Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization

2018 ◽  
Vol 47 (1) ◽  
pp. 175-199 ◽  
Author(s):  
Max E. Wilkinson ◽  
Pei-Chun Lin ◽  
Clemens Plaschka ◽  
Kiyoshi Nagai
Keyword(s):  
Messenger Rna ◽  
Model Building ◽  
De Novo ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Recent Developments ◽  
Nuclear Ribonucleoprotein ◽  
Model Visualization

The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.

Structures of the fully assembledSaccharomyces cerevisiaespliceosome before activation

Science ◽  
2018 ◽  
Vol 360 (6396) ◽  
pp. 1423-1429 ◽  
Author(s):  
Rui Bai ◽  
Ruixue Wan ◽  
Chuangye Yan ◽  
Jianlin Lei ◽  
Yigong Shi
Keyword(s):  
Messenger Rna ◽  
Small Nuclear Rna ◽  
U6 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Specific Proteins ◽  
Complex Structural ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo–electron microscopy structures of theSaccharomyces cerevisiaepre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5′ splice site (5′SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5′-exon–5′SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

scholarly journals In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells

1994 ◽  
Vol 297 (2) ◽  
pp. 297-301 ◽  
Author(s):  
J R Patton
Keyword(s):  
Cell Growth ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Significant Inhibition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Specific Step ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

scholarly journals Advancing Native-SAD Phasing at Synchrotron with 13 Real-life Case Studies

2014 ◽  
Vol 70 (a1) ◽  
pp. C601-C601
Author(s):  
Meitian Wang
Keyword(s):  
Data Collection ◽  
Single Crystal ◽  
Measurement Errors ◽  
Model Building ◽  
De Novo ◽  
Real Life ◽  
Data Sets ◽  
X Ray ◽  
Recent Developments ◽  
Sad Phasing

The key step in elucidating de novo 3D X-ray structures relies on the incorporation of heavy elements into proteins or crystals. Selenomethionine incorporation or heavy metal derivatization are however not always possible and require additional efforts. Exploiting anomalous signals from intrinsically present elements like S, P, and Ca2+ from proteins and nucleic acids, as well as Cl-, SO42-, and PO42- from crystallization solutions, is therefore an appealing alternative. Such a method has been shown to be valid by collecting data from several crystals and combining them(1). Recent developments at macromolecular crystallography beamlines are however pushing the limits of what could be obtained out of a single crystal. Here we introduce a novel data collection routine for native-SAD phasing, which distributes tolerable X-ray life-doses to very high multiplicity X-ray diffraction data sets measured at 6 keV energy and at different crystal orientations on a single crystal. This allows the extraction of weak anomalous signals reliably by reducing both systematic and random measurement errors. The data collection method has been applied successfully to thirteen real-life examples including membrane proteins, a protein/DNA complex, and a large protein complex. In addition to de novo structure determination, we advocate such a data collection protocol for molecular replacement solvable structures where unbiased phase information is crucial in objective map interpretation and model building, especially for medium and low-resolution cases.

scholarly journals Structure of an active human histone pre-mRNA 3′-end processing machinery

Science ◽  
2020 ◽  
Vol 367 (6478) ◽  
pp. 700-703 ◽  
Author(s):  
Yadong Sun ◽  
Yixiao Zhang ◽  
Wei Shen Aik ◽  
Xiao-Cui Yang ◽  
William F. Marzluff ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Messenger Rnas ◽  
Small Nuclear Rna ◽  
Processing Machinery ◽  
Small Nuclear Ribonucleoprotein ◽  
Cryo Electron Microscopy ◽  
Nuclear Rna ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Nuclear Ribonucleoprotein

The 3′-end processing machinery for metazoan replication-dependent histone precursor messenger RNAs (pre-mRNAs) contains the U7 small nuclear ribonucleoprotein and shares the key cleavage module with the canonical cleavage and polyadenylation machinery. We reconstituted an active human histone pre-mRNA processing machinery using 13 recombinant proteins and two RNAs and determined its structure by cryo–electron microscopy. The overall structure is highly asymmetrical and resembles an amphora with one long handle. We captured the pre-mRNA in the active site of the endonuclease, the 73-kilodalton subunit of the cleavage and polyadenylation specificity factor, poised for cleavage. The endonuclease and the entire cleavage module undergo extensive rearrangements for activation, triggered through the recognition of the duplex between the authentic pre-mRNA and U7 small nuclear RNA (snRNA). Our study also has notable implications for understanding canonical and snRNA 3′-end processing.

