Cas6f (also called Csy4) is the pre-crRNA endoribonuclease of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system. It recognises the hairpin formed by each 28-nucleotide CRISPR repeat in the primary array transcript, clamping the RNA stem between the protein body and an arginine-rich helix, and cleaves immediately downstream of the hairpin using a serine-histidine catalytic dyad. Catalysis is metal-ion independent and leaves 5'-hydroxyl and 3'-phosphate ends, releasing single-spacer guide RNAs. Cas6f does not release its product: it remains clamped on the crRNA 3' hairpin, and that retention nucleates assembly of the Csy surveillance complex (one Cas8f/Csy1, one Cas5f/Csy2, six Cas7f/Csy3, one Cas6f and a single 60-nucleotide crRNA), in which Cas6f occupies the PAM-distal head. The protein is therefore both the crRNA-biogenesis enzyme and a structural subunit of the effector it loads, and loss of it abolishes mature crRNA and with it both target interference and new spacer acquisition.
Definition: A ribonucleoprotein complex composed of CRISPR-associated (Cas) proteins assembled around a single mature CRISPR RNA (crRNA), which uses the crRNA as a guide to locate nucleic acid complementary to its spacer sequence. In type I systems the complex additionally recognises a protospacer-adjacent motif, forms an R-loop at the target, and recruits a trans-acting nuclease for target destruction. The Pseudomonas aeruginosa type I-F complex, Csy, has the composition Cas8f(1)/Cas5f(1)/Cas7f(6)/Cas6f(1) with one 60-nucleotide crRNA; the type I-E equivalent is Cascade.
Justification: GO's entire CRISPR vocabulary is five biological-process terms (GO:0099048, GO:0043571, GO:0098672 and the obsolete GO:0110132 and GO:0110133), with no cellular-component term, so every subunit of every CRISPR surveillance complex can currently only be placed on the bare GO:0032991 root or on its generic child GO:1990904. The complex is a well-defined, stoichiometrically determined, structurally solved entity that ComplexPortal already models for the type I-E case (CPX-1005, Cascade complex), and the Cas7f backbone in particular is the surface targeted by several families of phage-encoded anti-CRISPR proteins, so a specific term would make a large body of structural and counter-defence curation expressible. This is the same request made from the Escherichia coli type I-E Cascade subunit reviews, so that the two systems ask for one term rather than two.
Parent term: ribonucleoprotein complex
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004519 endonuclease activity | IEA GO_REF:0000002 | MODIFY | Summary: The right branch but one level too general. Cas6f/Csy4 is a ribonuclease, not a general endonuclease: its substrate is the pre-crRNA transcript and cleavage was measured directly on RNA. The type I-E counterpart Cas6e/CasE (UniProtKB:Q46897) carries GO:0004521 RNA endonuclease activity by IDA, so the specific term is the established convention for this enzyme family. Reason: Substrate specificity is directly established for this protein and the family comparator carries the child term; the generic parent discards the one fact that was measured. Proposed replacements: RNA endonuclease activity Supporting Evidence: PMID:20829488 we concluded that Csy4 is the endoribonuclease responsible for crRNA biogenesis PMID:22522703 crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript PMID:20829488 CRISPR transcript cleavage is a rapid, metal ion-independent reaction |
| GO:0005515 protein binding | IPI PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | MODIFY | Summary: Uninformative as a molecular function. The Cas6f-Csy3 interaction records a contact made inside the Csy ribonucleoprotein surveillance complex. Cas6f clamps the 3' stem-loop of the crRNA at the PAM-distal head of the spiral and abuts the Cas7f/Csy3 backbone from there, and because it never releases the product it cleaved, that clamp is what nucleates the whole assembly. The contribution is therefore structural: it holds one end of the guide and fixes the head of the complex. Reason: The term "protein binding" conveys nothing about function. The underlying observation is a contact internal to the Csy surveillance complex, so the honest subunit-level function is GO:0005198 structural molecule activity, defined as the action of a molecule that contributes to the structural integrity of a complex. None of its 22 children fits a non-ribosomal ribonucleoprotein, so the parent is the correct level. This keeps the replacement in the molecular-function aspect, which the GOA row's enables qualifier requires: a gene product cannot enable a cellular component, so the complex-membership reading of this row, however well supported, cannot be expressed by replacing an enables MF term with a CC term. Complex membership is instead carried by a dedicated NEW part_of GO:1990904 ribonucleoprotein complex row and by core_functions in_complex, and the missing specific term is requested in proposed_new_terms. The same replacement is used for the equivalent intra-complex rows on cas6f, csy1, csy2 and csy3, and matches how the five Escherichia coli type I-E Cascade subunits were treated. Proposed replacements: structural molecule activity Supporting Evidence: PMID:32170016 the maps exhibit an asymmetric spiral, with one Cas6f subunit in the head, one Cas5f and one Cas8f in the tail, and six Cas7f comprising the spiral backbone PMID:22522703 We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition PMID:22522703 Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product |
