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Andrew J MacRae, Megan Mayerle, Eva Hrabeta-Robinson, Robert J Chalkley, Christine Guthrie, A L Burlingame, Melissa Jurica
Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as scaffolding the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in 5' splice site recognition...
February 27, 2018: RNA
Cathleen M Green, Olga Novikova, Marlene Belfort
Background: Inteins are mobile, self-splicing sequences that interrupt proteins and occur across all three domains of life. Scrutiny of the intein landscape in prokaryotes led to the hypothesis that some inteins are functionally important. Our focus shifts to eukaryotic inteins to assess their diversity, distribution, and dissemination, with the aim to comprehensively evaluate the eukaryotic intein landscape, understand intein maintenance, and dissect evolutionary relationships. Results: This bioinformatics study reveals that eukaryotic inteins are scarce, but present in nuclear genomes of fungi, chloroplast genomes of algae, and within some eukaryotic viruses...
2018: Mobile DNA
Wojciech P Galej, Navtej Toor, Andrew J Newman, Kiyoshi Nagai
Nuclear pre-mRNA splicing and group II intron self-splicing both proceed by two-step transesterification reactions via a lariat intron intermediate. Recently determined cryo-electron microscopy (cryo-EM) structures of catalytically active spliceosomes revealed the RNA-based catalytic core and showed how pre-mRNA substrates and reaction products are positioned in the active site. These findings highlight a strong structural similarity to the group II intron active site, strengthening the notion that group II introns and spliceosomes evolved from a common ancestor...
January 29, 2018: Chemical Reviews
Xiaofeng Zhang, Chuangye Yan, Xiechao Zhan, Lijia Li, Jianlin Lei, Yigong Shi
During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the branching reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex...
January 23, 2018: Cell Research
Hans Garcia Garces, Raquel Theodoro Cordeiro, E Bagagli
Dermatophytes are keratinophilic fungi belonging to the family Arthrodermataceae. Despite having a monophyletic origin, its systematics has always been complex and controversial. Sequencing of nuclear ribosomal ITS and D1/D2 rDNA has been proposed as an efficient tool for identifying species in this group of fungi, while multilocus analyses have been used for phylogenetic species recognition. However, the search for new markers, with sequence and size variation, which enable species identification in only one polymerase chain reaction (PCR) step, is very attractive...
December 8, 2017: Medical Mycology: Official Publication of the International Society for Human and Animal Mycology
Julian Vosseberg, Berend Snel
ᅟ: The spliceosome is a eukaryote-specific complex that is essential for the removal of introns from pre-mRNA. It consists of five small nuclear RNAs (snRNAs) and over a hundred proteins, making it one of the most complex molecular machineries. Most of this complexity has emerged during eukaryogenesis, a period that is characterised by a drastic increase in cellular and genomic complexity. Although not fully resolved, recent findings have started to shed some light on how and why the spliceosome originated...
December 1, 2017: Biology Direct
Rui Bai, Chuangye Yan, Ruixue Wan, Jianlin Lei, Yigong Shi
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...
November 4, 2017: Cell
Jennifer L Stamos, Alfred M Lentzsch, Alan M Lambowitz
Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs as well as the spliceosomal protein Prp8 in eukaryotes. Here we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming deoxynucleotide triphosphate (dNTP) at 3.0-Å resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs but remarkably similar to viral RNA-dependent RNA polymerases...
December 7, 2017: Molecular Cell
M Murshida Mahbub, Saiful M Chowdhury, Shawn M Christensen
Background: R2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs. Results: Using limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE)...
2017: Mobile DNA
Ruixue Wan, Chuangye Yan, Rui Bai, Jianlin Lei, Yigong Shi
The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5' exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly...
September 21, 2017: Cell
Karl Bertram, Dmitry E Agafonov, Olexandr Dybkov, David Haselbach, Majety N Leelaram, Cindy L Will, Henning Urlaub, Berthold Kastner, Reinhard Lührmann, Holger Stark
Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step...
August 10, 2017: Cell
Yigong Shi
Pre-mRNA splicing is executed by the ribonucleoprotein machinery spliceosome. Nearly 40 years after the discovery of pre-mRNA splicing, the atomic structure of the spliceosome has finally come to light. Four distinct conformational states of the yeast spliceosome have been captured at atomic or near-atomic resolutions. Two catalytic metal ions at the active site are specifically coordinated by the U6 small nuclear RNA (snRNA) and catalyze both the branching reaction and the exon ligation. Of the three snRNAs in the fully assembled spliceosome, U5 and U6, along with 30 contiguous nucleotides of U2 at its 5'-end, remain structurally rigid throughout the splicing reaction...
August 18, 2017: Journal of Molecular Biology
Clemens Plaschka, Pei-Chun Lin, Kiyoshi Nagai
Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2...
June 29, 2017: Nature
Chen Zhao, Anna Marie Pyle
The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex...
December 2017: Current Opinion in Structural Biology
Xiaofeng Zhang, Chuangye Yan, Jing Hang, Lorenzo I Finci, Jianlin Lei, Yigong Shi
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...
May 18, 2017: Cell
Megan Mayerle, Madhura Raghavan, Sarah Ledoux, Argenta Price, Nicholas Stepankiw, Haralambos Hadjivassiliou, Erica A Moehle, Senén D Mendoza, Jeffrey A Pleiss, Christine Guthrie, John Abelson
Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown...
May 2, 2017: Proceedings of the National Academy of Sciences of the United States of America
John Abelson
Major developments in cryo-electron microscopy in the past three or four years have led to the solution of a number of spliceosome structures at high resolution, e.g., the fully assembled but not yet active spliceosome (Bact), the spliceosome just after the first step of splicing (C), and the spliceosome activated for the second step (C*). Therefore 30 years of genetics and biochemistry of the spliceosome can now be interpreted at the structural level. I have closely examined the RNase H domain of Prp8 in each of the structures...
April 25, 2017: Proceedings of the National Academy of Sciences of the United States of America
Tatsuo Kanno, Wen-Dar Lin, Jason L Fu, Antonius J M Matzke, Marjori Matzke
To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana In wild-type plants, three major splice variants issue from the GFP gene but only one represents a translatable GFP mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFP expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype depending on the ratio of the three splice variants...
July 2017: RNA
Tanuza Das, Joon Kyu Park, Jinyoung Park, Eunji Kim, Michael Rape, Eunice EunKyeong Kim, Eun Joo Song
Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3...
May 5, 2017: Nucleic Acids Research
Karl Bertram, Dmitry E Agafonov, Wen-Ti Liu, Olexandr Dybkov, Cindy L Will, Klaus Hartmuth, Henning Urlaub, Berthold Kastner, Holger Stark, Reinhard Lührmann
Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains...
February 16, 2017: Nature
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