Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus
[Display omitted] •The length of RNA duplex required for Type III CRISPR-Cas immunity in Thermus thermophilus depends on target RNA abundance;•Type III effectors bind targets through a simple bimolecular reaction, where extensive crRNA-target base pairing compensates lower target abundance;•The mini...
Saved in:
| Published in: | Journal of molecular biology Vol. 436; no. 6; p. 168448 |
|---|---|
| Main Authors: | , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Netherlands
Elsevier Ltd
15-03-2024
|
| Subjects: | |
| Online Access: | Get full text |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | [Display omitted]
•The length of RNA duplex required for Type III CRISPR-Cas immunity in Thermus thermophilus depends on target RNA abundance;•Type III effectors bind targets through a simple bimolecular reaction, where extensive crRNA-target base pairing compensates lower target abundance;•The minimal RNA duplex sufficient for interference can occupy multiple positions within the Type III-A effector;•The HD nuclease activity is crucial for efficient Type III-A immunity against plasmid transformation. The absence of HD nuclease in Type III-B effectors suggests that it relies on alternative mechanisms of interference.
Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity. |
|---|---|
| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-2836 1089-8638 1089-8638 |
| DOI: | 10.1016/j.jmb.2024.168448 |