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Chaperoning 5S RNA assembly by structural probing

ABSTRACT: In eukaryotes, three of the four ribosomal RNAs (rRNAs), the 5.8S, 18S and 25S/28S rRNAs, are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (5S RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into pre-ribosomes. In mammals the 5S RNP is also central regulator of the homeostasis of the tumour suppressor p53. The nucleolar localisation of the 5S RNP and its assembly into pre-ribosomes is performed by a specialised complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterisation of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialised 5S RNA E loop binding module, contacts the Rpl5 protein and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in pre-ribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.

ORGANISM(S): Saccharomyces cerevisiae BY4741

PROVIDER: GSE68431 | GEO | 2015/07/20

SECONDARY ACCESSION(S): PRJNA282731

REPOSITORIES: GEO

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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.

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Chaperoning 5S RNA assembly by structural probing

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