id: A0A9L0IK51 gene_symbol: RBM41 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9793 label: Equus asinus description: RBM41 (RNA-binding motif protein 41) is an X-linked nuclear RNA-binding protein of the minor (U12-type) spliceosome, with a conserved, largely helical N-terminal region that lacks annotated domains and a single C-terminal RNA recognition motif (RRM). It is the paralog of U11/U12-65K (RNPC3) within the RBM41/RNPC3 family, and its RRM is homologous to the 65K C-terminal RRM. In human cells, the ortholog's RRM binds the 3'-terminal stem-loops of U12 and U6atac snRNAs, and the protein associates with the U12 mono-snRNP rather than the U11/U12 di-snRNP. It also associates with excised U12-type intron lariats and, through its N-terminal region, with the DEAH-box helicase DHX8, and it concentrates in Cajal bodies. These findings place RBM41 in the post-splicing steps of the minor spliceosome assembly/disassembly cycle, where it is thought to replace 65K on the U12 snRNA 3' stem-loop. Loss of RBM41 is tolerated by cultured cells but shifts 3' splice site choice at a subset of U12-type introns. The donkey protein (432 aa) is a full-length ortholog, 95.8% identical to human RBM41 over paired residues, with a 24-residue insertion in the N-terminal region and an RRM that is 78 of 79 residues identical. existing_annotations: - term: id: GO:0000398 label: mRNA splicing, via spliceosome evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in supporting_entities: - PANTHER:PTN009029508 review: summary: mRNA splicing, via spliceosome (TreeGrafter transfer from the PANTHER PTHR16105 family node). action: ACCEPT reason: Donkey RBM41 is classified in PANTHER subfamily PTHR16105:SF2 (RNA-BINDING PROTEIN 41), the same subfamily as human RBM41 (Q96IZ5), so the human experimental data transfer to it as an ortholog. The PAINT family node behind this term is seeded by the 65K paralogs (RNPC3, Arabidopsis SNRNP65, fly and fish orthologs), and the term holds for the RBM41 branch on its own evidence. Human RBM41 is a specific component of the minor spliceosome, binds U12 snRNA through its RRM, co-precipitates excised U12-type intron lariats and DHX8, and its knockout alters 3' splice site usage at U12-type introns. The protein therefore does part of the work of spliceosomal splicing, in the post-catalytic steps of the minor spliceosome cycle, rather than only being required for it. supported_by: - reference_id: PMID:38499487 supporting_text: providing evidence that RBM41 functions in post-splicing steps of the minor spliceosome assembly/disassembly cycle - reference_id: PMID:38499487 supporting_text: RBM41 preferentially co-immunoprecipitated U12-type but not U2-type intron lariats - reference_id: PMID:38499487 supporting_text: suggesting that the loss of RBM41 has a weak, but nevertheless statistically significant effect on the splicing of a subset of U12-type introns - reference_id: file:EQUAS/RBM41/RBM41-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: PTHR16105:SF2; RNA-BINDING PROTEIN 41 - reference_id: file:interpro/panther/PTHR16105/PTHR16105-entries.csv supporting_text: Q96IZ5,RNA-binding protein 41,protein,9606,Homo sapiens,Homo sapiens (Human),RBM41,413,PTHR16105:SF2 - term: id: GO:0003676 label: nucleic acid binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR035979 review: summary: Nucleic acid binding (InterPro RNA-binding domain superfamily mapping). action: TOO_GENERAL reason: True, since the RRM binds RNA, but uninformative beside the accepted U12 snRNA binding and the proposed U6atac snRNA binding, which both entail it. supported_by: - reference_id: PMID:38499487 supporting_text: the RBM41 RRM has a low micromolar affinity to both U12 (Kd= 2.14 μM) and U6atac hairpins (Kd= 3.78 μM; Figure 2D) - term: id: GO:0003723 label: RNA binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - InterPro:IPR000504 - UniRule:UR000414619 review: summary: RNA binding (RRM domain mapping). action: TOO_GENERAL reason: Correct. The human ortholog's RRM binds RNA directly, and alanine substitutions of the