of function is poorly understood. humans (21), it contains no recognizable structural motifs. To Our previous studies implicate Prp8 in control of spliceosome define domains important for the function of Prp8 in catalytic activation for the first catalytic step of splicing, because substitu- activation for the first transesterification reaction, we isolated tions in five distinct regions (a–e) of Prp8 suppress a cold-sensitive over 40 additional single amino acid substitutions in Prp8 that block to activation caused by a mutation in U4 RNA. Catalytic suppress U4-cs1 (11), here collectively called prp8-cat mutations, activation of the spliceosome is thought to require unwinding of because of their positive effect on catalytic activation. These the U1 RNA͞5؅ splice site and U4͞U6 RNA helices by the Prp28 and mutations cluster in five regions named a–e (see Fig. 4) and are Prp44͞Brr2 DExD͞H-box helicases, respectively. Here we show that distinct from mutations in Prp8 that suppress defects in the mutations in regions a, d, and e of Prp8 exhibit allele-specific second transesterification reaction. genetic interactions with mutations in Prp28, Prp44͞Brr2, and U6 We hypothesized that Prp8 regulates spliceosome activation RNA, respectively.
    [Show full text]
  • The RNA Splicing Response to DNA Damage
    Biomolecules 2015, 5, 2935-2977; doi:10.3390/biom5042935 OPEN ACCESS biomolecules ISSN 2218-273X www.mdpi.com/journal/biomolecules/ Review The RNA Splicing Response to DNA Damage Lulzim Shkreta and Benoit Chabot * Département de Microbiologie et d’Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-819-821-8000 (ext. 75321); Fax: +1-819-820-6831. Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 12 August 2015 / Accepted: 16 October 2015 / Published: 29 October 2015 Abstract: The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.
    [Show full text]
  • Group II Intron Rnps and Reverse Transcriptases: from Retroelements to Research Tools
    Downloaded from http://cshperspectives.cshlp.org/ on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Group II Intron RNPs and Reverse Transcriptases: From Retroelements to Research Tools Marlene Belfort1 and Alan M. Lambowitz2 1Department of Biological Sciences and RNA Institute, University at Albany, State University of New York, Albany, New York 12222 2Institute for Cellular and Molecular Biology and Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712 Correspondence: [email protected]; [email protected] SUMMARY Group II introns, self-splicing retrotransposons, serve as both targets of investigation into their structure, splicing, and retromobility and a source of tools for genome editing and RNA anal- ysis. Here, we describe the first cryo-electron microscopy (cryo-EM) structure determination, at 3.8–4.5 Å, of a group II intron ribozyme complexed with its encoded protein, containing a reverse transcriptase (RT), required for RNA splicing and retromobility. We also describe a method called RIG-seq using a retrotransposon indicator gene for high-throughput integration profiling of group II introns and other retrotransposons. Targetrons, RNA-guided gene targeting agents widely used for bacterial genome engineering, are described next. Finally, we detail thermostable group II intron RTs, which synthesize cDNAs with high accuracy and processiv- ity, for use in various RNA-seq applications and relate their properties to a 3.0-Å crystal structure of the protein poised for reverse transcription. Biological insights from these group II intron revelations are discussed. Outline 1 Introduction 5 Technique 4. RTs with novel enzymatic properties as potent research tools 2 Technique 1.
    [Show full text]
  • Structural Studies of the Spliceosome: Zooming Into the Heart Of
    Available online at www.sciencedirect.com ScienceDirect Structural studies of the spliceosome: zooming into the heart of the machine Wojciech P Galej, Thi Hoang Duong Nguyen, Andrew J Newman and Kiyoshi Nagai Spliceosomes are large, dynamic ribonucleoprotein complexes canonical subunits: U1, U2, U4, U5 and U6 small nuclear that catalyse the removal of introns from messenger RNA ribonucleoprotein particles (snRNPs) and various other precursors via a two-step splicing reaction. The recent crystal non-snRNP factors [2]. The assembly process begins with 0 0 structure of Prp8 has revealed Reverse Transcriptase-like, the recognition of the 5 splice site (5 SS) and the branch Linker and Endonuclease-like domains. The intron branch- point sequence (BP) by U1 and U2 snRNPs, respectively, point cross-link with the Linker domain of Prp8 in active and further recruitment of the U4/U6.U5 tri-snRNP leads 0 0 spliceosomes and together with suppressors of 5 and 3 splice to the formation of the pre-catalytic complex B. A major site mutations this unambiguously locates the active site cavity. remodeling event catalyzed by the two DExD/H box Structural and mechanistic similarities with group II self- helicases Prp28 and Brr2 [3] leads to formation of the splicing introns have encouraged the notion that the catalytically competent complex B*, in which the first spliceosome is at heart a ribozyme, and recently the ligands for step of splicing occurs. two catalytic magnesium ions were identified within U6 snRNA. They position catalytic divalent metal ions in the same way as The enormous complexity and highly dynamic nature of Domain V of group II intron RNA, suggesting that the the spliceosome present a formidable challenge for struc- spliceosome and group II intron use the same catalytic tural and mechanistic studies.
