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Proteomic evaluation of yeast pre-60S ribosomes_2022

ABSTRACT: The Nsa1-TAP and Rlp7-TAP purified pre-60S ribosomes from WT or tif4631Δ cell were subjected to LC-MS/MS analysis.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Yun-Ting Tseng

LAB HEAD: Lo, Kai-Yin

PROVIDER: PXD033082 | Pride | 2022-06-23

REPOSITORIES: Pride

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			Action	DRS
	20200117_NTUKYLo_4G1_Nsa1__1_.mzML	Mzml
	20200117_NTUKYLo_4G1_Nsa1__1_.raw	Raw
	20200117_NTUKYLo_PeptideGroups.txt	Txt
	20200117_NTUKYLo_Proteins.txt	Txt
	20200117_NTUKYLo_WT_Nsa1__1_.mzML	Mzml

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Project description:The eukaryotic ribosome biogenesis is a highly orchestrated multistep process that starts at the nucleolus with the transcription of pre-rRNAs 5S and 35S. The latter comprises the mature 18S, 5.8S and 25S rRNAs separated by internal transcribed spacers (ITS1 and ITS2) and externally flanked by the 5’ETS and 3’ETS. The 35S pre-rRNA undergoes several co- and post-transcriptional processing events, which will enable the pre-60S and pre-40S particles to take independent maturation routes. Hundreds of assembly factors (AF) are required, being recruited and released hierarchically, for the proper folding of the rRNAs and correct positioning of the ribosomal proteins. One of the most intricate events that have recently been described is the removal of the ITS2-containing structure called pre-60S foot, which happens in a step-wise manner. Nop53 is an essential 60S AF that binds close to the ITS2 and plays a fundamental role in recruiting the RNA exosome for the 7S pre-rRNA processing, thereby dismantling the foot structure. Here we characterize the impact of Nop53 binding to the pre-60S on the compositional changes that happen during 60S assembly. For this purpose, preribosomes were affinity-purified with TAP-tagged 60S AFs (Nop7, Erb1, Rsa4, Arx1, Nmd3, Yvh1, and Lsg1) representative of different maturation stages both in the presence and absence of Nop53. Nop7 particles were also coimmunoprecipitated in the presence of Nop53 mutants incapable of recruiting the exosome (Nop53∆1-71, Nop53∆48-98) to compare with Nop53 depletion. The isolated preribosomes were analyzed by label-free MS/MS-based quantitative proteomics, revealing early and late-stage specific effects of Nop53 depletion.

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