conserved Gln352 and Phe354 in the RNP1 motif abolish its snRNA associations. The specific targets are known, however, and U12 snRNA binding (accepted) and U6atac snRNA binding (proposed) both entail this term. supported_by: - reference_id: PMID:38499487 supporting_text: The Q352A and F354A mutations and deletion of the RRM led to dramatic loss of the U12, U4atac and U6atac interactions - reference_id: file:EQUAS/RBM41/RBM41-bioinformatics/RESULTS.md supporting_text: 'DOMAIN RRM: human [309, 387] KN -> donkey [333, 411] KN; 78 of 79 human residues in this span are identical' - term: id: GO:0005689 label: U12-type spliceosomal complex evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: part_of supporting_entities: - PANTHER:PTN009029508 review: summary: U12-type spliceosomal complex (TreeGrafter). action: ACCEPT reason: Human RBM41, in the same PANTHER subfamily (SF2) as the donkey protein, specifically associates with minor spliceosomal snRNAs (U12, U4atac, U6atac, not U11) and with excised U12-type intron lariats, which places it in U12-type spliceosomal complexes. Its association with the U12 mono-snRNP is proposed separately as U12 snRNP. supported_by: - reference_id: PMID:38499487 supporting_text: In contrast, V5-RBM41 co-immunoprecipitated the U12 snRNA, U4atac and U6atac snRNAs, but not the U11 snRNA - reference_id: PMID:38499487 supporting_text: RBM41 preferentially co-immunoprecipitated U12-type but not U2-type intron lariats - reference_id: file:EQUAS/RBM41/RBM41-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: PTHR16105:SF2; RNA-BINDING PROTEIN 41 - term: id: GO:0030626 label: U12 snRNA binding evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: enables supporting_entities: - PANTHER:PTN009029508 review: summary: U12 snRNA binding (TreeGrafter). action: ACCEPT reason: This is the core molecular function. The family-level PAINT node rests on the 65K paralog, and the RBM41 branch now has its own direct evidence. The isolated human RBM41 RRM binds the U12 snRNA 3'-terminal stem-loop in EMSA (Kd about 2 uM) but not a complementary control hairpin. In cells, endogenous and tagged RBM41 co-precipitate U12 snRNA in an RRM-dependent way. The donkey RRM is 78 of 79 residues identical to human, including the RNA-contacting Tyr/Gln/Phe triad positions. supported_by: - reference_id: PMID:38499487 supporting_text: the RBM41 RRM has a low micromolar affinity to both U12 (Kd= 2.14 μM) and U6atac hairpins (Kd= 3.78 μM; Figure 2D) - reference_id: PMID:38499487 supporting_text: The Q352A and F354A mutations and deletion of the RRM led to dramatic loss of the U12, U4atac and U6atac interactions - reference_id: file:EQUAS/RBM41/RBM41-bioinformatics/RESULTS.md supporting_text: 'DOMAIN RRM: human [309, 387] KN -> donkey [333, 411] KN; 78 of 79 human residues in this span are identical' - term: id: GO:0097157 label: pre-mRNA intronic binding evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: enables supporting_entities: - PANTHER:PTN009029508 review: summary: Pre-mRNA intronic binding (TreeGrafter). action: UNDECIDED reason: This term comes from the family-level PAINT node (Arabidopsis SNRNP65 among its donors) and not from RBM41 data. For human RBM41, the characterized RNA contacts are the 3' stem-loops of U12 and U6atac snRNAs, and the RRM did not bind a non-cognate control hairpin. RBM41 does co-precipitate excised U12-type intron lariats, but this was shown by immunoprecipitation from cells and is consistent with indirect association through the post-splicing complex. Direct contact with intronic sequence has not been shown. No evidence against inheritance has been found either, so the row is left undecided rather than removed. supported_by: - reference_id: PMID:38499487 supporting_text: In contrast, no complex formation was observed with either RRM when a control hairpin (complementary to the U12 hairpin) was used as a ligand - reference_id: PMID:38499487 supporting_text: RBM41 preferentially co-immunoprecipitated U12-type but not U2-type intron lariats - term: id: GO:0030624 label: U6atac snRNA