mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations

. Author manuscript; available in PMC: 2024 Aug 3.

Published in final edited form as: Mol Cell. 2023 Jul 27;83(15):2726–2738.e9. doi: 10.1016/j.molcel.2023.06.036

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
rabbit anti-GFP	ThermoFisher	Cat#A-11122
rabbit anti-NET1	Abcam	Cat#ab 113202
rabbit anti-Importin beta 1	Cell Signaling Technology	Cat#60769
mouse anti-CASK	Santa Cruz	Cat#sc-13158
rabbit anti-CASK	Invitrogen	Cat#PA1544
Mouse anti-tubulin	Sigma	Cat#T6199
rabbit anti-GAPDH	Cell Signaling Technology	Cat#2118L
IR Dye 680RD donkey anti-mouse	Li-Cor	Cat#926–68072
IR Dye 800CW donkey anti-rabbit	Li-Cor	Cat#926–32213
mouse anti-paxillin	BD Transduction Laboratories	Cat#610619
Alexa48 8-Phalloidin	Invitrogen	Cat#A12379
anti-mouse Alexa647	Invitrogen	Cat#A28181
mouse anti-FLAG M2	Sigma	Cat#F1804
Chemicals, Peptides, and Recombinant Proteins
Lipofectamine RNAiMAX	ThermoFisher	Cat#13778–150
PolyJet In Vitro DNA Transfection Reagent	SignaGen	Cat# SL100688
Leibovitz L-15	Gibco	Cat#11415064
Penicillin-Streptomycin	Gibco	Cat#15070063
DMEM, high glucose, pyruvate	Invitrogen	Cat#11995073
Trypsin-EDTA (0.05%)	Gibco	Cat#25300054
Blasticidin	ThermoFisher	Cat#A1113903
Geneticin	Gibco	Cat#10131035
Doxycycline Hydrochloride	ThermoFisher	Cat#BP26535
Puromycin Dihydrochloride	Gibco	Cat#A1113803
Recombinant Human TGF-B1	Peprotech	Cat#100–21
Endoporter	Genetools LLC	Cat#OT-EP-PEG-1
Collagen IV	Sigma	Cat#C5533–5MG
Fibronectin bovine plasma	Sigma	Cat#F1141
GFP-Trap magnetic agarose beads	Chromotek	Cat#gtma-10
Paraformaldehyde	Electron Microscope Sciences	Cat#15710
Intercept Blocking Buffer	Li-Cor	Cat#927–60001
ProLong Gold Antifade reagent with DAPI	Invitrogen	Cat#P36931
Triton X-100	Sigma	Cat#93443–100ML
Halt Protease and Phosphatase Inhibitor Cocktail	ThermoFisher	Cat#78444
HCS Green CellMask Stain	ThermoFisher	Cat# H32714
GST-KPNA1	Abnova	Cat#H00003836-P01
GST-KPNA2	Abnova	Cat#H00003838-P01
GST-KPNA4	Abnova	Cat#H00003840-P01
JFX554 HaloTag ligand	Janelia Research Campus	N/A
DRAQ5	ThermoFisher	Cat#62254
RNase I	ThemorFisher	Cat#AM2295
Superase-In	ThermoFisher	Cat#AM2694
TRIzol	ThermoFisher	Cat#15596026
Critical Commercial Assays
DuoLink In Situ Red PLA Kit	Sigma	Cat#DUO92008
ViewRNA ISH Cell Assay Kit	ThermoFisher	Cat#QVC0001
Quant-it RiboGreen RNA Assay Kit	ThermoFisher	Cat#R11490
Qiagen FastSelect Kit	Qiagen	Cat#334386
High Sensitivity DNA Kit	Agilent	Cat#5067–4626
Deposited Data
Ribo-seq and Disome-seq-HEK293 cells	This study	GEO: GSE207146
Ribosome profiling-A549 cells	Karasik et al, 2021⁴⁴	GEO: GSE158044
Ribosome profiling-HEK293 cells	Han et al, 20 20⁴⁵	GEO: GSE133393
Imaging data	This study	http://dx.doi.org/10.17632/f4mr55trf6.1
Experimental Models: Cell Lines
MDA-MB-231	ATCC	Cat#CRM-HTB-26
MCF7	ATCC	Cat#HTB-22
Flp-In T-REx HEK293	Gibco	Cat#R780–07
NIH/3T3	ATCC	Cat#CRL-1658
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A WT UTR + pLV-PGK-hCA SK-FLAG	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_WT UTR + pLV-PGK-hCA SK-FLAG-mCherry	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_AGA UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A AGA UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A AGA UTR + pLV-PGK-hCASK-FLAG	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_ AGA UTR + pLV-PGK-hCASK-FLAG-mCherry	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1 A_slow2_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A slow2 WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1 A_slow2_AGA UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A slow2 AGA UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_stallmutant(fast)_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A stallmutant(fast) WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A_stallmutant(fast)_ AGA UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A stallmutant(fast) AGA UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A C-term NLS_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A C-term NLS WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A C-term NLS_AGA UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A C-term NLS AGA UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-mutantNLS_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-mutantNLS WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-ADH_hNET1 UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-ADH WT UTR + pCDH-CMV-hKPNBl	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-APH_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-APH WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-APDZ_WT UTR	This study	N/A
