Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast

Grzegorz Kudla, Sander Granneman, Daniela Hahn, Jean D Beggs, David Tollervey

Research output: Contribution to journalArticlepeer-review

Abstract

Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3' side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3' domain and long-range contacts between the 3' and 5' domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.
Original languageEnglish
Pages (from-to)10010–10015
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume108
Issue number24
DOIs
Publication statusPublished - 2011

Keywords

  • pre-rRNA
  • RNA structure
  • ribosome synthesis
  • UV cross-linking

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