Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs.
A hallmark of defense mechanisms based on clustered regularly interspaced short palindromic repeats (CRISPR) and associated sequences (Cas) are the crRNAs that guide these complexes in the destruction of invading DNA or RNA. Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Based on genetic and transcriptomic evidence, two associated endoribonucleases, Cas6-1 and Cas6-2a, were postulated to be involved in crRNA maturation from CRISPR1 or CRISPR2, respectively. Here, we report a promiscuity of both enzymes to process in vitro not only their cognate transcripts, but also the respective non-cognate precursors, whereas they are specific in vivo Moreover, while most of the repeats serving as substrates were cleaved in vitro, some were not. RNA structure predictions suggested that the context sequence surrounding a repeat can interfere with its stable folding. Indeed, structure accuracy calculations of the hairpin motifs within the repeat sequences explained the majority of analyzed cleavage reactions, making this a good measure for predicting successful cleavage events. We conclude that the cleavage of CRISPR1 and CRISPR2 repeat instances requires a stable formation of the characteristic hairpin motif, which is similar between the two types of repeats. The influence of surrounding sequences might partially explain variations in crRNA abundances and should be considered when designing artificial CRISPR arrays.
Genetics and Experimental Bioinformatics group, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Viktoria
V
Reimann
Bioinformatics group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany.
Omer S
OS
Alkhnbashi
Bioinformatics group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany.
Sita J
SJ
Saunders
Genetics and Experimental Bioinformatics group, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Ingeborg
I
Scholz
Genetics and Experimental Bioinformatics group, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Stephanie
S
Hein
Bioinformatics group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany backofen@informatik.uni-freiburg.de.|Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Habsburgerstrasse 49, D-79104 Freiburg, Germany.|BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, D-79104 Freiburg, Germany.
Rolf
R
Backofen
Genetics and Experimental Bioinformatics group, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany wolfgang.hess@biologie.uni-freiburg.de.|Centre for Biological Systems Analysis (ZBSA), University of Freiburg, Habsburgerstrasse 49, D-79104 Freiburg, Germany.
Wolfgang R
WR
Hess
D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004722:Endoribonucleases / Q000378:metabolism; D055029:Inverted Repeat Sequences; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000235:genetics*; D059367:RNA Cleavage; D012260:Ribonucleases / Q000378:metabolism; D013379:Substrate Specificity
D009843:Oligoribonucleotides; D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases