Dataset Information

XL-MS of 5S RNP preribosomal complexes

ABSTRACT: The 5S RNP is assembled from its three components (5S rRNA, Rpl5/uL18, and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2–p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-EM structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1–uL18–uL5, and upon further recruitment of two nucleolar factors Rpf2–Rrs1 develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unraveled how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Henrik Hammarén

LAB HEAD: Martin Beck

PROVIDER: PXD040087 | Pride | 2023-06-13

REPOSITORIES: Pride

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Publications

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

Castillo Duque de Estrada Nestor Miguel NM Thoms Matthias M Flemming Dirk D Hammaren Henrik M HM Buschauer Robert R Ameismeier Michael M Baßler Jochen J Beck Martin M Beckmann Roland R Hurt Ed E

Nature structural & molecular biology 20230608 8

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial ...[more]

PMID: 37291423

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Project description:Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and rRNA remodeling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors (AFs), organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these AFs function are largely unknown. Here, we use cryo-electron microscopy (cryo-EM), to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged AF Nog2. Our data pinpoint the locations and determine the structures of over 20 AFs, which are enriched in two areas, an arc region extending from the central protuberance (CP) to the polypeptide tunnel exit (PTE), and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S rRNAs. These structural data suggest that the arc-located factors might function to chaperone formation of RNA helices found in the active sites of the subunit, including helices from the CP, peptidyl-transferase center (PTC) and intersubunit bridge. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant AFs and functional rRNA elements, manifesting their critical roles in structural remodeling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodeling events before nuclear export: rotation of the 5S RNP, construction of the active center, and ITS2 removal. Therefore, our structures provide a framework to understand the molecular roles of diverse AFs, and potentially the atomic information therein constitutes a resource to generalize principles governing the elusive functions of nuclear RNA-binding proteins.

Dataset Information

XL-MS of 5S RNP preribosomal complexes

Publications

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

Similar Datasets

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