| GO:0005515 protein binding | IPI PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | MODIFY | Summary: Second IntAct record of the same Cas6f-Csy3 contact, from the study of anti-CRISPR mechanisms. Treated identically to the PMID:21536913 row: what the interaction shows is this subunit's structural contribution to the surveillance complex, not a binding activity. Reason: Consistency with the other protein binding row on this protein and with the four Csy subunits and the five Escherichia coli Cascade subunits; the paper's subject is inhibition of the assembled complex, which presupposes the structural contribution being asserted. GO:0005198 keeps the replacement in the molecular-function aspect that the enables qualifier requires. Proposed replacements: structural molecule activity Supporting Evidence: PMID:26416740 Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits PMID:32170016 The Csy complex is comprised of four types of Cas proteins (Cas5f-8f) and a single 60-nt crRNA |
| GO:0043571 maintenance of CRISPR repeat elements | IEA GO_REF:0000002 | ACCEPT | Summary: Correct. The term's definition explicitly includes transcription of the CRISPR repeat arrays into RNA and processing, which is the step Cas6f catalyses, and the InterPro signature IPR013396 is the Cas6f/Csy4 family itself. Supporting Evidence: PMID:20829488 we concluded that Csy4 is the endoribonuclease responsible for crRNA biogenesis |
| GO:0043571 maintenance of CRISPR repeat elements | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | ACCEPT | Summary: Well grounded and correctly hedged. A csy4 H29A catalytic-dyad mutant abolished new spacer acquisition in the transplanted Pseudomonas aeruginosa type I-F system, and the requirement runs through loss of mature crRNA rather than through a Cas6f step in the integration chemistry itself, which is what the acts_upstream_of_or_within qualifier records. Supporting Evidence: PMID:26586803 Deleting the csy3 gene (lane 6), or mutating the catalytic residue His29 of Csy4 nuclease (lane 7) that is needed for generation of mature crRNA also abolished spacer acquisition PMID:26586803 both modes require, in addition to Cas1 and Cas2, intact Csy complex, an ortholog of the E. coli Cascade, and crRNA |
| GO:0003723 RNA binding | IDA PMID:20829488 Sequence- and structure-specific RNA processing by a CRISPR ... | NEW | Summary: Sequence- and structure-specific binding of the CRISPR repeat hairpin, separable from catalysis: active-site mutants that abolish cleavage retain RNA binding. The retained product interaction is what makes Cas6f a complex subunit as well as an enzyme. Reason: Missing from GOA. All five Escherichia coli Cascade subunits carry GO:0003723 by IDA, including Cas6e/CasE (Q46897). Here the activity is demonstrated independently of the endonuclease activity by the H29A and S148C mutants, which lose cleavage but not binding, so it is a separate assertion rather than an implication of GO:0004521. Supporting Evidence: PMID:20829488 Mutations of His 29 or Ser 148 (to alanine and cysteine, respectively) completely abolished cleavage activity without disrupting RNA binding PMID:20829488 the protein makes extensive interactions with the ssRNA-dsRNA junction at the base of the crRNA stem as well as with the major groove of the RNA hairpin PMID:22522703 Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product |
| GO:0099048 CRISPR-cas system | IDA PMID:20829488 Sequence- and structure-specific RNA processing by a CRISPR ... | NEW | Summary: Cas6f performs the crRNA biogenesis stage named in this term's own definition, and its product retention couples that stage to assembly of the interference effector. Reason: Comparator check: every subunit of the Escherichia coli Cascade complex (casA, casB, casC, casD, casE) carries involved_in GO:0099048 by IDA from PMID:18703739, and Cas1 and Cas2 carry it too, yet no Pseudomonas aeruginosa PA14 Csy subunit does. The absence is a curation lag for this strain, not a convention. GO:0099048 was checked against QuickGO and is neither an ancestor nor a descendant of GO:0043571 or GO:0051607, so it is not redundant with the existing process annotation. Supporting Evidence: PMID:20829488 we concluded that Csy4 is the endoribonuclease responsible for crRNA biogenesis PMID:22522703 We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition PMID:21398535 proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs |
