Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | CLUH | NCBI | NM_001366661.1 | |
| Gene (Homo sapiens) | SPAG5 | NCBI | NM_006461.3 | |
| Gene (Homo sapiens) | KNSTRN | NCBI | NM_033286.4 | |
| Cell line (Homo sapiens) | CLUH KO HeLa cells | Wakim et al., 2017 | Exon 4 has been deleted with CRISPR-Cas9 technique | |
| Cell line (Homo sapiens) | Doxycyline inducible WT HeLa cells | McKinley and Cheeseman, 2017 | Cas9 expression is induced by doxycycline treatment; constitutive sgRNA expression | |
| Cell line (Homo sapiens) | Inducible SPAG5 KO HeLa cells | McKinley and Cheeseman, 2017; Kern et al., 2017 | Cas9 expression is induced by doxycycline treatment; constitutive sgRNA expression targeting SPAG5 | |
| Cell line (Homo sapiens) | Inducible KNSTRN KO HeLa cells | McKinley and Cheeseman, 2017; Kern et al., 2017 | Cas9 expression is induced by doxycycline treatment; constitutive sgRNA expression targeting KNSTRN | |
| Cell line (Homo sapiens) | HEK293T cells expressing FLAG-kinastrin | This paper | Doxycyline inducible | |
| Cell line (Homo sapiens) | SPAG5 KO HeLa cells | This paper | Stable KO cells; derived by monoclonal selection of inducible SPAG5 KO HeLa cells | |
| Cell line (Homo sapiens) | KNSTRN KO HeLa cells | This paper | Stable KO cells; derived by monoclonal selection of inducible KNSTRN KO HeLa cells | |
| Cell line (Mus musculus) | Cluh KO MEFs | Gao et al., 2014 | Exon 10 has been deleted with Cre/LoxP system | |
| Recombinant DNA reagent | p3xFLAG-CMV-14 (plasmid) |
Sigma-Aldrich | Backbone of all SPAG5 constructs; used as empty vector control | |
| Transfected construct (human) | ATG1-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 from ATG1 | |
| Transfected construct (human) | Δ151-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 lacking the first 151 amino acids | |
| Transfected construct (human) | 5UTR-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR; used to overexpress astrin-1 and 2 together | |
| Transfected construct (human) | uATGGGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with uATG mutated to GGG | |
| Transfected construct (human) | ATG1GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG1 mutated to GGG; used to overexpress only astrin-2 | |
| Transfected construct (human) | ATG3GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG3 mutated to GGG | |
| Transfected construct (human) | ATG4GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG4 mutated to GGG | |
| Transfected construct (human) | ATG5GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG5 mutated to GGG | |
| Transfected construct (human) | ATG6GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG6 mutated to GGG | |
| Transfected construct (human) | ATG3 + 5GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG3 and 5 mutated to GGG | |
| Transfected construct (human) | ATG3 + 4 + 5GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG3, 4, and 5 mutated to GGG; used to overexpress only astrin-1 | |
| Transfected construct (human) | ATG3 + 4 + 5 + 6GGG-SPAG5 | This paper | Allows the expression of a FLAG-tagged human SPAG5 including its 5′ UTR with ATG3, 4, 5, and 6 mutated to GGG | |
| Transfected construct (human) | ctrl siRNA | Invitrogen | #12935115 | Transfected construct (human) |
| Transfected construct (human) | siRNA targeting CLUH | Invitrogen | CLUHHSS118510 | Transfected construct (human) |
| Transfected construct (human) | siRNA targeting CLUH | Invitrogen | CLUHHSS118511 | Transfected construct (human) |
| Transfected construct (human) | siRNA targeting CLUH | Invitrogen | CLUHHSS177299 | Transfected construct (human) |
| Antibody | Anti-CLUH (rabbit polyclonal) | Novus Biologicals | #NB100-93305 | WB (1:1000) IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-CLUH (rabbit polyclonal) | Novus Biologicals | #NB100-93306 | IF(1:1000), WB (1:1000) IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-RPS6 (rabbit polyclonal) | Novus Biologicals | #NB100-1595 | WB (1:1000) |
| Antibody | Anti-RPL7 (rabbit polyclonal) | Novus Biologicals | #NB100-2269 | WB (1:1000) |
| Antibody | Anti-astrin (rabbit polyclonal) | ProteinTech | #14726-1-AP | WB (1:1000) IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-FLAG (rabbit polyclonal) |
Sigma-Aldrich | #F7425 | IF (1:1000), WB (1:1000) IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-FLAG (mouse monoclonal) |
