Structural basis for RNA surveillance by the human Nuclear Exosome Targeting (NEXT) complex

. Author manuscript; available in PMC: 2023 Jun 9.

Published in final edited form as: Cell. 2022 Jun 9;185(12):2132–2147.e26. doi: 10.1016/j.cell.2022.04.016

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-ROS1 (phospho-Y2274)	Thermo Fisher Scientific	Cat#PA5-64608; RRID:AB_2663812
Mouse monoclonal anti-ROS1	Origene	Cat#TA805734; RRID:AB_2627841
Mouse polyclonal anti-ZCCHC8	Abcam	Cat#ab68739; RRID:AB_1271512
Mouse monoclonal anti-GFP	Thermo Fisher Scientific	Cat#MA5-15256; RRID:AB_10979281
Mouse monoclonal Anti-mCherry	Thermo Fisher Scientific	Cat#MA5-32977
Mouse monoclonal anti-beta-actin	Thermo Fisher Scientific	Cat#MA5-15739; RRID:AB_10979409
Sheep monoclonal anti-mouse IgG	GE Healthcare	Cat#NA931V; RRID:AB_772210
Bacterial and virus strains
E. coli BL21CodonPlus(DE3)-RIL competent cells	Agilent Technologies	Cat#230245
E. coli E. cloni 10G competent cells	Lucigen	Cat#60106-2
Chemicals, peptides, and recombinant proteins
AdvanBlock-Chemi blocking solution	Advansta	Cat#R-03726-E10
Adenosine 5′(β,γ-imido) triphosphate lithium salt hydrate	Sigma	Cat#A2647
GFP-Trap magnetic agarose	Chromotek	Cat#gtma-20; RRID:AB_2631358
IGEPAL CA-630	Sigma	Cat#I8896
Iscove’s Modified Dulbecco’s medium	Thermo Fisher Scientific	Cat#12440053
Laemmli Sample buffer (4X)	Bio-Rad	Cat#1610747
Magnesium chloride hexahydrate	Sigma	Cat#M9272
Tris(2-carboxyethyl)phosphine (TCEP)	Soltec Ventures	Cat#M115
RIPA	Thermo Fisher Scientific	Cat#89901
RNase inhibitor, human placenta	New Engaland Biolabs	Cat#M0307S
Proteinase K	New England Biolabs	Cat#P8107S
Superdex 200 Increase 10/300 GL	GE Healthcare	Cat#28990944
SuperSep Phos-tag (50 μmol/L), 12.5%, 17-well	FUJIFILM Wako	Cat#195-17991
SuperSignal West Dura Extended Duration substrate	Thermo Fisher Scientific	Cat#34075
TBE (10X)	Thermo Fisher Scientific	Cat#AM9864
NU7741	StemCell Technologies	Cat#74082
NuPAGE MES SDS Running Buffer (20X)	Thermo Fisher Scientific	Cat#NP0002
NuPAGE LDS Sample Buffer (4X)	Thermo Fisher Scientific	Cat#NP0007
Novex TBE gel, 20%	Thermo Fisher Scientific	Cat#EC63152BOX
Novex TBE gel, 4-40%	Thermo Fisher Scientific	Cat#EC62252BOX
NuPage 4-12% Bis-Tris protein gels	Thermo Fisher Scientific	Cat#NP0322BOX
NuPage Transfer Buffer (20X)	Thermo Fisher Scientific	Cat#NP006
Ni-NTA agarose	Qiagen	Cat#30250
HiTrap Heparin HP	GE Healthcare	Cat#17040601
HiLoad 26/600 Superdex 200 pg	GE Healthcare	Cat#289289336
Ulp1 protease	In-house	N/A
Critical commercial assays
Expi293 Expression System Kit	Thermo Fisher Scientific	Cat#A14635
Gel filtration calibration kit high molecular weight	GE Healthcare	Cat#28403842
iBind Flex solution kit	Thermo Fisher Scientific	Cat#SLF2020
Lipofectamine 3000 transfection reagents	Thermo Fisher Scientific	Cat#L3000001
LIVE/DEAD Fixable Violet Dead Cell Stain Kit	Thermo Fisher Scientific	Cat#L34955
NucSpot Live 650 kit	Biotium	Cat#40082
RNeasy Pus Mini Kit	Qiagen	Cat#74034
Superscript IV VILO master mix	Thermo Fisher Scientific	Cat#11756050
TruSeq Standard Total RNA LT Kit	Illumina	Cat#RS-122-1202
