Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 52; pp. 21218 - 21222
• Main Authors: Hatoum-Aslan, Asma, Maniv, Inbal, Marraffini, Luciano A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27.12.2011; National Acad Sciences
• Subjects: Antisense; Bacterial proteins; Biological Sciences; Blotting, Northern; Cellular apoptosis susceptibility protein; DNA; DNA Primers - genetics; Gene loci; genes; Genetic loci; Genetic mutation; Genetics; Inverted Repeat Sequences - genetics; loci; Measuring rulers; MicroRNA; Models, Genetic; Molecular modelling; Nucleotides; Oligonucleotides, Antisense - genetics; Plasmids; Plasmids - genetics; Prokaryotes; proteins; RNA; RNA precursors; RNA processing; RNA Processing, Post-Transcriptional - genetics; RNA Processing, Post-Transcriptional - physiology; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA-mediated interference; RNA-protein interactions; Staphylococcus epidermidis; Staphylococcus epidermidis - genetics; Staphylococcus epidermidis - physiology; Staphylococcus infections
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1112832108

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.