Csy3, also called Cas7f, is the backbone subunit of the Csy crRNA-guided surveillance complex of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system. Six copies oligomerise head to tail along the spacer portion of a single 60-nucleotide crRNA, forming the helical spiral between the Cas6f head and the Cas8f-Cas5f tail. Each copy grips the guide through an arginine-rich surface that contacts the sugar-phosphate backbone rather than the bases, so recognition is sequence-independent, and a thumb from each subunit kinks the crRNA at six-nucleotide intervals, dividing the guide into the short segments that are presented for base pairing. When a complementary target strand invades, a beta-hairpin from the adjacent subunit threads through each kink and pins the crRNA-DNA heteroduplex to the backbone, so Cas7f holds the hybrid while target verification proceeds along the guide. A lysine-rich patch on the same surface is critical for the complex to bind target DNA, and that patch is the principal target surface of the phage anti-CRISPR proteins AcrIF8 and AcrIF9.
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:0005515 protein binding | IPI PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | MODIFY | Summary: Uninformative as a molecular function. The Csy3-Cas6f interaction records a contact made inside the Csy ribonucleoprotein surveillance complex. Cas7f/Csy3 is the structural element of the complex: six copies interlock thumb-into-palm along the guide through roughly 2,000 square Angstrom interfaces, and the resulting spiral runs from the Cas6f head to the Cas8f-Cas5f tail, which is the contact this IntAct record samples. Holding the complex together is the whole of what this subunit's protein-protein contacts do. 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 thumb of Cas7.5f traverses the palm of Cas7.4f, and the interface area between them was about 2,000 Å2, involving extensive salt bridges and hydrogen bonds PMID:32170016 The six copies of the hand-shaped backbone protein Cas7 are arranged in the same direction along the spiral, creating five equivalent interfaces between them PMID:28985564 Cas6f is located at the 3′ stem-loop of the crRNA, Cas8f and Cas5f are located at the 5′ handle of crRNA, and six interlocking copies of Cas7f are located along the length of the crRNA spacer |
| GO:0005515 protein binding | IPI PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | MODIFY | Summary: Second IntAct record of the same Csy3-Cas6f 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 | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | ACCEPT | Summary: Well grounded and correctly hedged. A clean csy3 deletion abolished new spacer acquisition in the transplanted Pseudomonas aeruginosa type I-F system, in which both naive and primed adaptation require the intact Csy complex and crRNA. The acts_upstream_of_or_within qualifier is the right one, because the requirement runs through complex integrity rather than through a Csy3 catalytic step in the integration chemistry. Note that this annotation is earned by the acquisition phenotype, not by a crRNA-biogenesis phenotype: Csy3 is not strictly required for crRNA to accumulate. 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:32170016 Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against typ... | NEW | Summary: Each Cas7f copy binds the crRNA through an arginine-rich surface making eleven resolved contacts, almost all to the sugar-phosphate backbone, and a thumb that kinks the guide at six-nucleotide intervals. Recognition is explicitly sequence-independent, which is what allows one backbone to carry any spacer. Reason: Missing from GOA, which holds only two protein binding rows and one process row for this protein. Comparator check: casC, the type I-E Cas7 (UniProtKB:Q46899), carries GO:0003723 by IDA from PMID:25103409, as do casA, casD and casE, so RNA binding is annotated across the Cascade subunits. Here the contacts are resolved residue by residue in the structure of this complex. Supporting Evidence: PMID:32170016 Cas7f and crRNA form multiple hydrogen bonds, which mostly occur between the arginine-rich region (F32, R34, R68, Q95, R168, Q247, Q276, K277, R283, S308, R350) and the sugar-phosphate backbone of crRNA PMID:32170016 This finding indicates the nonsequence-specific crRNA recognition mode of Cas7f PMID:32170016 The thumbs of the spiral backbone proteins (Cas7f) distort the crRNA at 6-nt intervals |
| GO:0071667 DNA/RNA hybrid binding | IDA PMID:28985564 Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Ta... | NEW | Summary: Cas7f does not merely hold the guide; once the target strand has invaded, a beta-hairpin from each adjacent subunit threads through the kinks and pins the crRNA-DNA heteroduplex to the backbone. This is the contact that makes directional target verification along the guide possible, and it is a hybrid contact rather than an RNA or a DNA one. Reason: Missing from GOA. Comparator check: this is precisely the term that casC, the type I-E Cas7 subunit, carries by IDA from PMID:25123481, and casD carries it as well; of all the proposals in this gene set it has the tightest subunit-to-subunit comparator match. Cas7f is the entity making the contact, resolved directly in cryo-EM of the target-bound Pseudomonas aeruginosa complex. Supporting Evidence: PMID:28985564 hairpin emanating from the adjacent Cas7f subunit threads through each of these gaps spaced along the backbone, effectively pinning the RNA:DNA heteroduplex to the Csy complex backbone PMID:28985564 The structural studies we describe here are focused on the type I-F Csy (CRISPR system yersinia) found in Pseudomonas aeruginosa |