    [Show full text]
  • The Chloroplast Trans-Splicing RNA–Protein Supercomplex from the Green Alga Chlamydomonas Reinhardtii
    cells Review The Chloroplast Trans-Splicing RNA–Protein Supercomplex from the Green Alga Chlamydomonas reinhardtii Ulrich Kück * and Olga Schmitt Allgemeine und Molekulare Botanik, Faculty for Biology and Biotechnology, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-234-32-28951 Abstract: In eukaryotes, RNA trans-splicing is a significant RNA modification process for the end-to- end ligation of exons from separately transcribed primary transcripts to generate mature mRNA. So far, three different categories of RNA trans-splicing have been found in organisms within a diverse range. Here, we review trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. We discuss the origin of intronic sequences and the evolutionary relationship between chloroplast ribonucleoprotein complexes and the nuclear spliceosome. Finally, we focus on the ribonucleoprotein supercomplex involved in trans-splicing of chloroplast group II introns from the green alga Chlamydomonas reinhardtii. This complex has been well characterized genetically and biochemically, resulting in a detailed picture of the chloroplast ribonucleoprotein supercomplex. This information contributes substantially to our understanding of the function of RNA-processing machineries and might provide a blueprint for other splicing complexes involved in trans- as well as cis-splicing of organellar intron RNAs. Keywords: group II intron; trans-splicing; ribonucleoprotein complex; chloroplast; Chlamydomonas reinhardtii Citation: Kück, U.; Schmitt, O. The Chloroplast Trans-Splicing RNA–Protein Supercomplex from the Green Alga Chlamydomonas reinhardtii. 1. Introduction Cells 2021, 10, 290. https://doi.org/ One of the unexpected and outstanding discoveries in 20th century biology was the 10.3390/cells10020290 identification of discontinuous eukaryotic genes [1,2].
    [Show full text]
  • Allele-Specific Genetic Interactions Between Prp8 and RNA Active Site Residues Suggest a Function for Prp8 at the Catalytic Core of the Spliceosome
    Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome Catherine A. Collins1 and Christine Guthrie2,3 1Graduate Group in Biophysics, 2Department of Biochemistry and Biophysics, University of California San Francisco (UCSF), San Francisco, California 94143-0448 USA The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5؅ GU) and 3؅ (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of) .suppressing exon ligation defects due to mutations in position 2 of the 5؅ GU, and all positions of the 3؅ YAG The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5؅ GU. Other mutations in the 5؅ splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5؅ and 3؅ ends of the intron. This model suggests a mechanism for positioning the 3؅ splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. [Key Words: Pre-mRNA splicing; Prp8; U6 snRNA; yeast] Received April 29, 1999; revised version accepted June 24, 1999. The removal of introns from eukaryotic genes to gener- sequences that define the 5ЈSS, the branch-point (BP), ate mRNA is catalyzed by the spliceosome, a complex and the 3Ј splice site (3ЈSS) (MacMillan et al.
    [Show full text]
  • Review Pre-Mrna Splicing and Retinitis Pigmentosa
    Molecular Vision 2006; 12:1259-71 <http://www.molvis.org/molvis/v12/a143/> ©2006 Molecular Vision Received 1 August 2005 | Accepted 14 June 2006 | Published 26 October 2006 Review Pre-mRNA splicing and retinitis pigmentosa Daniel Mordes,1,2,3 Xiaoyan Luo,1,2,3 Amar Kar,1,2,3 David Kuo,1,2,3 Lili Xu,1,2,3 Kazuo Fushimi,1,2,3 Guowu Yu,1,2,3 Paul Sternberg, Jr,4 Jane Y. Wu1,2,3,4 Departments of 1Pediatrics, and 2Cell and Developmental Biology, and 3Pharmacology, John F. Kennedy Center for Research on Human Development; 4Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous retinal diseases and a common cause of blindness. Among the 12 autosomal dominant RP (adRP) genes identified, four encode ubiquitously expressed proteins involved in pre-mRNA splicing, demonstrating the important role that pre-mRNA splicing plays in the pathogenesis of retinal degeneration. This review focuses on recent progress in identifying adRP mutations in genes encoding pre-mRNA splicing factors and the potential underlying molecular mechanisms. A major cause of blindness is retinal degeneration. One proteins essential for pre-mRNA splicing, pre-mRNA process- of the most common forms of retinal degeneration is retinitis ing factors (PRPF), including PRPF31 (also named PRP31; pigmentosa (RP), affecting 1 in 4000 people worldwide and for RP11) [10], PRPF8 (also named PRP8 or PRPC8; for RP13) leaving more than 1.5 million people visually handicapped. [11] and PRPF3 (also named HPRP3; for RP18) [12].
