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The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

ABSTRACT: Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center and is responsible for stabilizing interactions between the 5’ and 3’ ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

INSTRUMENT(S): Q Exactive HF

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

SUBMITTER: Kuan-Ting Pan

LAB HEAD: Henning Urlaub

PROVIDER: PXD022625 | Pride | 2021-08-23

REPOSITORIES: Pride

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			Action	DRS
	G_Aquino_070918_021018_R2_L3_21.raw	Raw
	G_Aquino_070918_021018_R2_L3_22.raw	Raw
	G_Aquino_070918_021018_R2_L3_23.raw	Raw
	G_Aquino_070918_021018_R2_L4_1.raw	Raw
	G_Aquino_070918_021018_R2_L4_10.raw	Raw

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The RNA helicase Dbp7 promotes domain VI compaction and stabilization of inter-domain interactions during early 60S assembly

Project description:Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center and is responsible for stabilizing interactions between the 5’ and 3’ ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

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The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly

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Project description:Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here we introduce Rbp95, a constituent of early pre-60S particles, as a novel ribosome assembly factor that likely already binds before the 90S to pre-60S transition. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members, and to ribosomal protein Rpl3. Notably, the RNA-binding protein Rbp95 associates with helix H95 in the 3’ region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. In the absence of Rbp95, proteins binding in proximity to helix H95 are less efficiently recruited to early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the formation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex in the 90S to pre-60S transition and in promoting pre-rRNA folding events within pre-60S particles that facilitate the recruitment of subsequent assembly factors.

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