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Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

ABSTRACT: CRISPR-Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR-Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacterium Synechocystis sp. PCC 6803 encodes three separate CRISPR-Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions such as nitrogen starvation or changing light intensities. Here, we found that the promoter of the respective six-gene cas operon is controlled by the light- and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif 53 to 70 nt upstream of the transcriptional start site, leading to the activation of transcription at low light intensities. However, the strong promoter driving transcription of the cognate repeat-spacer array is not under RpaB control. Instead, we found the 125 nt transcribed leader to be bound by the redox-sensitive RNA helicase CrhR, and crosslinking coupled to mass spectrometry analysis revealed six residues specifically interacting with it. From these, L103, F104, H225, and C371 were predicted on the surface of a dimeric CrhR model. These results show that the expression of the CRISPR-Cas system is linked to the redox status of the photosynthetic cyanobacterial cell and that the elements involved are unique in that they act at two different levels. While RpaB affects transcription, CrhR engages with the transcribed leader at the post-transcriptional level. These results highlight the complex nature of the interrelationships between a CRISPR-Cas system and its host cell.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Synechocystis Sp. (strain Pcc 6803 / Kazusa)

SUBMITTER: Olexandr Dybkov

LAB HEAD: Olexandr Dybkov

PROVIDER: PXD047440 | Pride | 2024-07-15

REPOSITORIES: Pride

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			Action	DRS
	AWulf_050719_combined_validated_results.xlsx	Xlsx
	AWulf_050719_results.zip	Other
	MRaabe_AW_230519_020719_19XL-xyz_CrhR_Mut_DEB_rep1.raw	Raw
	MRaabe_AW_230519_020719_19XL-xyz_CrhR_Mut_DEB_rep2.raw	Raw
	MRaabe_AW_230519_020719_19XL-xyz_CrhR_Mut_UV_rep1.raw	Raw

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Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs

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Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

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