| GO:0099048 CRISPR-cas system | IDA PMID:28985564 Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Ta... | NEW | Summary: Cas7f does work in the target interference stage named in this term's definition: it presents the guide in base-pairing segments and holds the resulting heteroduplex while verification proceeds. That is participation in the process, not merely a structural requirement for it. Reason: Comparator check: all five Escherichia coli Cascade subunits carry involved_in GO:0099048 by IDA from PMID:18703739, including the Cas7 backbone subunit casC, yet no Pseudomonas aeruginosa PA14 Csy subunit does, which marks a per-strain curation lag rather than a convention about structural subunits. Checked against QuickGO: GO:0099048 is neither an ancestor nor a descendant of GO:0043571 or GO:0051607, so it does not duplicate the existing process annotation. Supporting Evidence: PMID:28985564 hairpin emanating from the adjacent Cas7f subunit threads through each of these gaps spaced along the backbone, effectively pinning the RNA:DNA heteroduplex to the Csy complex backbone PMID:32170016 a lysine-rich region of the Cas7f subunit (K76, K78, K84, and K256) has been reported to be critical for DNA binding by the Csy complex 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 |
| GO:1990904 ribonucleoprotein complex | IDA PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | NEW | Summary: Cas7f/Csy3 is an integral subunit of the Csy surveillance complex, which supplies the six-copy spiral backbone between the head and the tail. 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:32170016 The six copies of the hand-shaped backbone protein Cas7 are arranged in the same direction along the spiral, creating five equivalent interfaces between them |
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Download this section (compressed HTML)Q: Cas7f recognises the crRNA sequence-independently but pins the heteroduplex sequence-dependently through the kinks. Should GO distinguish these as separate functions of the same surface, given that one is a scaffolding contact and the other is the verification contact?
Q: AcrIF8 contacts both the Cas7f backbone and the crRNA at a kink, while AcrIF9 competes for the lysine patch. Do the six Cas7f copies differ functionally by position, such that occluding one specific subunit is enough to block verification?
Q: Csy3 is not strictly required for crRNA accumulation yet a csy3 deletion abolishes spacer acquisition. Is the acquisition defect due to loss of target engagement by the complex, or does the Cas7f backbone have a further role in generating the adaptation substrate?
Experiment: Build Cas7f K76A, K78A, K84A and K256A variants, confirm Csy complex assembly and crRNA loading, then measure target DNA binding by electrophoretic mobility shift and Cas3-dependent cleavage in the reconstituted system. Compare the loss of function with that caused by saturating AcrIF9.
Hypothesis: The lysine-rich Cas7f patch is required for target DNA binding and not merely for complex assembly, so patch mutants give an assembled but targeting-dead complex.
Type: structure-guided mutagenesis with reconstituted binding and cleavage assays
Experiment: Delete or shorten the threading beta-hairpin in individual Cas7f positions using a mosaic complex assembled from differently tagged subunits, and use single-molecule DNA unzipping or magnetic-tweezer R-loop assays to ask whether verification stalls at the modified segment.
Hypothesis: Heteroduplex pinning by the Cas7f beta-hairpins enforces directional target verification, so hairpin mutants permit binding but not licensing of Cas3.
Type: single-molecule R-loop assay with mosaic complexes
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: GO has no molecular-function term for crRNA-guided, PAM-licensed target recognition, the activity the assembled Csy complex has and in which the Cas7f backbone does the segment-by-segment base-pairing work. The terms that can be asserted (RNA binding, DNA/RNA hybrid binding) do not distinguish this protein from an unguided nucleic-acid-binding protein.
OPEN ONTOLOGY
What is known: The structural mechanism is resolved to near-atomic detail, including the kink spacing, the hairpin threading, the helical pitch change on target binding, and the lysine patch required for DNA engagement. What is missing is a term for the guide dependence that defines the system.
Significance: The Cas7f backbone is the host surface that most type I-F anti-CRISPR proteins attack, so the absence of a term for what that surface does leaves both sides of a well-characterised arms race unstatable in GO.
What would resolve it: Request a molecular-function term for guide-RNA-dependent nucleic acid recognition, as proposed in modules/crispr_cas_adaptive_immunity.yaml and modules/anti_crispr_suppression.yaml. No existing id has been substituted for the missing concept here.
Provenance (the field's own admissions):
Gap: Whether the lysine-rich Cas7f patch binds target DNA directly, or only as part of the complex-level DNA-binding surface, has not been separated experimentally; isolated Cas7f has not been assayed for DNA binding.
OPEN BIOLOGY
What is known: Established: the patch is required for DNA binding by the complex, and it is the surface AcrIF8 and AcrIF9 occupy. Not established: whether a Cas7f monomer binds DNA, and whether the requirement is for affinity or for correct heteroduplex geometry.
Significance: This determines whether Cas7f should eventually carry a DNA-binding term in its own right or only contributes_to one, and it is the same distinction that separates competitive from allosteric anti-CRISPR mechanisms.
What would resolve it: Assay purified Cas7f and the Cas7f-Cas6f-crRNA subcomplex for DNA binding, and test lysine-patch substitutions for target affinity versus R-loop geometry in the complete complex.
Provenance (the field's own admissions):
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