cas6f / Csy4 (Q02MM2) — Pseudomonas aeruginosa UCBPP-PA14 — curation notes

Deep research was unavailable in this environment (no provider API keys), so this
file is my own literature journal. Every assertion carries an inline PMID plus a
verbatim quote from the cached record in publications/.

System context

PA14 carries a type I-F (Yersinia-subtype, "Csy") CRISPR-Cas system at
PA14_33300–PA14_33350: cas6f/csy4, csy3, csy2, csy1, cas2-3 (annotated
cas3), cas1, flanked by two CRISPR arrays. Unusually for type I, Cas2 is not a
separate protein: PMID:26586803.

The interference effector is the Csy surveillance complex:
PMID:32170016, arranged as
PMID:32170016. In the PA14 nomenclature Cas8f = Csy1, Cas5f = Csy2, Cas7f = Csy3,
Cas6f = Csy4. This matches UniProt's stoichiometry
Csy1(1),Csy2(1),Csy3(6),Cas6/Csy4(1)-crRNA(1) and the original biochemistry
PMID:21536913.

This is the host side of the anti-CRISPR system reviewed in
genes/BPZF4/AcrF8/AcrF8-ai-review.yaml: AcrIF8 binds the Cas7f (Csy3) backbone and
the crRNA; AcrIF3 sequesters Cas3.

Cas6f/Csy4 is the pre-crRNA endoribonuclease

Csy4 is the enzyme that makes mature crRNA:
PMID:20829488. The substrate is RNA, not DNA, and catalysis is metal-independent:
PMID:20829488.
Independently confirmed: PMID:22522703.

Recognition is sequence- and structure-specific for the repeat hairpin:
PMID:20829488.
Catalysis uses a Ser/His dyad; the catalytic residues are separable from binding:
PMID:20829488. That last
result is the cleanest evidence that Csy4 has two separable activities — RNA
endonuclease and high-affinity crRNA binding.

Dual role: enzyme and retained complex subunit

Like E. coli Cas6e/CasE, Csy4 does not release its product:
PMID:22522703, and this retention is what builds the effector:
PMID:22522703. Structurally it sits
at the PAM-distal head on the crRNA 3' hairpin
PMID:28985564. The IntAct
interaction behind GOA's two GO:0005515 rows is Csy4–Csy3 (Q02MM2–Q02MM1, NbExp=12),
i.e. the head-to-backbone contact of this same complex.

In vivo requirement

Loss of Csy4 abolishes crRNA:
PMID:21398535. The catalytic residue is required for spacer
acquisition as well as interference:
PMID:26586803 — the adaptation defect is downstream of the crRNA
defect, since PMID:26586803.

Curation decisions

• GO:0004519 endonuclease activity (IEA, InterPro IPR013396) is the correct branch
  but one level too general. The substrate is RNA and the measurement is direct, so
  GO:0004521 RNA endonuclease activity is better supported. Comparator check: the
  type I-E counterpart Cas6e/CasE (Q46897) carries exactly GO:0004521 by IDA from
  PMID:18703739 (genes/ECOLI/casE/casE-goa.tsv). So the specific term is the
  convention for this family, and PA14 Cas6f is simply lagging. → MODIFY.
• Both GO:0005515 protein binding rows are uninformative per project policy. The
  Csy4–Csy3 IPI samples a contact internal to the Csy complex: Cas6f clamps the crRNA
  3' stem-loop at the PAM-distal head and abuts the Cas7f backbone from there, and
  because it never releases the product it cleaved, that clamp nucleates the whole
  assembly. → MODIFY both to GO:0005198 structural molecule activity.

Aspect correction (follow-up). My first pass proposed
part_of GO:0032991 protein-containing complex as the replacement, reasoning from the
E. coli comparator where all five Cascade subunits carry exactly that. That was wrong
in form: these GOA rows carry qualifier: enables, so they are molecular-function
rows, and a gene product cannot enable a cellular component. However
well-supported complex membership is, it cannot be expressed by swapping a CC term
into an enables MF row. The E. coli reviews were not making this mistake — their
GOA already contained separate part_of GO:0032991 rows, which they MODIFY to
GO:1990904; their enables GO:0005515 rows went to GO:0005198 instead.
Reconciled to the same convention:

• MODIFY the enables rows to GO:0005198 structural molecule activity ("the
  action of a molecule that contributes to the structural integrity of a complex").
  Aspect-correct, and none of its 22 children fits a non-ribosomal ribonucleoprotein,
  so the parent is the right level. Each row is backed by the verbatim structural
  quote naming the contact this subunit actually makes.
• Carry complex membership on its own NEW part_of GO:1990904 ribonucleoprotein complex row. PA14 has no part_of complex row for any Csy subunit, unlike all
  five E. coli Cascade subunits, so this is a real missing annotation rather than a
  reformulation. GO:1990904 rather than the bare GO:0032991 root because the
  60-nt crRNA is an integral component, not a ligand.
• core_functions[].in_complex → GO:1990904 for the same reason.
• Added a proposed_new_terms entry for a "CRISPR RNA-guided surveillance
  complex" CC term under GO:1990904, identical in substance to the one the five
  E. coli Cascade reviews carry, so the two systems request one term rather than two.
  GO's entire CRISPR vocabulary is five BP terms (GO:0099048, GO:0043571,
  GO:0098672 and the obsolete GO:0110132/GO:0110133); ComplexPortal models the
  type I-E complex as CPX-1005, GO does not.
• GO:0043571 maintenance of CRISPR repeat elements — both the IEA and the CACAO IMP
  are right. The term's definition explicitly includes "transcription of the CRISPR
  repeat arrays into RNA and processing" and "capture of new spacer elements", which is
  precisely what Csy4 does and what the H29A phenotype shows. → ACCEPT both.

Gaps identified (missing annotations)

• GO:0003723 RNA binding — held by all five E. coli Cascade subunits by IDA; here it
  is separately demonstrated from catalysis by the H29A/S148C result. → NEW.
• GO:0099048 CRISPR-cas system — held by all five E. coli Cascade subunits by IDA
  (PMID:18703739). Not an ancestor or descendant of GO:0043571 or GO:0051607
  (checked via QuickGO ancestors), so not redundant. Csy4 performs the crRNA-biogenesis
  stage named in the term's own definition. → NEW.
• No molecular-function term exists for crRNA-guided target recognition, the activity
  the assembled Csy complex actually has. This is recorded as an open ontology gap in
  modules/crispr_cas_adaptive_immunity.yaml and I have not substituted an ill-fitting
  id; raised in suggested_questions instead.
• Not proposed: GO:0051607 defense response to virus. The PA14 native array's
  antiviral role is contested in the primary literature
  PMID:21398535 versus
  PMID:23242138 (ΔCR/cas).
  The second is a whole-locus deletion, not a cas6f allele, so there is no
  gene-specific antiviral evidence to annotate. GO:0099048 already carries the
  immunity role without over-claiming.
• Deliberately not annotated: the use of Csy4 as a laboratory RNA-processing reagent.
  That is a biotechnology application, not a function of the gene in its host.