Journal: Sci Adv / Year: 2023 Title: Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins. Authors: Olexandr Dybkov / Marco Preußner / Leyla El Ayoubi / Vivi-Yun Feng / Caroline Harnisch / Kilian Merz / Paula Leupold / Peter Yudichev / Dmitry E Agafonov / Cindy L Will / Cyrille Girard / ...Authors: Olexandr Dybkov / Marco Preußner / Leyla El Ayoubi / Vivi-Yun Feng / Caroline Harnisch / Kilian Merz / Paula Leupold / Peter Yudichev / Dmitry E Agafonov / Cindy L Will / Cyrille Girard / Christian Dienemann / Henning Urlaub / Berthold Kastner / Florian Heyd / Reinhard Lührmann / Abstract: Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by ...Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details
4 microliter sample was applied to a grid. After 15 s, the grid was blotted for 3 s with a blot force of 5 and vitrified by plunging into liquid ethane using FEI Vitrobot Mark IV.
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 14388 / Average exposure time: 1.465 sec. / Average electron dose: 44.24 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
-
Image processing
Particle selection
Number selected: 1643674
Startup model
Type of model: INSILICO MODEL
Initial angle assignment
Type: MAXIMUM LIKELIHOOD
Final angle assignment
Type: MAXIMUM LIKELIHOOD
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1150057
In the structure databanks used in Yorodumi, some data are registered as the other names, "COVID-19 virus" and "2019-nCoV". Here are the details of the virus and the list of structure data.
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)
EMDB accession codes are about to change! (news from PDBe EMDB page)
The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
The EM Navigator/Yorodumi systems omit the EMD- prefix.
Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator
Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.
Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi