{"gene":"PF3D7_1136300","run_date":"2026-09-08T12:51:28","timeline":{"discoveries":[{"year":2007,"finding":"P. falciparum TSN (PF3D7_1136300) possesses nuclease activity that preferentially cleaves single-stranded RNAs, and its Tudor domain functions as the RNA-binding domain. The nuclease and RNA-binding activities are inhibited by 3',5'-deoxythymidine bisphosphate (pdTp), a specific inhibitor of micrococcal nucleases. TSN shows predominantly nuclear localization in the parasite.","method":"In vitro nuclease/RNA-binding activity assays; pdTp inhibition; immunolocalization","journal":"International journal for parasitology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct biochemical activity assay with domain mapping, inhibitor validation, and subcellular localization, multiple orthogonal methods in a single study","pmids":["18035357"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Transient silencing of TSN (PF3D7_1136300) by siRNA caused significant reduction in TSN mRNA and protein, inhibited parasite growth, and blocked parasite development at the trophozoite stage. Pharmacological inhibition with pdTp produced the same developmental block, consistent with an essential role for TSN at the trophozoite stage.","method":"siRNA knockdown; pdTp chemical inhibition; parasite growth assay; morphological examination","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function (siRNA + chemical inhibitor) with specific phenotypic readout (trophozoite-stage block), two orthogonal methods, single lab","pmids":["18486605"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"TSN (PF3D7_1136300) interacts with multiple P. falciparum nuclear proteins identified in a bacterial two-hybrid screen, including Histone H2A, SPT2 (a transcriptional regulator), and a Cold-shock DNA-binding protein. Yeast two-hybrid domain mapping showed that the SN domains of TSN mediate interactions with Plasmodium N-methyl-D-aspartate receptor associated protein, PfUbiquitin conjugating enzyme, and Cold-shock DNA-binding protein. The Tudor domain of TSN interacts with the spliceosomal Sm protein PfSmD1, implicating TSN in spliceosome assembly.","method":"Bacterial two-hybrid screen; yeast two-hybrid domain mapping; immunolocalization of interacting partners","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two-hybrid screens with domain mapping, single lab, multiple interaction partners identified but limited in vitro validation","pmids":["20713134"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"The Tudor domain of TSN (PF3D7_1136300; referred to as PfTu-TSN in this study) interacts with the spliceosomal Sm protein PfSmD1, and this interaction is methylation-dependent, as demonstrated by pull-down assays.","method":"Yeast two-hybrid analysis; in vitro pull-down assay; immunofluorescence co-localization","journal":"Malaria journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal pull-down and yeast two-hybrid for the TSN–SmD1 interaction, with methylation-dependence demonstrated, single lab","pmids":["24047207"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"Under persistent cellular stress inducing apoptosis-like cell death in P. falciparum, activation of VAD-FMK-binding proteases leads to proteolytic degradation of TSN (PF3D7_1136300), a phylogenetically conserved metacaspase target. Degradation of TSN and other spliceosomal components impairs mRNA splicing, causing accumulation of unprocessed RNAs and dysregulation of vital cellular functions.","method":"Quantitative global proteomics; detection of TSN degradation under stress; functional readout of mRNA splicing impairment","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic identification of TSN degradation linked to splicing defects under defined stress conditions, single lab, multiple readouts","pmids":["26136076"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"finding":"P. falciparum metacaspase-2 (PfMCA-2) directly cleaves TSN (PF3D7_1136300) in vitro. Proteolysis of TSN was abolished by treatment with the Z-FA-FMK inhibitor of PfMCA-2, establishing TSN as a natural substrate of PfMCA-2 in the context of parasite cell death.","method":"In vitro cleavage assay (recombinant PfMCA-2 + TSN substrate); pharmacological inhibition with Z-FA-FMK; western blot detection of cleavage products","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro cleavage assay establishing TSN as PfMCA-2 substrate, with inhibitor confirmation, but single lab and limited mutagenesis at this stage","pmids":["29317266"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"finding":"PfMCA-2 cleaves TSN (PF3D7_1136300) at multiple sites both in vitro and in vivo. Active-site mutagenesis showed that Cys157 and His205 (catalytic dyad) of PfMCA-2 are essential for its general enzymatic activity, while Tyr224 and Cys157 are specifically required for interaction with and cleavage of TSN. The Tyr224A mutant retained activity on an artificial substrate but abolished TSN cleavage, indicating that PfMCA-2 engages TSN through a non-canonical mechanism using non-conserved residues.","method":"In vitro and in vivo cleavage assays; active-site mutagenesis of PfMCA-2 (Cys157A, His205A, Tyr224A); artificial peptide substrate (z-GRR-AMC) activity assay","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro cleavage combined with site-directed mutagenesis of catalytic residues and orthogonal in vivo confirmation, multiple mutants tested","pmids":["35670460"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P. falciparum TSN (PF3D7_1136300) is a nuclear, multifunctional protein with intrinsic nuclease activity (cleaving single-stranded RNA via its SN domains) and RNA-binding activity (via its Tudor domain); the Tudor domain also mediates methylation-dependent interaction with the spliceosomal Sm protein PfSmD1, implicating TSN in spliceosome assembly, while its SN domains interact with transcriptional regulators and other nuclear proteins; TSN is essential for parasite survival at the trophozoite stage, and during apoptosis-like cell death it is proteolytically cleaved by metacaspase-2 (PfMCA-2) at multiple sites through a non-canonical mechanism requiring Tyr224 and Cys157 of PfMCA-2, leading to impaired mRNA splicing and parasite death."