Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA

PLOS ONE, Nov 2019

The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.

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Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA

April Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA Weidong An 0 1 Yifei Du 1 Keqiong Ye 1 0 College of Biological Sciences, China Agricultural University , Beijing , China , 2 National Institute of Biological Sciences , Beijing , China , 3 Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing , China , 4 University of Chinese Academy of Sciences , Beijing , China 1 Editor: Katrin Karbstein, Scripps Research Institute , UNITED STATES The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity. - Data Availability Statement: All coordinates and structural factors files are available from the Protein Data Bank database (accession number 5YZ4). Introduction Assembly of yeast ribosome requires processing and modification of rRNAs and association of 79 ribosomal proteins [ 1, 2 ]. This process begins with the transcription of a 35S precursor rRNA (pre-rRNA) that encodes 18S, 5.8S and 25S rRNAs and the external (5' ETS and 3' ETS) and internal (ITS1 and ITS2) transcribed spacers. The four spacers need to be removed by a series of endo- and exo-nucleolytic activities in the context of various pre-ribosomal particles [3]. Early processing of 18S rRNA occurs in the nucleolus within the 90S pre-ribosome or the small subunit processome [ 4, 5 ], which is the early assembly intermediate of small ribosomal subunits. The 90S pre-ribosome is co-transcriptionally assembled from ~70 non-ribosomal assembly factors (AFs), U3, U14, snR30 and snR10 snoRNAs and ~ 20 ribosomal proteins in a stepwise and dynamic manner [6±8]. At the final stage of 90S formation, the U14 and snR30 snoRNAs and a dozen labile AFs recruited by the 18S region are released, resulting in a fully study design, data collection and analysis, decision to publish, or preparation of the manuscript. assembled 90S [ 6, 9 ]. The cryo-EM structures of 90S have been recently determined, revealing that the nascent 40S ribosome is assembled into several isolated subdomains and stabilized by numerous AFs [10±14]. The pre-rRNA is sequentially cleaved at the A0 and A1 sites of the 5' ETS and at the A2 site of the ITS1. Consequently, the 90S is transformed into a pre-40S ribosome that contains a 20S pre-rRNA [ 15 ]. The 20S pre-rRNA is further processed at the D site in the cytoplasm to give rise to the mature 18S rRNA. In an alternative pathway, the 35S pre-rRNA is first cleaved at the A3 site in the ITS1, yielding a 23S intermediate for small subunits. The 23S pre-rRNA is also considered as a non-functional intermediate that strongly accumulates under stress conditions [ 16 ]. It can be targeted by an RNA surveillance system that involves oligoadenylation by the TRAMP complex and degradation by the nuclear exosome [ 17 ]. The PIN (PilT N-terminus) domain is a small domain with ~ 130 amino acid residues and is widely distributed in all three kingdoms of life [ 18 ]. It typically functions as an endonuclease in degradation and processing of various RNAs. The activity of PIN domain depends on four conserved acidic residues and the presence of Mn2+ or Mg2+ ion [ 19, 20 ]. Three PIN domain proteins, Utp24, Utp23 and Nob1, have been found to be required for 18S rRNA processing. Nob1 cleaves the D site, generating the 3' end of 18S rRNA [21]. Utp23 contains a degenerated PIN domain and functions together with snR30 snoRNA in the assembly of 90S [22±25]. Utp24 is the endonuclease proposed to cleave the A1 and A2 sites. Depletion of Utp24, aka Fcf1 (Faf1 copurifying factor 1), inhibits cleavage at sites A0, A1 and A2 in yeast [ 24, 26 ], whereas mutations of the active site of Utp24 specifically block cleavage at sites A1 and A2, but not at site A0 [24]. This suggests that Utp24 plays both a structural and catalytic role in 90S. In humans, inactivation of UTP24 similarly inhibits cleavage at the equivalent 1 and 2a sites [ 27, 28 ]. Structurall (...truncated)


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Weidong An, Yifei Du, Keqiong Ye. Structural and functional analysis of Utp24, an endonuclease for processing 18S ribosomal RNA, PLOS ONE, 2018, Volume 13, Issue 4, DOI: 10.1371/journal.pone.0195723