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IGBMC-human-HeLa-TAF10 and TBP-HeLa-polysome RNA Ips

ABSTRACT: RNA immunoprecipitation using mouse monoclonal antibody against human TAF10 protein from HeLa polysome extracts Cells dedicate significant energy to building proteins1, which are often organized in multiprotein assemblies with tightly regulated stoichiometries2. Cotranslational assembly, a process of synchronous translation and protein heterodimerization, is a potential mechanism for efficient matching of partner subunits and avoiding the negative effects of protein aggregation3. Recent studies in bacteria demonstrate that cotranslational assembly of the LuxA-LuxB dimer follows the order established by operon structure and is more efficient than post-translational assembly4. As genes encoding protein complex subunits are dispersed among chromosomes in eukaryotes, it is unclear how cotranslational assembly is accomplished mechanistically, but studies in yeast have nevertheless suggested it as a potential assembly pathway5,6,7. Here we show that mammalian transcription complexes, such as the RNA polymerase II general transcription factor TFIID and the TRanscription and EXport complex-2 (TREX-2) assemble co-translationally. Moreover, we show that the position of heterodimerization domains determines the order of cotranslational assembly in mammalian TFIID. In polysomes, the TATA binding protein associated factor 10 (TAF10), which contains a C-terminal histone-fold dimerization domain (HFD) is recruited cotranslationally to its HFD-containing binding partner TAF8. This interaction is established unidirectionally and determined by the position rather than the sequence of the dimerization domain. We further show that similar mechanisms guide the assembly of other TFIID subunits. Our results thus predict that cotranslational assembly of eukaryotic multisubunit complexes is a general principle in building multiprotein machines. We used microarrays to assess globally the mRNAs associated with TAF10 and TBP immunoprecipitations from HeLa polysomes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106299 | GEO | 2017/10/28

SECONDARY ACCESSION(S): PRJNA416143

REPOSITORIES: GEO

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Project description:Folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, since non-productive interactions can lead to misfolding, aggregation and degradation1. Cells cope with this challenge by coupling synthesis with polypeptide folding and by employing molecular chaperones to safeguard folding already cotranslationally2. However, little is known about the final step of folding, the assembly of polypeptides into complexes, although most of the cellular proteome forms oligomeric assemblies3. In prokaryotes, a proof-of-concept study showed that assembly of heterodimeric luciferase is an organized cotranslational process, facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA4. In eukaryotes, however, fundamental differences such as rarity of polycistronic mRNAs and different chaperone constellations raise the question whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of protein complex assembly in eukaryotes using ribosome profiling. We determined the in vivo nascent subunits interactions of 12 hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find 9 complexes assemble cotranslationally; the 3 complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones5-7. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit(s), thereby counteracting its aggregation propensity. The onset of cotranslational subunit association coincides sharply with full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb8 is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains, then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for hetero-oligomers assembly in yeast and indicates that translation, folding and assembly of protein complexes are integrated processes in eukaryotes.

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IGBMC-human-HeLa-TAF10 and TBP-HeLa-polysome RNA Ips

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