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

Annual Review of Biophysics
Max E WilkinsonKiyoshi Nagai

Abstract

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 ...Continue Reading

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Citations

Sep 6, 2018·Genome Biology and Evolution·Wenzhen ChengHongbo Gao
Feb 16, 2019·Cold Spring Harbor Perspectives in Biology·Clemens PlaschkaKiyoshi Nagai
Apr 27, 2019·ELife·Leonhard WachutkaPatrick Cramer
Sep 4, 2019·Acta Crystallographica. Section D, Structural Biology·Max E WilkinsonAna Casañal
Nov 14, 2018·Biochemical Society Transactions·Wojciech P Galej
Jul 23, 2020·Cell Reports·Nele Merret HollmannJanosch Hennig
Mar 7, 2021·Nature Communications·Lisa M StrittmatterKiyoshi Nagai
Jun 23, 2021·Blood·Sisi ChenOmar Abdel-Wahab

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Methods Mentioned

BETA
X-ray
nuclear magnetic resonance
electron microscopy
affinity purification
in vitro transcription
Electron
NMR
pull-down
cross-linking studies

Software Mentioned

MODELLER
MODEL
REFMAC5
RELION
Coot
PyMOL
UCSF Chimera
MDFF
TASSER
GeneSilico metaserver

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