scholarly journals Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fu-Lung Yeh ◽  
Shang-Lin Chang ◽  
Golam Rizvee Ahmed ◽  
Hsin-I Liu ◽  
Luh Tung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Messenger Rna ◽  
Dead Box ◽  
Eukaryotic Gene Expression ◽  
U1 Snrnp ◽  
Eukaryotic Gene ◽  
Snrnp Protein

AbstractSplicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5′ splice-site (5′SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28’s ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

scholarly journals Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein

Cell ◽  
1983 ◽  
Vol 35 (1) ◽  
pp. 101-107 ◽  
Author(s):  
Richard A. Padgett ◽  
Stephen M. Mount ◽  
Joan A. Steitz ◽  
Phillip A. Sharp
Keyword(s):  
Messenger Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein

Science ◽  
1985 ◽  
Vol 230 (4732) ◽  
pp. 1344-1349 ◽  
Author(s):  
B Chabot ◽  
D. Black ◽  
D. LeMaster ◽  
J. Steitz
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

scholarly journals Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core

2017 ◽  
Vol 73 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Eric J. Montemayor ◽  
Allison L. Didychuk ◽  
Honghong Liao ◽  
Panzhou Hu ◽  
David A. Brow ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Messenger Rna ◽  
Rna Recognition Motif ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Conformational Plasticity ◽  
Gel Shift Assay ◽  
Nuclear Ribonucleoprotein

U6 small nuclear RNA (snRNA) is a key component of the active site of the spliceosome, a large ribonucleoprotein complex that catalyzes the splicing of precursor messenger RNA. Prior to its incorporation into the spliceosome, U6 is bound by the protein Prp24, which facilitates unwinding of the U6 internal stem-loop (ISL) so that it can pair with U4 snRNA. A previously reported crystal structure of the `core' of the U6 small nuclear ribonucleoprotein (snRNP) contained an ISL-stabilized A62G mutant of U6 bound to all four RNA-recognition motif (RRM) domains of Prp24 [Montemayoret al.(2014),Nature Struct. Mol. Biol.21, 544–551]. The structure revealed a novel topology containing interlocked rings of protein and RNA that was not predicted by prior biochemical and genetic data. Here, the crystal structure of the U6 snRNP core with a wild-type ISL is reported. This complex crystallized in a new space group, apparently owing in part to the presence of an intramolecular cross-link in RRM1 that was not observed in the previously reported U6-A62G structure. The structure exhibits the same protein–RNA interface and maintains the unique interlocked topology. However, the orientation of the wild-type ISL is altered relative to the A62G mutant structure, suggesting inherent structural dynamics that may facilitate its pairing with U4. Consistent with their similar architectures in the crystalline state, the wild-type and A62G variants of U6 exhibit similar Prp24-binding affinities and electrophoretic mobilities when analyzed by gel-shift assay.

scholarly journals Sexual lineage specific DNA methylation regulates Arabidopsis meiosis

2017 ◽  
Author(s):  
James Walker ◽  
Hongbo Gao ◽  
Jingyi Zhang ◽  
Billy Aldridge ◽  
Martin Vickers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Arabidopsis Thaliana ◽  
De Novo ◽  
Cell Lineage ◽  
Flowering Plants ◽  
Animal Development ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
A Cell

SUMMARYDNA methylation controls eukaryotic gene expression and is extensively reprogrammed to regulate animal development. However, whether developmental methylation reprogramming during the sporophytic life cycle of flowering plants regulates genes is presently unknown. Here we report a distinctive, gene-targeted RNA-directed DNA methylation (RdDM) activity in the Arabidopsis thaliana male sexual lineage that regulates gene expression in meiocytes. Loss of sexual lineage-specific RdDM causes mis-splicing of the MPS1/PRD2 gene, thereby disrupting meiosis. Our results establish a regulatory paradigm in which de novo methylation creates a cell-lineage-specific epigenetic signature that controls gene expression and contributes to cellular function in flowering plants.

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