A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.
Department of Biology II, Ulm University, 89069 Ulm, Germany.
Jutta
J
Brendel
Department of Biology II, Ulm University, 89069 Ulm, Germany.
Britta
B
Stoll
Bioinformatics Group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany.
Sita J
SJ
Lange
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Kundan
K
Sharma
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Bioanalytics, Institute of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
Christof
C
Lenz
Department of Biology II, Ulm University, 89069 Ulm, Germany.
Aris-Edda
AE
Stachler
Department of Biology II, Ulm University, 89069 Ulm, Germany.
Lisa-Katharina
LK
Maier
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany.
Hagen
H
Richter
Institute for General Microbiology, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany.
Lisa
L
Nickel
Institute for General Microbiology, Christian-Albrechts-Universität Kiel, 24118 Kiel, Germany.
Ruth A
RA
Schmitz
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany.
Lennart
L
Randau
School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
Thorsten
T
Allers
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Bioanalytics, Institute of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
Henning
H
Urlaub
Bioinformatics Group, Department of Computer Science, University of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), Cluster of Excellence, University of Freiburg, 79110 Freiburg, Germany.
Rolf
R
Backofen
Department of Biology II, Ulm University, 89069 Ulm, Germany. Electronic address: anita.marchfelder@uni-ulm.de.
Anita
A
Marchfelder
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D019622:Haloferax volcanii / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020871:RNA Stability; D019642:RNA, Archaeal / Q000737:chemistry*
D019843:Archaeal Proteins; D019642:RNA, Archaeal
Archaea; CRISPR/Cas; Cas6; Haloferax volcanii; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; Type I-B; crRNA