Csy2, also called Cas5f, is the Cas5-family subunit of the Csy crRNA-guided surveillance complex of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system. The complex comprises one Cas8f/Csy1, one Cas5f/Csy2, six Cas7f/Csy3 and one Cas6f/Csy4 wrapped around a single 60-nucleotide crRNA in a helically twisted G shape; Csy2 sits with Cas8f in the PAM-proximal tail. Its defining job is to cap and read the 5' handle of the crRNA: together with Cas8f it makes base-specific contacts that bend the first eight nucleotides into an S shape and fix the register of the guide within the complex, and its thumb domain also contributes to the pocket that grips the protospacer-adjacent-motif-containing target duplex. Csy2 is one of the two subunits strictly required for mature crRNA to accumulate in the cell, since an unincorporated guide is degraded, and loss of it therefore abolishes both target interference and the acquisition of new spacers.
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 Csy2-Csy1 interaction records a contact made inside the Csy ribonucleoprotein surveillance complex. Cas5f/Csy2 and Cas8f/Csy1 together build the PAM-proximal tail of the spiral: the two subunits jointly clamp the 5' handle of the crRNA into its bent conformation, and the Cas5f thumb and the Cas8f hook form two of the three walls of the pocket that holds the target duplex. The contribution is therefore structural, and it is mutual. 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 Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle PMID:28985564 the duplex region of the target DNA (including the G-C/G-C PAM) is sandwiched between the Cas8f N-terminal hook domain (residues 1-166), the Cas5f thumb domain (residues 48-109), and Cas7 |
| GO:0005515 protein binding | IPI PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | MODIFY | Summary: Second IntAct record of the same Csy2-Csy1 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:0003723 RNA binding | IDA PMID:32170016 Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against typ... | NEW | Summary: Cas5f, with Cas8f, clamps the 5' handle of the crRNA, and the contacts are made predominantly to the nucleobases rather than only to the sugar-phosphate backbone, so the recognition is sequence-specific. This is what sets the register of the guide and, in vivo, what protects the mature crRNA from degradation. Reason: Missing from GOA, which holds only two protein binding rows for this protein. Comparator check: casD, the type I-E Cas5 (UniProtKB:Q46898), carries GO:0003723 by IDA twice plus an InterPro IEA, and casA, casC and casE carry it too, so RNA binding is annotated across the Cascade subunits. Here the sequence-specific 5'-handle recognition is directly resolved in the structure of this complex. Supporting Evidence: PMID:32170016 the Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle PMID:32170016 supporting a sequence-specific recognition of the crRNA 5′ handle by Cas5f and Cas8f |
| GO:0043571 maintenance of CRISPR repeat elements | IMP PMID:21398535 Non-identity-mediated CRISPR-bacteriophage interaction media... | NEW | Summary: Csy2 is one of only two Csy subunits whose loss abolishes, rather than merely reduces, the mature crRNA pool in Pseudomonas aeruginosa PA14. The term's definition explicitly covers transcription of the CRISPR repeat arrays into RNA and processing, which is the step a csy2 null breaks, and the mechanism is clear: Cas5f capping of the 5' handle is part of the assembly that protects the guide from degradation. Reason: A genuine per-gene gap, not a pathway-completeness argument. The paralogous PA14 subunits cas6f and csy3 already carry GO:0043571, and the type I-E Cas5 casD carries it too, yet csy2 does not, even though the PA14 gene-by-gene data place csy2 in the strictly-essential class for crRNA alongside csy4 and ahead of csy1 and csy3. The entity doing the work is Cas5f itself, which holds the handle; this is participation in crRNA maturation and stability, not merely a requirement for a process another protein carries out. Supporting Evidence: 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 PMID:22522703 We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition PMID:32170016 supporting a sequence-specific recognition of the crRNA 5′ handle by Cas5f and Cas8f |
| GO:0099048 CRISPR-cas system | IDA PMID:32170016 Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against typ... | NEW | Summary: Cas5f does work in two of the three stages this term's definition names: it holds the 5' handle that completes crRNA biogenesis into a loaded effector, and it contributes the thumb domain to the target-duplex pocket used in interference. Reason: Comparator check: all five Escherichia coli Cascade subunits carry involved_in GO:0099048 by IDA from PMID:18703739, including casD, 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 nothing is duplicated. Supporting Evidence: PMID:32170016 the Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle PMID:28985564 the duplex region of the target DNA (including the G-C/G-C PAM) is sandwiched between the Cas8f N-terminal hook domain (residues 1-166), the Cas5f thumb domain (residues 48-109), and Cas7 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: Cas5f/Csy2 is an integral subunit of the Csy surveillance complex, which forms the PAM-proximal tail together with Cas8f/Csy1. 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 Csy complex is comprised of four types of Cas proteins (Cas5f-8f) and a single 60-nt crRNA |
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Download this section (compressed HTML)Q: Does Cas5f protect the mature crRNA directly, by occluding its 5' end from exonucleases, or indirectly, by being required for the complex to assemble at all? The two predict different fates for a crRNA bound by a Cas7f-Cas6f subcomplex in a csy2 null.
