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The Adaptive Immune System of Haloferax volcanii.

Maier LK, Dyall-Smith M, Marchfelder A - Life (Basel) (2015)

Bottom Line: To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated).A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system.For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology II, Ulm University, 89069 Ulm, Germany. lisa-katharina.maier@uni-ulm.de.

ABSTRACT
To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

No MeSH data available.


The different forms of the crRNA. (A) The long form of the crRNA found in vivo contains the spacer sequence, an eight nucleotide long 5' handle and a 22 nucleotide long 3' handle. (B) The short form of the crRNA found in vivo contains only five repeat derived nucleotides at the 3' end resulting in a shorter 3' handle. (C) The shortest functional version of the crRNA does not contain a 3' handle at all.
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life-05-00521-f004: The different forms of the crRNA. (A) The long form of the crRNA found in vivo contains the spacer sequence, an eight nucleotide long 5' handle and a 22 nucleotide long 3' handle. (B) The short form of the crRNA found in vivo contains only five repeat derived nucleotides at the 3' end resulting in a shorter 3' handle. (C) The shortest functional version of the crRNA does not contain a 3' handle at all.

Mentions: The central molecule of the defence reaction is the crRNA, consisting of spacer and repeat sequences [25]. The repeat sequences of the haloarchaeal CRISPR RNAs are highly conserved and can form a stem loop structure with a three bp stem (Figure 4A) [36]. Analysis of the Haloferax crRNA population by high-throughput sequencing revealed that cleavage of the pre-crRNA takes place between nucleotides 22 and 23, right at the base of the potential hairpin motif (Figure 2 and Figure 4) [36]. This leaves an eight-nucleotide 5' handle originating from the upstream repeat sequence (that precedes the spacer sequence), and a 22-nucleotide 3' handle downstream of the spacer. As mentioned above, the CRISPR repeats of Haloferax are identical except for one position, nucleotide 23 (Figure 2B), and after processing this would result in mature crRNAs that differ at the first base (the 5' nucleotide, Figure 4). RNAseq and northern blot analysis showed that the majority of the stably maintained crRNA population are between 64 and 69 nt in length (due to spacer length differences), with an average of 66 nucleotides [36]. The crRNAs consist of the eight nucleotide 5' handle, the 34–39 nt long spacer and the 22 nt long 3' handle. Further analysis revealed a second population of crRNAs with a 3' handle of only five nucleotides. This differs from crRNA maturation in other type I systems, where after the initial cleavage by Cas6 no further processing is observed (types I-A, I-E and I-F) [25]. A similar shortening of the 3' end has been reported for the type I-B systems in two other microorganisms, Methanococcus maripaludis and Clostridium thermocellum [35]. Together, these data clearly show that type I-B crRNA maturation is different from the same process in type I-A, -E and -F systems.


The Adaptive Immune System of Haloferax volcanii.

Maier LK, Dyall-Smith M, Marchfelder A - Life (Basel) (2015)

The different forms of the crRNA. (A) The long form of the crRNA found in vivo contains the spacer sequence, an eight nucleotide long 5' handle and a 22 nucleotide long 3' handle. (B) The short form of the crRNA found in vivo contains only five repeat derived nucleotides at the 3' end resulting in a shorter 3' handle. (C) The shortest functional version of the crRNA does not contain a 3' handle at all.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390866&req=5

life-05-00521-f004: The different forms of the crRNA. (A) The long form of the crRNA found in vivo contains the spacer sequence, an eight nucleotide long 5' handle and a 22 nucleotide long 3' handle. (B) The short form of the crRNA found in vivo contains only five repeat derived nucleotides at the 3' end resulting in a shorter 3' handle. (C) The shortest functional version of the crRNA does not contain a 3' handle at all.
Mentions: The central molecule of the defence reaction is the crRNA, consisting of spacer and repeat sequences [25]. The repeat sequences of the haloarchaeal CRISPR RNAs are highly conserved and can form a stem loop structure with a three bp stem (Figure 4A) [36]. Analysis of the Haloferax crRNA population by high-throughput sequencing revealed that cleavage of the pre-crRNA takes place between nucleotides 22 and 23, right at the base of the potential hairpin motif (Figure 2 and Figure 4) [36]. This leaves an eight-nucleotide 5' handle originating from the upstream repeat sequence (that precedes the spacer sequence), and a 22-nucleotide 3' handle downstream of the spacer. As mentioned above, the CRISPR repeats of Haloferax are identical except for one position, nucleotide 23 (Figure 2B), and after processing this would result in mature crRNAs that differ at the first base (the 5' nucleotide, Figure 4). RNAseq and northern blot analysis showed that the majority of the stably maintained crRNA population are between 64 and 69 nt in length (due to spacer length differences), with an average of 66 nucleotides [36]. The crRNAs consist of the eight nucleotide 5' handle, the 34–39 nt long spacer and the 22 nt long 3' handle. Further analysis revealed a second population of crRNAs with a 3' handle of only five nucleotides. This differs from crRNA maturation in other type I systems, where after the initial cleavage by Cas6 no further processing is observed (types I-A, I-E and I-F) [25]. A similar shortening of the 3' end has been reported for the type I-B systems in two other microorganisms, Methanococcus maripaludis and Clostridium thermocellum [35]. Together, these data clearly show that type I-B crRNA maturation is different from the same process in type I-A, -E and -F systems.

Bottom Line: To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated).A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system.For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology II, Ulm University, 89069 Ulm, Germany. lisa-katharina.maier@uni-ulm.de.

ABSTRACT
To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

No MeSH data available.