    [Show full text]
  • Viewed In: Nielsen and Johansen, 2009)
    University of Alberta Structural and Functional Studies of the Core Splicing Factor Prp8 by Tao Wu A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemistry ©Tao Wu Spring 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Dedicated to my wife Na Wei, who has suffered a lot and will continue to suffer during my career. i Abstract: More than 90% of human genes undergo a processing step called splicing, whereby noncoding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Splicing involves two sequential transesterification reactions catalyzed by a large RNA/protein complex called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. However, the precise architecture of the spliceosome active site and whether it includes protein components remain unknown. Numerous genetic and biochemical analyses have placed one of the largest and most highly conserved of nuclear proteins, Prp8, at the heart of the catalytic core of the splicing machinery during spliceosome assembly and through catalysis.
    [Show full text]
  • The Prp8 Rnase H-Like Domain Inhibits Brr2-Mediated U4/U6 Snrna Unwinding by Blocking Brr2 Loading Onto the U4 Snrna
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA Sina Mozaffari-Jovin,1 Karine F. Santos,2 He-Hsuan Hsiao,3,5 Cindy L. Will,1 Henning Urlaub,3,4 Markus C. Wahl,2,6 and Reinhard Lu¨ hrmann1,6 1Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Go¨ ttingen, Germany; 2Laboratory of Structural Biochemistry, Freie Universita¨t Berlin, D-14195 Berlin, Germany; 3Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, D-37077 Go¨ ttingen, Germany; 4Bioanalytics, Department of Clinical Chemistry, University Medical Center Go¨ ttingen, D-37075 Go¨ ttingen, Germany The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2’s interaction with the U4 snRNA.
    [Show full text]
  • The Mammalian Analogue of the Yeast PRP8 Splicing Protein Is Present In
    Proc. Nail. Acad. Sci. USA Vol. 86, pp. 8742-8746, November 1989 Biochemistry The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome (RNA processing/pre-mRNA/antibody) ANN L. PINTO AND JOAN A. STEITZ Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06510 Contributed by Joan A. Steitz, September 5, 1989 ABSTRACT HeLa cell nuclear extracts contain a protein essential for spliceosome assembly, but neither appears to be reactive with antibodies against PRP8, a polypeptide essential tightly associated with any snRNP. for pre-mRNA splicing in yeast and a specific component ofthe Immunoprecipitation with antibodies raised against PRP8 yeast U5 small nuclear ribonucleoprotein (snRNP) [Lossky, fusion proteins has shown that PRP8 is associated with the M., Anderson, G. J., Jackson, S. P. & Beggs, J. (1987) Cell 51, yeast U5 snRNP (11). Preincubation ofyeast splicing extracts 1019-10261. The mammalian protein appears as a doublet at with ATP results in enhanced precipitation of U5 and the ==200 kDa, smaller than the 260-kDa yeast protein, and additional precipitation of U4 and U6 snRNAs. Anti-PRP8 possesses an Sm epitope as determined by immunoblotting. Its antibodies inhibit splicing of an actin pre-mRNA in yeast association with a snRNP of the Sm class other than Ul or U2 extracts and also precipitate splicing intermediates (11, 12). is indicated by its immunoprecipitation by anti-Sm and anti- Using antibodies against yeast PRP8, we have identified trimethylguanosine antibodies but not by anti-(U1) or anti-(U2) the mammalian analogue of the 260-kDa yeast PRP8 protein.
    [Show full text]
  • Structural Toggle in the Rnaseh Domain of Prp8 Helps Balance Splicing Fidelity and Catalytic Efficiency
    Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency Megan Mayerlea,1, Madhura Raghavanb,1, Sarah Ledouxa,1, Argenta Pricea,1,2, Nicholas Stepankiwb, Haralambos Hadjivassilioua, Erica A. Moehlea,3, Senén D. Mendozaa, Jeffrey A. Pleissb,4, Christine Guthriea,4, and John Abelsona,4 aDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and bDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853 Contributed by John Abelson, March 22, 2017 (sent for review January 26, 2017; reviewed by Harry F. Noller and Joan A. Steitz) Pre-mRNA splicing is an essential step of eukaryotic gene expression loop forms of RH correlate with distinct genetic phenotypes previously that requires both high efficiency and high fidelity. Prp8 has long proposed to arise from unique first-step and second-step catalytic been considered the “master regulator” of the spliceosome, the mo- conformations of the spliceosome (Fig. 1 and Table S1)(17–20). lecular machine that executes pre-mRNA splicing. Cross-linking and However, recent structural and biochemical studies of the spli- structural studies place the RNaseH domain (RH) of Prp8 near the ceosome and its evolutionary precursor, the group II intron, reveal spliceosome’s catalytic core and demonstrate that prp8 alleles that that the spliceosome’s catalytic core is similar at both catalytic steps map to a 17-aa extension in RH stabilize it in one of two mutually (21, 22). Structural data further suggest the group II intron passes exclusive structures, the biological relevance of which are unknown. through an obligatory intermediate transitional structure between We performed an extensive characterization of prp8 alleles that map two highly similar first- and second-step catalytic structures (22).
    [Show full text]