| GO:1990904 ribonucleoprotein complex | IDA PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | NEW | Summary: Cas6f/Csy4 is an integral subunit of the Csy surveillance complex, which clamps the crRNA 3' stem-loop at the PAM-distal head. The complex contains a single mature 60-nucleotide crRNA as an integral component, not as a ligand, so it is a ribonucleoprotein complex rather than merely a protein-containing one. Reason: Complex membership is not currently annotated for any Pseudomonas aeruginosa PA14 Csy subunit, whereas all five Escherichia coli type I-E Cascade subunits (casA, casB, casC, casD, casE) carry a part_of protein-complex row by IDA or IPI. The GOA rows that record this contact for the PA14 proteins carry the enables qualifier and are therefore molecular-function rows, which cannot be converted to a cellular-component term; the membership claim consequently needs its own row, which is what this is. GO:1990904 is used rather than the bare GO:0032991 root because the guide RNA is an integral component of the assembly, and because GO has no cellular-component term for any CRISPR surveillance complex - its entire CRISPR vocabulary is five biological-process terms - so GO:1990904 is the most specific existing term. The missing specific term is requested in proposed_new_terms. Supporting Evidence: PMID:21536913 the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences PMID:32170016 the maps exhibit an asymmetric spiral, with one Cas6f subunit in the head, one Cas5f and one Cas8f in the tail, and six Cas7f comprising the spiral backbone PMID:22522703 We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition |
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Download this section (compressed HTML)Q: Cas6f has two separable activities, repeat cleavage and product retention, and the second is the one that builds the effector. Should GO express the retained-subunit role as a distinct function, or is RNA binding plus complex membership sufficient?
Q: GO has no molecular-function term for crRNA-guided target recognition, the activity the assembled Csy complex has. Cas6f contributes the guide and the 3' anchor but not the target contacts. If such a term were created, should a biogenesis subunit retained in the complex carry it, contribute to it, or neither?
Q: In the PA14 strain background, is the mature crRNA pool limited by Cas6f cleavage rate or by the availability of Csy1, Csy2 and Csy3 to protect the product from degradation?
Experiment: Complement a PA14 csy4 H29A strain with pre-cleaved mature crRNA supplied in trans (for example from a tRNA-flanked expression construct that needs no Cas6f processing) and test whether spacer acquisition and interference are restored. Restoration would confirm that the adaptation requirement is purely upstream.
Hypothesis: The H29A catalytic mutant abolishes spacer acquisition solely by failing to make mature crRNA, not by any separate role of Cas6f in adaptation.
Type: genetic complementation
Experiment: Use the separation-of-function mutant set from the structural work (binding competent, cleavage dead versus cleavage competent, release accelerated) in pulse-chase northern blots and quantitative pull-downs of the Csy complex to measure the partition of crRNA between free, Cas6f-bound and complex-bound pools.
Hypothesis: Cas6f product retention, rather than cleavage per se, is what commits a crRNA to complex assembly.
Type: biochemical kinetics
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: GO has no cellular-component term for a CRISPR RNA-guided surveillance complex, so Csy complex membership can only be asserted with the generic GO:1990904 ribonucleoprotein complex, which does not distinguish it from a ribosome, a spliceosome or any other RNA-protein assembly.
OPEN ONTOLOGY
What is known: The composition and stoichiometry are settled to near-atomic resolution (Cas8f(1), Cas5f(1), Cas7f(6), Cas6f(1), crRNA(1)), and the Escherichia coli Cascade subunits are annotated the same uninformative way, so this is a shared ontology limitation rather than a gap in knowledge about this protein. ComplexPortal does model the type I-E equivalent as CPX-1005; GO does not.
Significance: Every anti-CRISPR protein acts on this complex; without a term for it the host target of a whole class of phage counter-defence proteins is not machine-readable.
What would resolve it: Request a cellular-component term for a CRISPR RNA-guided surveillance complex under GO:1990904, with subtype children for Cascade and the Csy complex; the request is made in this review's proposed_new_terms.
Provenance (the field's own admissions):
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