binding evidence_type: ISS original_reference_id: PMID:38499487 review: summary: Proposed. The human ortholog's RRM binds the U6atac snRNA 3'-terminal stem-loop. action: NEW reason: The isolated human RBM41 RRM binds the U6atac 3' stem-loop with low-micromolar affinity, about half its affinity for U12. U6atac snRNA co-precipitates with RBM41 in cells, and this depends on the RRM. The donkey protein is an SF2 ortholog with an essentially identical RRM. The authors judge the U12 interaction to be the more significant one in vivo, so this term is an additional activity, not the core one. supported_by: - reference_id: PMID:38499487 supporting_text: the RBM41 RRM has a low micromolar affinity to both U12 (Kd= 2.14 μM) and U6atac hairpins (Kd= 3.78 μM; Figure 2D) - reference_id: PMID:38499487 supporting_text: The Q352A and F354A mutations and deletion of the RRM led to dramatic loss of the U12, U4atac and U6atac interactions - term: id: GO:0005693 label: U12 snRNP evidence_type: ISS original_reference_id: PMID:38499487 review: summary: Proposed. Human RBM41 cosediments with the U12 mono-snRNP, not the U11/U12 di-snRNP. action: NEW reason: In glycerol gradients of HeLa nuclear extract, RBM41 peaks with the U12 mono-snRNP and cosediments with U11/U12-31K. It co-precipitates U12 snRNA but not U11 snRNA. This separates it from its paralog 65K, which sits in the U11/U12 di-snRNP. The donkey protein is in the same PANTHER subfamily as human RBM41. supported_by: - reference_id: PMID:38499487 supporting_text: RBM41 peak co-migrated with the U12 mono-snRNP - reference_id: PMID:38499487 supporting_text: In contrast, V5-RBM41 co-immunoprecipitated the U12 snRNA, U4atac and U6atac snRNAs, but not the U11 snRNA - term: id: GO:0015030 label: Cajal body evidence_type: ISS original_reference_id: PMID:38499487 review: summary: Proposed. Endogenous human RBM41 localizes to Cajal bodies. action: NEW reason: Endogenous RBM41 colocalizes with coilin-GFP in HEK293 nuclear bodies, and coilin is a major RBM41 BioID partner. Cajal body localization depends on the RRM, consistent with RBM41 entering Cajal bodies as part of a U12 snRNA-containing particle. This holds for the SF2 ortholog of the donkey protein. supported_by: - reference_id: PMID:38499487 supporting_text: we found that endogenous RBM41 localizes to Cajal bodies, as shown by colocalization with coilin-GFP in HEK293 cells - reference_id: PMID:38499487 supporting_text: This suggests that RBM41 localizes to Cajal bodies in an RNA binding-dependent manner. references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:38499487 title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome. findings: - statement: The human RBM41 RRM binds the 3'-terminal stem-loops of U12 and U6atac snRNAs in vitro, with about 2-fold weaker affinity than the U11/U12-65K C-terminal RRM. supporting_text: the RBM41 RRM has a low micromolar affinity to both U12 (Kd= 2.14 μM) and U6atac hairpins (Kd= 3.78 μM; Figure 2D) - statement: RBM41 partitions into the U12 mono-snRNP, separately from 65K in the U11/U12 di-snRNP. supporting_text: RBM41 peak co-migrated with the U12 mono-snRNP - statement: RBM41 associates with excised U12-type intron lariats and, through its N-terminal region, with DHX8. supporting_text: RBM41 preferentially co-immunoprecipitated U12-type but not U2-type intron lariats - statement: Endogenous RBM41 localizes to Cajal bodies. supporting_text: we found that endogenous RBM41 localizes to Cajal bodies, as shown by colocalization with coilin-GFP in HEK293 cells - statement: RBM41 knockout HEK293 cells are viable but show altered 3' splice site usage at a subset of U12-type introns. supporting_text: suggesting that the loss of RBM41 has a weak, but nevertheless statistically significant effect on the splicing of a subset of U12-type introns - statement: Phylogenetic profiling links RBM41 to minor spliceosome components across eukaryotes. supporting_text: three minor spliceosome-specific proteins, CENATAC, U11-48K (SNRP48) and U11/U12-31K (ZCRB1) were found among the proteins showing the