ArticleRNA (New York, N.Y.)2011
Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies.
Article in RNA (New York, N.Y.), 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed.
- Nucleoplasmic checkpoint of the 40S ribosomal decoding center maturation.Cell reports · 2026Article
- LBR and LAP2 mediate heterochromatin tethering to the nuclear periphery to preserve genome homeostasis.Nature cell biology · 2026Article
- The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).Molecular and cellular biology · 2024Article
- Ribosome biogenesis factors-from names to functions.The EMBO journal · 2023Review
- Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism.Nucleic acids research · 2022Article
- ATRX proximal protein associations boast roles beyond histone deposition.PLoS genetics · 2021Article
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- RIOK2 phosphorylation by RSK promotes synthesis of the human small ribosomal subunit.PLoS genetics · 2021Article
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- Structural basis for DEAH-helicase activation by G-patch proteins.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.eLife · 2020Article
- Identification of distinct maturation steps involved in human 40S ribosomal subunit biosynthesis.Nature communications · 2020Article
- In vitro dimerization of human RIO2 kinase.RNA biology · 2019Article
- Single-cell RNA-sequencing of herpes simplex virus 1-infected cells connects NRF2 activation to an antiviral program.Nature communications · 2019Article
- Interactions and activities of factors involved in the late stages of human 18S rRNA maturation.RNA biology · 2019Article
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- Low-level miR-646 in colorectal cancer inhibits cell proliferation and migration by targeting NOB1 expression.Oncology letters · 2017Article
- Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation.Nucleic acids research · 2017Article
Corrections and comments
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Authors and funding
7 authors.
Funding
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Abstract
Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells. As a proof of principle, we have applied this method to the purification of human pre-ribosomal particles. Using inducible expression of ribosome assembly factors as bait proteins, different pre-40S particles could be isolated and characterized, revealing high conservation of the ribosome biogenesis pathway from yeast to human cells. Besides known ribosome maturation factors, C21orf70 was identified as a novel pre-40S component. By combining TAP of pre-40S particles with shRNA-mediated depletion of the pre-40S-associated protein kinase Rio2, we observed that increased levels of the nuclear HEAT-repeat protein Rrp12 are associated with 40S precursors in absence of Rio2. Further analyses revealed that Rrp12 is partially mislocalized to the cytoplasm and trapped on late 40S precursors upon loss of Rio2, and therefore fails to efficiently recycle to the nucleus. Thus, the combination of tandem affinity purification and shRNA induction provided further insights into late cytoplasmic 40S maturation steps, demonstrating the high potential of this method.
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