Q: Cas5f reads the crRNA 5' handle sequence-specifically. Does that make the repeat-derived handle a second specificity determinant alongside the protospacer-adjacent motif, and would handle mutations be tolerated by engineered guides in this system?
Q: Type I-E Cas5 (casD) is annotated with DNA/RNA hybrid binding. Does PA14 Cas5f also contact the crRNA-target heteroduplex, or is that role carried entirely by the Cas7f thumbs in type I-F?
Experiment: Compare small-RNA northern blots and 5'-end sequencing of a PA14 csy2 deletion with a csy4 H29A catalytic mutant, in strains with and without major cytoplasmic ribonucleases depleted. Processing-competent but unprotected intermediates in the csy2 strain would separate the two models.
Hypothesis: Cas5f is required for crRNA stability rather than for processing, so a csy2 null accumulates cleaved but unprotected crRNA that is degraded.
Type: RNA stability and processing analysis
Experiment: Mutate the handle nucleotides that contact Cas5f and, reciprocally, the Cas5f residues that read them, and measure Csy complex assembly, crRNA loading and plasmid-targeting efficiency for each pair. Compensatory double mutants would establish the contacts as a direct specificity code.
Hypothesis: Base-specific Cas5f contacts to the crRNA 5' handle are required for correct guide register and for interference.
Type: reciprocal mutagenesis with targeting assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether Cas5f contributes to target DNA engagement in its own right, beyond donating one thumb residue to a pocket dominated by Cas8f, is undetermined. No Cas5f DNA-contact mutant has been tested, and no term is asserted for a Cas5f DNA-binding activity.
OPEN BIOLOGY
What is known: Established: the Cas5f thumb is part of the structural sandwich around the PAM-containing duplex, and the type I-E Cas5 orthologue casD carries DNA/RNA hybrid binding rather than DNA binding. Not established: whether isolated Cas5f binds DNA, or whether its thumb contacts are load-bearing for target engagement.
Significance: Distinguishing structural proximity from functional contribution is exactly what separates a defensible subunit annotation from an over-annotation, and the answer determines whether Cas5f should carry a DNA-binding term at all.
What would resolve it: Test Cas5f thumb substitutions (for example R90A) for target binding and interference in an otherwise wild-type complex, and assay isolated Cas5f for DNA binding.
Provenance (the field's own admissions):
Gap: GO has no molecular-function term for crRNA-guided, PAM-licensed target recognition, the activity the assembled Csy complex has, so Cas5f can only be annotated with generic nucleic-acid-binding terms.
OPEN ONTOLOGY
What is known: The nuclease chemistry of the system is covered by existing terms and the structural steps are resolved to near-atomic detail; what is missing is a term for the guide dependence that defines the system.
Significance: Without such a term the defining property of every Cas effector is invisible to GO-based reasoning.
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. No existing id has been substituted for the missing concept here.
Provenance (the field's own admissions):
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