strongest co-occurrence with RBM41 reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached from PMC (PMC11039992); PMID resolved through Europe PMC. This is the only primary functional study of RBM41, done on the human protein, which is in the same PANTHER subfamily (PTHR16105:SF2) as the donkey protein. - id: file:EQUAS/RBM41/RBM41-uniprot.txt title: UniProt record for donkey RBM41 (A0A9L0IK51) findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Used only for the PANTHER subfamily classification (PTHR16105:SF2) and domain features. The FUNCTION line here is ARBA-derived and is not used as support. - id: file:interpro/panther/PTHR16105/PTHR16105-entries.csv title: PANTHER PTHR16105 family member list (InterPro export) findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Shows that human RBM41 (Q96IZ5) and mouse Rbm41 (Q8JZV4) are in subfamily SF2, apart from the RNPC3/65K subfamily SF0. - id: file:EQUAS/RBM41/RBM41-bioinformatics/human-Q96IZ5-uniprot.txt title: UniProt record for human RBM41 (Q96IZ5) findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: The 2026-10 re-review no longer uses this as support. Its FUNCTION line ("May bind RNA") predates the 2024 functional study. Retained for provenance. - id: file:EQUAS/RBM41/RBM41-bioinformatics/RESULTS.md title: 'RBM41: donkey-human sequence comparison' findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Global alignment of donkey to human RBM41 showing near-identity across the RRM. Pairwise identity alone does not establish orthology; the PANTHER SF2 placement does. - id: file:EQUAS/RBM41/RBM41-bioinformatics/interpro-IPR045164.md title: InterPro IPR045164 RBM41/RNPC3 family description findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Describes the 65K paralog. Its statement that RBM41 function "remains unclear" is superseded by PMID:38499487. No longer used as support but retained. core_functions: - description: RRM-containing protein of the minor (U12-type) spliceosome. Its C-terminal RRM binds the 3'-terminal stem-loop of U12 snRNA, and it associates with the U12 mono-snRNP and post-splicing complexes containing excised U12-type intron lariats and DHX8. It is concentrated in Cajal bodies and acts in the post-catalytic phase of U12-type intron splicing. molecular_function: id: GO:0030626 label: U12 snRNA binding directly_involved_in: - id: GO:0000398 label: mRNA splicing, via spliceosome locations: - id: GO:0015030 label: Cajal body in_complex: id: GO:0005689 label: U12-type spliceosomal complex supported_by: - reference_id: PMID:38499487 supporting_text: the RBM41 RRM has a low micromolar affinity to both U12 (Kd= 2.14 μM) and U6atac hairpins (Kd= 3.78 μM; Figure 2D) - reference_id: PMID:38499487 supporting_text: providing evidence that RBM41 functions in post-splicing steps of the minor spliceosome assembly/disassembly cycle - reference_id: file:EQUAS/RBM41/RBM41-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: PTHR16105:SF2; RNA-BINDING PROTEIN 41 - reference_id: file:EQUAS/RBM41/RBM41-bioinformatics/RESULTS.md supporting_text: 'DOMAIN RRM: human [309, 387] KN -> donkey [333, 411] KN; 78 of 79 human residues in this span are identical' suggested_questions: - question: Does RBM41 contact intronic pre-mRNA directly in post-splicing complexes, or does its lariat association go entirely through U12 snRNA and DHX8? The answer decides whether the inherited pre-mRNA intronic binding term holds for the RBM41 branch. - question: At what step is U11/U12-65K exchanged for RBM41 on the U12 snRNA 3' stem-loop, and does RBM41 recruit or activate DHX8 to release spliced mRNA from minor spliceosomes? suggested_experiments: - description: Crosslinking immunoprecipitation (CLIP/eCLIP) of RBM41 to map its direct RNA contacts on U12 and U6atac snRNAs and on U12-type intron lariats. - description: In vitro minor-spliceosome disassembly assays with RBM41-depleted extracts, with or without recombinant RBM41 or an RBM41 N-terminal mutant that cannot bind DHX8, to test whether RBM41 is needed for mRNA release or lariat-intron complex disassembly.