RluA-1 (CG31719) encodes a 739-residue stand-alone pseudouridine synthase of the RluA family in Drosophila melanogaster, with a C-terminal RsuA/RluA-like catalytic domain (residues ~385-531, catalytic Asp428) preceded by a long, partly disordered N-terminal extension. It belongs to the eukaryotic RPUSD2 subfamily (PANTHER PTHR21600:SF40), whose characterized members in yeast (Pus9, Rib2/Pus8) and human (RPUSD2) isomerize the first unpaired uridine of the tRNA anticodon loop, chiefly position 32 (and 31 or 34 in some tRNAs), and have lost the ancestral bacterial RluA activity on rRNA. Drosophila has two members of this subfamily, RluA-1 and RluA-2. RluA-1 is expressed specifically in larval peripheral sensory neurons, where an endogenously tagged protein is nuclear; its own RNA substrates have not been mapped. Loss of RluA-1 from nociceptors causes thermal and mechanical hypersensitivity and hyperbranched class IV dendrites, phenotypes whose link to a particular modified RNA is unknown.
Summary: rRNA pseudouridylation is an ancestral bacterial RluA-family activity that the eukaryotic RPUSD2 subfamily, which contains RluA-1, does not appear to retain.
Reason: The IBA is placed at the deep node PTN002727951, whose rRNA-acting donors are mainly bacterial RluA/RluC/RluD/RsuA/RluE/RluF (several in other PANTHER subfamilies or even the separate PTHR47683 family) and yeast mitochondrial Pus5 (SF81). RluA-1 sits in PANTHER subfamily PTHR21600:SF40 (RPUSD2), and every experimentally characterized SF40 member modifies tRNA rather than rRNA. Yeast Rib2/Pus8 and Pus9 form Psi32 in cytoplasmic and mitochondrial tRNAs, and human RPUSD2 acts on tRNA anticodon-loop positions 31, 32 and 34; the human tRNA Psi mapping study concludes that eukaryotic RPUSD2 has lost activity towards rRNA. No rRNA modification has been reported for RluA-1. The term is therefore likely an over-propagation from a node deeper than the RPUSD2 branch, but it is not removed because the fly protein itself has not been tested.
Propagation Review
Root cause:PROPAGATION BAD
Failure modes:FUNCTIONAL DIVERGENCE
Sources checked:
PANTHER:PTN002727951
Deep RluA-family node spanning bacterial RluA/RluC/RluD and RPUSD-type eukaryotic subfamilies. rRNA pseudouridylation is experimentally grounded in bacterial and yeast Pus5 branches, but the RPUSD2 subfamily (SF40) containing RluA-1 has diverged to tRNA substrates and human RPUSD2 lacks rRNA activity, so the rRNA process should not be inherited by SF40.
Summary: Correct but general; the RPUSD2 subfamily acts on tRNA, so tRNA pseudouridine synthesis is the better-supported process.
Reason: RluA-1 retains the RluA-family catalytic motifs, including the catalytic Asp (Asp428 in UniProt numbering; Asp426 in the paper's homology model), so participation in pseudouridine synthesis is well founded. Its placement in PANTHER subfamily PTHR21600:SF40, whose characterized members in yeast and human are tRNA anticodon-loop pseudouridine synthases, supports refining the process to tRNA pseudouridine synthesis. The specific modified tRNAs in the fly remain to be mapped.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: RNA binding is implied by substrate engagement but is uninformative; the catalytic activity describes the molecular function.
Reason: Binding of the RNA substrate is part of the catalytic cycle of a stand-alone pseudouridine synthase rather than a separate function. The informative molecular function is tRNA pseudouridine(32) synthase activity, supported by the conserved catalytic domain and by RPUSD2-subfamily membership.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: Correct but very general; tRNA pseudouridine synthesis is the specific process.
Reason: Pseudouridylation is an RNA modification step within RNA processing. The RPUSD2-subfamily evidence supports refining this to tRNA pseudouridine synthesis.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: Correct but general; tRNA pseudouridine synthesis is the specific process.
Reason: RluA-1 is a pseudouridine synthase, so RNA modification is true. The specific supported process is tRNA pseudouridine synthesis, inferred from the conserved catalytic domain and the tRNA specificity shared by characterized RPUSD2-subfamily members.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: The generic catalytic activity is soundly inherited; the RPUSD2-subfamily members support the more specific tRNA pseudouridine(32) synthase activity.
Reason: PAINT placed pseudouridine synthase activity at the deep RluA-family node PTN002727951, and RluA-1 retains the catalytic residues, so the activity is well founded. Its donors include the SF40 members yeast Pus9 and Rib2 and human RPUSD2 (Q8IZ73), whose activity is Psi formation at the first unpaired uridine of the tRNA anticodon loop, chiefly position 32. Within SF40 that specificity is conserved from yeast to human, so tRNA pseudouridine(32) synthase activity is the more specific supported function. Fly-specific substrate mapping is lacking. Drosophila has two SF40 paralogs (RluA-1, RluA-2), but the yeast SF40 pair Rib2/Pus8 and Pus9 divides its substrates by compartment (cytoplasmic versus mitochondrial tRNA) and both form Psi32, so a paralog split does not argue against the Psi32 activity. The parent tRNA pseudouridine synthase activity (GO:0106029) is not used as the replacement because Psi32 is the one position modified by every characterized SF40 member; the Psi31 activity is kept separately as non-core (GO:0160152).
