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

被引:61
作者
Brendel, Jutta [1 ]
Stoll, Britta [1 ]
Lange, Sita J. [2 ]
Sharma, Kundan [3 ]
Lenz, Christof [3 ,8 ]
Stachler, Aris-Edda [1 ]
Maier, Lisa-Katharina [1 ]
Richter, Hagen [4 ]
Nickel, Lisa [5 ]
Schmitz, Ruth A. [5 ]
Randau, Lennart [4 ]
Allers, Thorsten [6 ]
Urlaub, Henning [3 ,8 ]
Backofen, Rolf [2 ,7 ]
Marchfelder, Anita [1 ]
机构
[1] Univ Ulm, Dept Biol 2, D-89069 Ulm, Germany
[2] Univ Freiburg, Dept Comp Sci, Bioinformat Grp, D-79110 Freiburg, Germany
[3] Max Planck Inst Biophys Chem, D-37077 Gottingen, Germany
[4] Max Planck Inst Terr Microbiol, D-35043 Marburg, Germany
[5] Univ Kiel, Inst Gen Microbiol, D-24118 Kiel, Germany
[6] Univ Nottingham, Sch Life Sci, Queens Med Ctr, Nottingham NG7 2UH, England
[7] Univ Freiburg, Ctr Biol Signalling Studies BIOSS, Cluster Excellence, D-79110 Freiburg, Germany
[8] Univ Med Ctr Gottingen, Inst Clin Chem, D-37075 Gottingen, Germany
关键词
Archaea; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; CRISPR; Cas; Cas6; Haloferax volcanii; crRNA; Type I-B; PROCESSES PRE-CRRNA; CRISPR RNA; ANTIVIRAL DEFENSE; IMMUNE-SYSTEM; SEQUENCE; DNA; RECOGNITION; MECHANISM; CLEAVAGE; INTERFERENCE;
D O I
10.1074/jbc.M113.508184
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: The Cas6 protein is required for generating crRNAs in CRISPR-Cas I and III systems. Results: The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax. Conclusion: A Cascade-like complex is required in the type I-B system for a stable crRNA population. Significance: The CRISPR-Cas system I-B has a similar Cascade complex like types I-A and I-E. 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.
引用
收藏
页码:7164 / 7177
页数:14
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