Critical roles for 'housekeeping' nucleases in Type III CRISPR-Cas immunity

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Abstract

CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e. phages). Type III systems launch a multi-layered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the Type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third 'housekeeping' nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation, and identify specific interactions with Csm5, a member of the Type III effector complex, which facilitate nuclease recruitment/stimulation. Further, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for Type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, 5, 6, Figure 1-figure supplement 1, Figure 2-figure supplement 1, Figure 3-figure supplement 1, Figure 3-figure supplement 2, Figure 5-figure supplement 1, and Figure 6-figure supplement 1. Corresponding source data files are called: Figure 1-source data 1, Figure 1-source data 2, Figure 1-source data 3, Figure 2-source data 1, Figure 2-source data 2, Figure 2-source data 3, Figure 2-source data 4, Figure 3-source data 1, Figure 3-source data 2, Figure 4-source data 1, Figure 4-source data 2, Figure 4-source data 3, Figure 5-source data 1, Figure 5-source data 2, Figure 5-source data 3, Figure 6-source data 1, Figure 6-source data 2, Figure 1-figure supplement 1-source data 1, Figure 1-figure supplement 1-source data 2, Figure 2-figure supplement 1-source data 1, Figure 3-figure supplement 1-source data 1, Figure 3-figure supplement 2-source data 1, Figure 3-figure supplement 2-source data 2, Figure 3-figure supplement 2-source data 3, Figure 5-figure supplement 1-source data 1, Figure 6-figure supplement 1-source data 1, Figure 6-figure supplement 1-source data 2, and Figure 6-figure supplement 1-source data 3.

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Author details

Lucy Chou-Zheng

Microbiology Department, University of Illinois Urbana-Champaign, Urbana, United States

Competing interests
The authors declare that no competing interests exist.
Asma Hatoum-Aslan

Microbiology Department, University of Illinois Urbana-Champaign, Urbana, United States

For correspondence
ahatoum@illinois.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2395-8900

Funding

National Science Foundation (MCB/2054755)

Asma Hatoum-Aslan

Burroughs Wellcome Fund

Asma Hatoum-Aslan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.