Propagation Review
Root cause:TERM SCOPING PROBLEM
Failure modes:GRANULARITY MISMATCH
Sources checked:
PANTHER:PTN002727951
Placement of generic pseudouridine synthase activity at this node is sound and RluA-1 retains the catalytic residues; the subfamily-level (SF40) evidence supports a more specific tRNA pseudouridine(32) synthase activity than the node carries.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: Same claim as the IBA row; correct but general, refined to tRNA pseudouridine(32) synthase activity.
Reason: The catalytic domain and catalytic Asp are conserved, supporting pseudouridine synthase activity. For consistency with the IBA row, the activity is refined to tRNA pseudouridine(32) synthase activity on the basis of RPUSD2-subfamily membership.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: tRNA Psi32 formation is the activity conserved across the characterized RPUSD2 subfamily, to which RluA-1 belongs; it is accepted as the best-supported specific activity.
Reason: The EC mapping itself stems from an ARBA-derived name, which is not evidence. The claim rests instead on evolutionary placement. RluA-1 is in PANTHER subfamily PTHR21600:SF40 together with yeast Rib2/Pus8 and Pus9 and human RPUSD2. Rib2 (cytoplasmic) and Pus9 (mitochondrial) are each required for tRNA Psi32, and human RPUSD2 forms Psi32 in mitochondrial tRNAs and other anticodon-loop Psi. Conservation of this activity across fungi and mammals, together with the retained catalytic residues in RluA-1, supports transferring it within the subfamily. The fly tRNA substrates, and whether RluA-1 and RluA-2 split cytosolic and mitochondrial targets, have not been tested.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Summary: Psi31 is a minor, tRNA-specific outcome of RPUSD2-subfamily specificity, seen so far only for one human mitochondrial tRNA.
Reason: Human RPUSD2 modifies the first unpaired uridine of the anticodon loop, which is position 31 in mt-tRNALeu(CUN) but position 32 in most substrates. Psi31 activity is therefore plausible for an SF40 enzyme but is a subsidiary consequence of the Psi32-type specificity and has no fungal or fly experimental counterpart. It is kept as non-core.
IDA PMID:33028630 Loss of Pseudouridine Synthases in the RluA Family Causes Hy...
NEW
Summary: An endogenous GFSTF exon-trap fusion of RluA-1 localizes to nuclei of larval sensory neurons.
Reason: The GFSTF (GFP exon-trap) cassette was inserted into a coding intron of the endogenous RluA-1 gene, so the signal reports the protein's location, unlike the membrane-targeted mCD8::GFP used with the RluA-1-GAL4 expression reporter. Nuclear localization fits a tRNA modification enzyme acting on pre-tRNAs, as human RPUSD2 does, though nuclear residence of a tagged protein in one cell type does not exclude other compartments.
For example, both Ξ¨34 and Ξ¨36 are induced by Pus1p in yeast pre-tRNAIle-TAT, while human pre-tRNAIle-TAT possesses only RPUSD2-dependent Ξ¨34.
Core Functions
Stand-alone RluA-family pseudouridine synthase of the RPUSD2 subfamily that isomerizes the first unpaired uridine of the tRNA anticodon loop (chiefly U32); activity inferred from conserved catalytic residues and subfamily-wide experimental data, with fly tRNA substrates not yet mapped.
Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2.
Q: Which tRNAs (cytosolic or mitochondrial) carry RluA-1-dependent pseudouridine in Drosophila sensory neurons, and do RluA-1 and RluA-2 split substrates as yeast Rib2 and Pus9 do?
Q: Is the nociceptive hypersensitivity and dendrite hyperbranching of RluA-1 mutants caused by loss of a tRNA modification, an mRNA modification, or a non-catalytic role? Phenotype-based process terms (e.g. negative regulation of sensory perception of pain) were not proposed because the gene's contribution may be indirect.
Suggested Experiments
Experiment: Map pseudouridine (e.g. BID-seq or PRAISE) in tRNA and poly(A) RNA from wild-type, RluA-1, RluA-2 and double-mutant larvae, and test catalytic-dead (D428A) RluA-1 for rescue of nociceptive hypersensitivity.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Pseudouridine synthesis is supported by target-resolved conservation of the RluA-family catalytic residues and is already in the annotation record; the RPUSD2 subfamily context points to tRNA substrates, but the fly protein's own modified RNAs are unmapped.
Prediction method: ProtNLM2 Β· Version: UniProt API snapshot 2026-09-08
Review rationale: The pseudouridine-synthesis prediction is correct. The published alignment of the exact Q9VKV0 protein shows that the conserved motif I residues and the residues needed for catalysis are retained, so the protein can be expected to carry out the RluA-family isomerization. The gene already carries this exact term in GOA, so the prediction is not novel. It is broader than the tRNA pseudouridine synthesis process supported by the gene's RPUSD2 subfamily placement, but that process is not yet an existing annotation on the gene, and the rRNA process term it does carry is an over-propagation; LSP therefore does not apply. The fly protein's endogenous substrates remain untested.
Supporting Evidence:
PMID:33028630: "Each of the fully conserved non-catalytic residues (i.e., motif I, Figure 1A) and critical residues thought to be important for catalysis (asterisks, Figure 1A) are conserved in both RluA-1 and RluA-2."
PMID:33028630: "leaving to future investigation the nature of their substrates and whether they interact with RNA similarly as other synthases"
FlyBase gene description for RluA-1 (DROME)FlyBase
FlyBase gene description for RluA-1 (DROME)
RluA pseudouridine synthase 1 (RluA-1) encodes a pseudouridine synthase, an enzyme catalyzing the formation of the most abundant nucleoside modification of RNA, pseudouridine.