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| Status |
Public on Apr 01, 2016 |
| Title |
The PIN domain endonuclease Utp24 cleaves pre-ribosomal RNA at two coupled sites in yeast and humans |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell.
Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately.
Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA.
Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2.
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| Overall design |
Examination of targets for pre-rRNA endonucleases in yeast cells.
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| Contributor(s) |
Wells GR, Weichmann F, Colvin D, Sloan KE, Kudla G, Tollervey D, Watkins NJ, Schneider C |
| Citation(s) |
27034467 |
| Submission date |
Dec 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Claudia Schneider |
| E-mail(s) |
Claudia.Schneider@ncl.ac.uk
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| Organization name |
Newcastle University
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| Street address |
Framlington Place
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| City |
Newcastle upon Tyne |
| ZIP/Postal code |
NE2 4HH |
| Country |
United Kingdom |
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| Platforms (2) |
| GPL13272 |
Illumina Genome Analyzer IIx (Saccharomyces cerevisiae) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA305911 |
| SRA |
SRP067353 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE75991_RAW.tar |
30.0 Kb |
(http)(custom) |
TAR (of WIG) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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