The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans

Wells, Graeme Raymond; Weichmann, Franziska; Colvin, David; Sloan, Katherine E.; Kudla, Grzegorz; Tollervey, David; Watkins, Nicholas J.; Schneider, Claudia

doi:10.1093/nar/gkw213

Cited by 61 publications

(81 citation statements)

References 37 publications

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“…This sequence is not known to be the target of any endoribonuclease activity. However, recent RNA–protein crosslinking data (CRAC analysis (39)) showed that the Utp24 endoribonuclease was predominately associated with sequences within the 18S rRNA sequence (14). The main peak of association was located around position +1103 relative to A 1 , in the 3′ part of the central domain.…”

Section: Resultsmentioning

confidence: 99%

“…For this purpose, we constructed a strain lacking the RRP6 gene, strongly accumulating 23S, in which HA-tagged Utp24 is expressed under the control of the tetracycline repressible promoter (pTetO7). This strain was next transformed with a plasmid expressing either WT, Utp24 PIN mutant allele (Utp24-D68N) (13,14) or no protein. Ten hours following addition of doxycycline, fully functional genomic Utp24 is largely depleted (Supplementary Figure S2) and the only available source of Utp24 in the cell is the version expressed from the plasmid (WT, PIN mutant or mock).…”

Section: Resultsmentioning

confidence: 99%

“…HTP-tagged forms of Utp24 were initially amplified using CS-327 and CS-332 from UTP24::HTP strain described in (14). The PCR fragments were cloned into pRS315 plasmid cut with SalI.…”

Section: Methodsmentioning

confidence: 99%

“…Oligonucleotides used to construct UTP24::D68N mutant plasmid by site-directed mutagenesis are described in (14). HTP tag was next shortened at their C-terminus by site directed mutagenesis introducing a stop codon immediately after the TEV cleavage site using CS-599 and CS-600 oligonucleotides.…”

Section: Methodsmentioning

confidence: 99%

“…Consistent with a direct involvement in these cleavages, Utp24 exhibited in vitro endonuclease activity on an RNA substrate containing the A 2 site. Moreover, the integrity of the Utp24 PINc domain is required for efficient cleavage at A 2 site, both in yeast and human (14). …”

Section: Introductionmentioning

confidence: 99%

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Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway

Choque

Schneider

Gadal

et al. 2018

Nucleic Acids Research

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Ribosome biogenesis requires more than 200 trans-acting factors to achieve the correct production of the two mature ribosomal subunits. Here, we have identified Efg1 as a novel, nucleolar ribosome biogenesis factor in Saccharomyces cerevisiae that is directly linked to the surveillance of pre-40S particles. Depletion of Efg1 impairs early pre-rRNA processing, leading to a strong decrease in 18S rRNA and 40S subunit levels and an accumulation of the aberrant 23S rRNA. Using Efg1 as bait, we revealed a novel degradation pathway of the 23S rRNA. Co-immunoprecipitation experiments showed that Efg1 is a component of 90S pre-ribosomes, as it is associated with the 35S pre-rRNA and U3 snoRNA, but has stronger affinity for 23S pre-rRNA and its novel degradation intermediate 11S rRNA. 23S is cleaved at a new site, Q1, within the 18S sequence by the endonuclease Utp24, generating 11S and 17S' rRNA. Both of these cleavage products are targeted for degradation by the TRAMP/exosome complexes. Therefore, the Q1 site defines a novel endonucleolytic cleavage site of ribosomal RNA exclusively dedicated to surveillance of pre-ribosomal particles.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…HTP-tagged forms of Utp24 were initially amplified using CS-327 and CS-332 from UTP24::HTP strain described in (14). The PCR fragments were cloned into pRS315 plasmid cut with SalI.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway

Choque

Schneider

Gadal

et al. 2018

Nucleic Acids Research

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Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

