Abstract
During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.
Highlights
The eukaryotic ribosomal RNA (rRNA) are processed from the 35S (Saccharomyces cerevisiae) or 47S (Homo sapiens) rRNA precursors by initial endonucleolytic cleavages and subsequent exonucleolytic trimming, with concomitant removal of external (5’-ETS, 3’-ETS)and internal (ITS1, ITS2) transcribed spacer sequences (Figures 1 and S1).Early pre-rRNA cleavages, at sites called A0, A1 and A2 in yeast and A0, A1/1 and 2a/E in humans, are important for 18S rRNA maturation
To dissect the roles of the two yeast candidate pre-rRNA endonucleases Rcl1 and Utp24, we applied in vivo RNA-protein crosslinking (CRAC) to identify their RNA binding sites [21]
We present data implicating Utp24 as the endonuclease responsible for early prerRNA cleavages at sites A1/1 and A2/2a that generate the major precursor to the 18S rRNA
Summary
The eukaryotic rRNAs are processed from the 35S (Saccharomyces cerevisiae) or 47S (Homo sapiens) rRNA precursors (pre-rRNAs) by initial endonucleolytic cleavages and subsequent exonucleolytic trimming, with concomitant removal of external (5’-ETS, 3’-ETS). Pre-rRNA cleavages, at sites called A0, A1 and A2 in yeast and A0, A1/1 and 2a/E in humans, are important for 18S rRNA maturation These events require a large ribonucleoprotein complex, the small subunit (SSU) processome (or 90S pre-ribosome). Mutation of Rcl inhibited site A2 cleavage, with less effect at A0 and A1 [5,9,10], and recombinant Rcl was reported to cleave pre-rRNA transcripts containing yeast site A2, consistent with direct endonuclease activity [9]. We established RNAi-rescue systems in HEK293 cells to study the effect of presumably catalytically inactive UTP24 and RCL1 mutants on pre-rRNA cleavage in the human system
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