Sigma-Aldrich | #F3165 | IF (1:1000), WB (1:1000) |
| Antibody | Pan-actin (mouse monoclonal) | EMD Millipore | #MAB1501 | WB (1:2000) |
| Antibody | Anti-GAPDH (mouse monoclonal) | EMD Millipore | #MAB374 | WB (1:2000) |
| Antibody | Anti-CLUH (rabbit polyclonal) | Aviva | #ARP70642_P050 | WB (1:1000) |
| Antibody | Anti-kinastrin (rabbit polyclonal) | Abcam | #ab122769 | WB (1:1000) |
| Antibody | pH3-Ser10 (rabbit polyclonal) | Abcam | #ab5176 | WB (1:1000) |
| Antibody | Anti-SDHA (mouse monoclonal) | Molecular probes | #459,200 | WB (1:1000) |
| Antibody | Anti-NDUFA9 (mouse monoclonal) | Molecular probes | #459,100 | WB (1:1000) |
| Antibody | Anti-UQCRC1 (mouse monoclonal) | Molecular probes | #459,140 | WB (1:1000) |
| Antibody | pRB1-Ser807/811 (rabbit polyclonal) | Cell Signaling | #9308 | WB (1:1000) |
| Antibody | pCDK1-Tyr15 (rabbit monoclonal) | Cell Signaling | #4539 | WB (1:1000) |
| Antibody | Anti-cyclin D3 (mouse monoclonal) | Cell Signaling | #2936 | WB (1:1000) |
| Antibody | pRPS6-Ser235/236 (rabbit polyclonal) | Cell Signaling | #2211 | WB (1:1000) |
| Antibody | Anti-CDK1 (mouse monoclonal) | Santa Cruz Biotechnologies | #sc-54 | WB (1:1000) |
| Antibody | Anti-kinastrin (rabbit polyclonal) | Sigma-Aldrich | #SAB1103031 | WB (1:1000) IP (0.5 µg /300–500 µg protein) |
| AntibodyA | Anti-astrin (rabbit polyclonal) | Novus Biologicals | #NB100-74638 | IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-AFG3L1 (rabbit polyclonal) | Koppen et al., 2007 | N/A | IP (0.5 µg /300–500 µg protein) |
| Antibody | Anti-TOMM20 (mouse monoclonal) | Santa Cruz Biotechnologies | #sc-17764 | IF (1:1000) |
| Antibody | Anti-PCM1 (mouse monoclonal) |
Santa Cruz Biotechnologies | #sc-398365 | IF (1:200) |
| Antibody | Anti-CEP43 (mouse monoclonal) |
Sigma-Aldrich | #WH0011116M1 | IF (1:500) |
| Antibody | Anti-rabbit Alexa594 (donkey polyclonal) | Invitrogen | #A21207 | IF (1:1000) |
| Antibody | Anti-mouse Alexa488 (goat polyclonal) | Invitrogen | #A11029 | IF (1:1000) |
| Antibody | Anti-mouse Alexa594 (goat polyclonal) |
Invitrogen | #A11005 | IF (1:1000) |
| Antibody | Anti-rabbit Alexa488 (goat polyclonal) | Invitrogen | #A11034 | IF (1:1000) |
| Sequence-based reagent | SPAG5 forward | This paper | PCR primers | 5′-CATCTCACAGTGGGATAACTAATAAAC-3′ |
| Sequence-based reagent | SPAG5 reverse | This paper | PCR primers | 5′-CAGGGATAGGTGAAGCAAGGATA-3′ |
| Sequence-based reagent | GAPDH forward | This paper | PCR primers | 5′-AATCCCATCACCATCTTCCA-3′ |
| Sequence-based reagent | GAPDH reverse | This paper | PCR primers | 5'-TGGACTCCACGACGTACTCA-3' |
| Sequence-based reagent | RPL13 forward | This paper | PCR primers | 5′-CGGACCGTGCGAGGTAT-3′ |
| Sequence-based reagent | RPL13 reverse | This paper | PCR primers | 5′-CACCATCCGCTTTTTCTTGTC-3′ |
| Sequence-based reagent | MT-TL1 forward | Rooney et al., 2015 | PCR primers | 5′-CACCCAAGAACAGGGTTTGT-3′ |
| Sequence-based reagent | MT-TL1 reverse | Rooney et al., 2015 | PCR primers | 5′-TGGCCATGGGTATGTTGTTA-3′ |
| Sequence-based reagent | B2M forward | Rooney et al., 2015 | PCR primers | 5′-TGCTGTCTCCATGTTTGATGTATCT-3′ |
| Sequence-based reagent | B2M reverse | Rooney et al., 2015 | PCR primers | 5′-TCTCTGCTCCCCACCTCTAAGT-3′ |
| Commercial assay or kit | Click-it Nascent RNA Capture Kit | Invitrogen | C10365 | |
| Commercial assay or kit | SuperScript VILO cDNA synthesis kit | Invitrogen | 11754250 | Used in combination with Click-it Nascent RNA Capture Kit |
| Commercial assay or kit | SuperScript First-Strand Synthesis System | Invitrogen | 11904018 | |
| Chemical compound, drug | Doxycycline | Sigma-Aldrich | #D5207 | Final conc.: 1 µg/ml |
| Chemical compound, drug | L-Arginine-HCl | Silantes | #201604102 | Final conc.: 28 µg/ml |
| Chemical compound, drug | L-Lysine-HCl | Silantes | #211604102 | Final conc.: 73 µg/ml |
| Chemical compound, drug | Cycloheximide | Sigma-Aldrich | #C4859 | Final conc.: 100 µg/ml |
| Chemical compound, drug | MG132 | Sigma-Aldrich | #M8699 | Final conc.: 20 µM |
| Chemical compound, drug | Thymidine | Sigma-Aldrich | #T1895 | Final conc.: 2 mM |
| Chemical compound, drug | Nocodazole | Sigma-Aldrich | #M1404 | Final conc.: 100 ng/µl |
| Chemical compound, drug | Benzonase HC nuclease | Sigma-Aldrich | #71206 | Final amount per samples: 25 U |
| Chemical compound, drug | Propidium iodide | Sigma-Aldrich | #P4864 | Final conc.: 50 µg/ml |