WesternEaze-Chemi kit	Advansta	Cat#K-12054-010
Zip Alexa Fluor 647 Rapid Antibody Labeling Kit	Thermo Fisher Scientific	Cat#Z11235
Deposited data
NEXT-RNA substrate 1 complex structure	This paper	PDB: 7S7B
NEXT-RNA substrate 1 complex EM maps	This paper	EMDB: EMD-24882
NEXT-RNA substrate 2 complex structure	This paper	PDB: 7S7C
NEXT-RNA substrate 2 complex EM maps	This paper	EMDB: EMD-24883
Apo NEXT EM map	This paper	EMDB: EMD-24884
RNA-seq data	This paper	GEO: GSE185374 Token: ytspiamilduxlsx
Experimental models: Cell lines
Human: Expi293F cells	Thermo Fisher Scientific	Cat#A14635
Human: HAP1	Horizon Discovery	Cat#C631
Human: HAP1 ZCCHC8 knockout	Horizon Discovery	Cat#HZGHC005158c012
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP-ZCCHC8, and PB-mCherry-ZCCHC8	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP-ZCCHC8^ΔHD, and PB-mCherry-ZCCHC8	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP-ZCCHC8^1-80-ROS1^kinase, and PB-mCherry-ZCCHC8^1-80-ROS1^kinase	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP-ZCCHC8^1-80dm-ROS1^kinase, and PB-mCherry-ZCCHC8^1-80-ROS1^kinase	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP-ROS1^kinase, and PB-mCherry-ROS1^kinase	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP- ZCCHC8^1-80-ROS1^kinase, and PB-mCherry-ZCCHC8	This work	N/A
Human: Expi293F stably transfected with PB-rtTA-Puro, PB-GFP- ROS1^kinase, and PB-mCherry-ZCCHC8	This work	N/A
Human: HAP1 ZCCHC8 CRISPR knockout stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8	This work	N/A
Human: HAP1 ZCCHC8 CRISPR knockout stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8^ΔHD	This work	N/A
Human: HAP1 ZCCHC8 CRISPR knockout stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8^ΔZK	This work	N/A
Human: HAP1 ZCCHC8 stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8^1-80-ROS1^kinase	This work	N/A
Human: HAP1 ZCCHC8 stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8^1-80dm-ROS1^kinase	This wok	N/A
Human: HAP1 stably transfected with PB-rtTA-Puro and PB-GFP-ZCCHC8^1-80-ROS1^kinase(dead)	This work	N/A
Human: HAP1 stably transfected with PB-rtTA-Puro and PB-GFP-ROS1^kinase	This work	N/A
Oligonucleotides
6-FAM-AGCACCGUAAAGACGC (gel-based assay RNA top strand)	IDT	N/A
GCGUCUUUACGGUGCUAAAAAAAAAAAAAAAAAAAA (gel-based assay bottom strand for duplex with 3′ 20-nt poly(A) overhang)	IDT	N/A
GCGUCUUUACGGUGCUU_pyAAAAAAAAAAAAAAAAA (gel-based assay bottom strand for duplex with 2′ pyrene modified uridine U_py)	Dharmacon	N/A
ACAUGAGGAUCACCCAUGUAAUCUCUUUCAAAAAAU_pyACAAAAAAAA (cryo-EM substrate 1, U_py= 2′ pyrene modified uridine)	Dharmacon	N/A
GGCGCGCGCCAAAAAUUUUUAAAAAAAAAA (cryo-EM substrate 2)	Dharmacon	N/A
6-FAM-AGUGCGCUGUAUCUUCAAGGCCACU (EMSA RNA top strand)	IDT	N/A
Iowa Black RQ-AGUGCGCUGUAUCUUCAAGGCCACU-Cy5 (molecular beacon helicase assay RNA top strand)	IDT	N/A
AGUGGCCUUGAAGAUACAGCGCACUAAAAAAAAAAAAAAAAAAAA (molecular beacon assay RNA bottom strand with 3′ 20-nt poly(A) overhang)	IDT	N/A
AGUGGCCUUGAAGAUACAGCGCACUAAAAAUUUUUAAAAAAAAAA (molecular beacon assay RNA bottom strand with 3′ A₅U₅A₁₀ overhang)	IDT	N/A
qPCR and CRISPR screening primers	See Table S2	N/A
