Project description:RNA polymerase II (Pol II) transcription initiation starts with the assembly of the preinitiation complex (PIC) on core promoters. The PIC is composed of six general transcription factors (GTFs). The recognition of the core promoter sequences by the TFIID GTF complex is the first step of the PIC assembly. In metazoans, holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Genetic depletion of different murine TAFs have shown that TAFs such as TAF7 or TAF10, can be either required, or dispensable for Pol II transcription, depending on different cellular contexts. In this report, we depleted TAF7 and/or TAF10 in the same cellular system; either in mesodermal progenitors during mouse development or in mESCs. In these two models, TAF7 depletion leads to a milder phenotype compared to TAF10 depletion. As TAF10 is also a subunit of the transcriptional co-activator Spt-Ada-Gcn5 acetyl transferase (SAGA), we first showed that the difference in phenotype between the Taf7 and Taf10 mutant is not due to the SAGA effect, at least for mESCs. Immunoprecipitations coupled with mass spectrometry analyses from mESCs lysates assembly of holo-TFIID complex is rapidly affected after induction of the depletion. In line with the model of holo-TFIID sequential assembly, TAF10 depletion leads to an early defect with formation of the core-TFIID, while TAF7 depletion results in the formation of a TAF7-less TFIID. Thus, the difference in phenotype severity correlates with the degree of TFIID disassembly. Surprisingly, no major global changes in Pol II transcription could be observed after either TAF7 or TAF10 depletion. Our data suggest that the inducible loss of fully assembled canonical TFIID does not correlate with the lack of global Pol II transcription activity changes suggesting that partially assembled TFIID complexes can participate in Pol II transcription initiation, with only limited effect on Pol II nascent transcription.

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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