MDA-MB-231 + pCDH-PGK-GFP-hNET1A-APDZ WT UTR + pCDH-CMV-hKPNB1	This study	N/A
MDA-MB-231 + pCDH-CMV-mTFP 1	This study	N/A
MDA-MB-231 + pIND20-RhoA-WT-biosensor	This study	N/A
MDA-MB-231 + pIND20-RhoA-Q63L-biosensor	This study	N/A
MDA-MB-231 + pIND20-RhoA-T19N-biosensor	This study	N/A
NIH/3T3 + NLS-HA-stdMCP-stdHalo + scFv-GCN4-sfGFP + pIND20-hNet1A 5UTR-24xGCN4-hNet1A CDS-24xMS2v7_hNet1 3UTR	This study	N/A
NIH/3T3 + NLS-HA-stdMCP-stdHalo + scFv-GCN4-sfGFP + pIND20-hNet1A 5UTR-24xGCN4-hNet1A SLOW2 CDS-24xMS2v7_hNet1 3UTR	This study	N/A
NIH/3T3 + NLS-HA-stdMCP-stdHalo + scFv-GCN4-sfGFP + pIND20-hNet1A 5UTR-24xGCN4-hNet1A STALLMUT(fast) CDS-24xMS2v7 hNet1 3UTR	This study	N/A
Oligonucleotides
Morpholino oligonucleotides (PMOs)	Table S2	GeneTools LLC
human NET1 FISH probes	ThermoFisher	Cat# VA6–3169338
human RHOA FISH probes	ThermoFisher	Cat#VA6–14829-01
GFP FISH probes	ThermoFisher	Cat#VF6–16198
AllStars siRNA Negative Control	Qiagen	Cat#1027281
human siNET1 #4 – 5’-ACGGAAAGAGACTTTGGTGTA-3’	Qiagen	Cat#SI00082040
human siCASK #5 – 5’-AACCAATGGGAATCACTTTAA-3’	Qiagen	Cat#SI02223368
human siCASK #10 – 5’-CAGACCGGTTTGCGTACCCTA-3’	Qiagen	Cat#SI04437720
Recombinant DNA
KPNB1 (NM_002265) Human Tagged ORF Clone	Origene	Cat#RC200659
pInducer20	Addgene	Cat#44012
pET263-pUC57 24xMS2V7	Addgene	Cat#140705
pLentiRhoA2G	Addgene	Cat#40179
pcDNA4TO-24xGCN4_v4-kif18b-24xPP7	Addgene	Cat#74928
phage UbiC NLS HA stdMCP stdHalo	Addgene	Cat#104999
pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE	Addgene	Cat#60906
pCDH-CMV-EF1-MCS-Puro	System Biosciences	Cat#CD510B-1
pCDH-CMV-hKPNB1	This study	N/A
pCDH-PGK-GFP-hNET1A-ADH WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A-APH WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A-APDZ WT UTR	This study	N/A
pCDH-CMV-mTFP1	This study	N/A
pIND20-RhoA-WT-biosensor	This study	N/A
pIND20-RhoA-Q63 L-biosensor	This study	N/A
pIND20-RhoA-T19N-biosensor	This study	N/A
pCDH-PGK-GFP-hNET1A_WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A_ AGA UTR	This study	N/A
pCDH-PGK-GFP-hNET1A-slow2 WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A-slow2 AGA UTR	This study	N/A
pCDH-PGK-GFP-hNET1A_stallmutant(fast)_WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A_stallmutant(fast)_ AGA UTR	This study	N/A
pCDH-PGK-GFP-hNET1A C-term NLS / WT UTR	This study	N/A
pCDH-PGK-GFP-hNET1A C-term NLS_AGA UTR	This study	N/A
pCDH-PGK-GFP-hNET1A-mutantNLS hNET1 UTR	This study	N/A
pIND20-hNet1A 5UTR-24xGCN4-hNet1A CDS-24xMS2v7_hNet1 3UTR	This study	N/A
pIND20-hNet1A 5UTR-24xGCN4-hNet1A CDS-24xMS2v7 hNeti A(GA) 3UTR	This study	N/A
pIND20-hNet1A 5UTR-24xGCN4-hNet1A SLOW2 CDS-24xMS2v7 hNet1 3UTR	This study	N/A
pIND20-hNet1A 5UTR-24xGCN4-hNet1A STALLMUT(fast) CDS-24xMS2v7_hNet1 3UTR	This study	N/A
pLV-PGK-hCASK-FLAG-mCherry	VectorBuilder	N/A
pLV-PGK-hCASK-FLAG	VectorBuilder	N/A
Software and Algorithms
Fiji	Schindelin et al., 2012⁴⁶	https://fiji.sc
Imaris	Oxford Instruments	https://imaris.oxinst.com
RibofootPrinter	Guydosh, 2021⁴⁷	https://github.com/guydoshlab/ribofootPrinter
Matlab	MathWorks	https://www.mathworks.com/products/matlab.html
Prism	GraphPad	https://www.graphpad.com
DiPer Macro	Gorelik and Gautreau, 2014⁴⁸	N/A
ADAPT ImageJ plugin	Barry et al., 2015⁴⁹	N/A
LAS X	Leica	https://www.leica.com
NIS-Elements	Nikon	https://www.microscope.healthcare.nikon.com/products/software/nis-elements
Other
35mm glass-bottom dishes	MatTek Corporation	Cat# P35G-1.5–14-C
TetraSpeck^™ Fluorescent microspheres sampler kit	Fisher Scientific	Cat#T7284
Coverslip, #1.5 thickness	Thomas Scientific	Cat#1217N81