Recombinant DNA
pET-28a-His₁₀-Smt3-RBM7^core	Puno and Lima, 2018	N/A
pET-28a- His₁₀-Smt3-MTR4	Puno and Lima, 2018	N/A
pRSF-Duet1-Smt3-ZCCHC8^core	Puno and Lima, 2018	N/A
pRSF-Duet1-Smt3-ZCCHC8^coreΔHD	This work	N/A
pRSF-Duet1-Smt3-ZCCHC8^coreΔZK	This work	N/A
pRSF-HiS₆-ncSmt3-MTR4	This work	N/A
pET-28a- His₁₀-Smt3-Strep-MTR4	This work	N/A
pET-28a- His₁₀-Smt3-Strep-MTR4-E253Q	This work	N/A
pRP[Exp]-mCherry-CAG>hyPBase	VectorBuilder	Cat#VB160216-10057
PB-TAG-ERPE	Addgene; Kim et al., 2015	Cat#80479, RRID:Addgene_80479
PB-rtTA-Puro	This work	N/A
PB-GFP-ZCCHC8	This work	N/A
PB-GFP-ZCCHC8ΔNTD	This work	N/A
PB-mCherry-ZCCHC8	This work	N/A
PB-GFP-ZCCHC8ΔZK	This work	N/A
PB-GFP-ZCCHC8^1-80-ROS1^kinase	This work	N/A
PB-GFP-ZCCHC8^1-80-ROS1^kinase(dead)	This work	N/A
PB-GFP-ZCCHC8^1-80dm-ROS1^kinase	This work	N/A
PB-GFP-ROS1^kinase	This work	N/A
pRP[2CRISPR]-mCherry-hCas9-U6>{hZCCHC8[gRNA#1]}-U6>{hZCCHC8[gRNA#2]})	This work	Cat#: VB200528-1045ndr
Software and algorithms
BD FACSDiva software	BD Biosciences	http://www.bdbiosciences.com/instruments/software/facsdiva/index.jsp; RRID:SCR_001456
Biorender	Biorender	http://biorender.com; #NI22WEQFBQ;#YI22XZN09U and #TM22XZN108 RRID:SCR_018361
Coot	Emsley et al., 2010	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/; RRID:SCR_014222
FCS Express Research version 7	De Novo software company	https://denovosoftware.com/; RRID:SCR_016431
Gctf	Zhang, 2016	https://www2.mrc-lmb.cam.ac.uk/research/locally-developed-software/zhang-software/#gctf; RRID:SCR_016500
Integrative Genomics Viewer (IGV)	Robinson et al., 2011	https://software.broadinstitute.org/software/igv/
ImageJ	Schneider et al., 2012	https://imagej.nih.gov/ij/
Jalview version 2	Waterhouse et al., 2009	www.jalview.org/: RRID:SCR_006459
Leginon data collection software	Suloway et al., 2005	https://emg.nysbc.org/redmine/projects/leginon; RRID:SCR_016731
MolProbity	Chen et al., 2010	http://molprobity.biochem.duke.edu; RRID:SCR_014226
Phenix suite	Adams et al., 2010	https://www.phenix-online.org/; RRID:SCR_014224
Prism version 9	Graphpad	https://www.graphpad.com/scientific-software/prism/; RRID:SCR_002798
Pymol version 2.4.1	Schroedinger LLC	https://pymol.org; RRID:SCR_000305
RELION	Scheres, 2012; Zivanov et al., 2018	http://www2.mrc-lmb.cam.ac.uk/relion; RRID:SCR_016274
Serial EM data collection software	Mastronarde, 2003	http://bio3d.colorado.edu/SerialEM/; RRID:SCR_017293
STAR	Dobin et al., 2013	http://code.google.com/p/rna-star/; RRID:SCR_004463
UCSF ChimeraX version 1.2.5	Pettersen et al., 2021	https://www.cgl.ucsf.edu/chimerax/; RRID:SCR_015872
UCSF MotionCor2	Zheng et al., 2017	https://emcore.ucsf.edu/ucsf-software
Other
Quantifoil R 1.2/1.3 300 mesh gold grids	Electron Microscopy Services	Cat#Q43841
Vitrobot Mark IV	FEI – Thermo Fisher	https://www.fei.com
Titan Krios	FEI – Thermo Fisher	https://www.fei.com
K2 Summit Camera	Gatan, Inc	www.gatan.com
K3 Camera	Gatan, Inc	www.gatan.com
Corning 3693 White Half-Area 96-well plate	Fisher Scientific	Cat#07-200-326
Trans-blot Turbo	Bio-Rad	Cat#1704150
iBind Flex Western device	Thermo Fisher Scientific	Cat#SLF2000