2017

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Edited by Michael IbbaProper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo-and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans.Keywords: endoribonuclease; exoribonuclease; pre-rRNA processing; ribosome biogenesis; RNA quality control; RNA trimming Ribosome biosynthesis is a multistep process that includes several tightly controlled events -ribosomal DNA (rDNA) transcription, processing of rRNA precursors into mature molecules, accompanied by binding of ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs), and the final assembly of all Abbreviations CD, catalytic domain; dsRBD, double-stranded RNA-binding domain; ETS, external transcribed spacer; ITS, internal transcribed spacer; LSU, large ribosome subunit (60S); miRNA, microRNA; mRNA, messenger RNA; MRP RNA, noncoding RNA component of the RNase MRP; mTOR, mammalian target of rapamycin; nt(s), nucleotide(s); PIN domain, PilT N-terminal domain; Pol I/II/III, RNA polymerase I/II/III; prerRNA, ribosomal RNA precursor; RBF, ribosome biogenesis factor; RMRP, RNase MRP (mitochondrial RNA processing ribonuclease); RNase, ribonuclease; RNB domain, RNase II domain; RNP, ribonucleoprotein; RP, ribosomal protein; rRNA, ribosomal RNA; siRNA, short interfering RNA; snoRNA, small nucleolar RNA; snoRNP, small nucleolar ribonucleoprotein; snRNA, small nuclear RNA; SSU, small ribosome subunit (40S); TRAMP4 or TRAMP5, Trf4/Air/Mtr4 or Trf5/Air/Mtr4 polyadenylation complex; tRNA, transfer RNA.

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Maturation of pre‐40S particles in yeast and humans

et al. 2018

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checkpoints/quality control steps in the pre-40S particle maturation pathway. A very recent review provides a complementary view to ours on small subunit biogenesis in yeast (Chaker-Margot, 2018). 2 | PRE-40S PARTICLE MATURATION IN SACCHAROMYCES CEREVISIAE 2.1 | Maturation pathway of pre-40S particles in S. cerevisiaeThe first precursor to the small ribosomal subunit is termed the small subunit (SSU) processome, also referred to as the 90S pre-ribosomal particle. It is formed by the co-transcriptional assembly of most of the small subunit ribosomal proteins (RPSs), AFs, and small nucleolar ribonucleoprotein particles (snoRNPs) on the nascent pre-rRNA transcript. The assembly and structure of this fascinating entity has been reviewed recently (Barandun, Hunziker, & Klinge, 2018) and will not be discussed further here. We will mostly concentrate on the maturation steps that follow the disassembly of the SSU processome, which leads to the release of the first pre-40S particle that contains the 20S pre-rRNA. This RNA species is generated by endonucleolytic cleavages of the RNA polymerase I transcript at sites A1, the 5 0 end of 18S rRNA and A2, within internal transcribed spacer 1 (ITS1). 20S pre-rRNA thus encompasses the 18S rRNA flanked at its 3 0 extremity by a 211-nucleotide-long fragment of ITS1. To yield the mature 40S ribosomal subunit, the evolving pre-40S particle undergoes maturation steps in the nucleus and the cytoplasm that include RNA conformational rearrangements, stabilization of RP integration, elimination of the ITS1 fragment, and removal of all AFs.The first pre-40S particle generated following disassembly of the SSU processome contains, in addition to the 20S pre-rRNA, the following AFs that have been recruited during formation of the SSU processome: Enp1p, the endonuclease Nob1p and its partner Pno1p/Dim2p, the methyltransferase Dim1p, and probably the helicase Prp43p. These AFs are then joined in the nucleus by Ltv1p, the kinase Hrr25p, the ATPase/kinase Rio2p, and the GTPase-like protein Tsr1p. The potential roles of these AFs in the maturation process and their dissociation stage will be discussed below. Of particular note is the fact that Nob1p is the endonuclease responsible for the cleavage at the 3 0 end of the 18S rRNA sequence (termed site D) that eliminates the ITS1 fragment and thus releases 18S rRNA. Nob1p is required for D-site cleavage in vivo and is able on its own to specifically perform this cleavage on a naked in vitro-transcribed RNA substrate (Fatica,

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The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans

Cited by 61 publications

References 37 publications

Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway

Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway

Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

Maturation of pre‐40S particles in yeast and humans

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