},"narrative":{"mechanistic_narrative":"TSN (PF3D7_1136300) is a multifunctional nuclear protein of P. falciparum that couples intrinsic nuclease activity to RNA metabolism and spliceosome assembly [PMID:18035357, PMID:20713134]. It preferentially cleaves single-stranded RNA and binds RNA through its Tudor domain, with both activities sensitive to the micrococcal nuclease inhibitor pdTp, indicating a staphylococcal-nuclease-type catalytic mechanism [PMID:18035357]. TSN engages the parasite nuclear machinery through domain-partitioned interactions: its SN domains bind transcriptional and other nuclear regulators including SPT2, a cold-shock DNA-binding protein, and a ubiquitin-conjugating enzyme, while its Tudor domain mediates a methylation-dependent interaction with the spliceosomal Sm protein PfSmD1, linking TSN to spliceosome assembly [PMID:20713134, PMID:24047207]. TSN function is essential at the trophozoite stage, as both siRNA silencing and pdTp inhibition block parasite development at this point [PMID:18486605]. During apoptosis-like cell death, TSN is a substrate of the parasite metacaspase PfMCA-2, which cleaves it at multiple sites through a non-canonical mechanism requiring the non-conserved Tyr224 (together with catalytic Cys157) of PfMCA-2; this proteolysis of TSN and other spliceosomal components impairs mRNA splicing and contributes to parasite death [PMID:26136076, PMID:29317266, PMID:35670460].","teleology":[{"year":2007,"claim":"Establishing whether the parasite TSN was catalytically active resolved that it is a bona fide ssRNA nuclease whose RNA-binding function maps to the Tudor domain, defining its core biochemical identity.","evidence":"In vitro nuclease/RNA-binding assays with pdTp inhibition and immunolocalization in P. falciparum","pmids":["18035357"],"confidence":"High","gaps":["Physiological RNA substrates in the cell not identified","Relative contribution of the individual SN domains to catalysis not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"claim":"Loss-of-function tests addressed whether TSN activity is required for the parasite, showing it is essential specifically at the trophozoite stage.","evidence":"siRNA knockdown plus pdTp chemical inhibition with growth and morphological readouts in P. falciparum","pmids":["18486605"],"confidence":"Medium","gaps":["Molecular cause of the trophozoite-stage arrest not defined","pdTp is not TSN-specific, so off-target contribution to phenotype not excluded"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Interaction screening addressed which nuclear partners TSN engages and through which domains, revealing SN-domain interactions with transcriptional/nuclear regulators and a Tudor-domain link to the spliceosomal Sm protein PfSmD1.","evidence":"Bacterial and yeast two-hybrid screens with domain mapping and partner immunolocalization in P. falciparum","pmids":["20713134"],"confidence":"Medium","gaps":["Interactions largely rest on two-hybrid data with limited in vitro validation","Functional consequence of each interaction not tested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Characterizing the Tudor domain–PfSmD1 interaction determined that it is methylation-dependent, mechanistically connecting TSN to spliceosome assembly.","evidence":"Yeast two-hybrid, in vitro pull-down, and immunofluorescence co-localization in P. falciparum","pmids":["24047207"],"confidence":"Medium","gaps":["The methyltransferase generating the PfSmD1 methyl mark not identified","Direct role of this interaction in splicing not functionally demonstrated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"Proteomic analysis of stress-induced death addressed the fate of TSN during apoptosis-like death, showing it is degraded by VAD-FMK-binding proteases with concurrent splicing impairment.","evidence":"Quantitative global proteomics with mRNA splicing readouts under apoptosis-inducing stress in P. falciparum","pmids":["26136076"],"confidence":"Medium","gaps":["Correlative link between TSN degradation and splicing defect not causally isolated","Specific protease responsible not identified in this study"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2018,"claim":"Reconstitution identified the responsible protease, establishing TSN as a direct natural substrate of the parasite metacaspase PfMCA-2.","evidence":"In vitro cleavage of recombinant TSN by PfMCA-2 with Z-FA-FMK inhibition and western blot detection in P. falciparum","pmids":["29317266"],"confidence":"Medium","gaps":["Cleavage sites within TSN not mapped","Catalytic residues of PfMCA-2 required for TSN cleavage not yet defined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2021,"claim":"Active-site mutagenesis dissected how PfMCA-2 recognizes TSN, showing a non-canonical mechanism in which Tyr224 (with catalytic Cys157) is required specifically for TSN engagement and cleavage.","evidence":"In vitro and in vivo cleavage assays with PfMCA-2 mutants (Cys157A, His205A, Tyr224A) and an artificial z-GRR-AMC substrate in P. falciparum","pmids":["35670460"],"confidence":"High","gaps":["Cleaved TSN fragments' downstream fate and effect on nuclease/splicing function not resolved","Structural basis of the Tyr224-dependent TSN recognition not determined"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How TSN's nuclease and spliceosome-assembly functions are integrated to sustain the essential trophozoite-stage role, and how their disruption by PfMCA-2 cleavage executes parasite death, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["In vivo RNA substrates of TSN nuclease activity unknown","Mechanistic connection between TSN loss and trophozoite arrest undefined","Whether PfMCA-2 cleavage inactivates the splicing versus nuclease function specifically is untested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["spliceosomal Sm complex (via Tudor-domain interaction with PfSmD1)"],"partners":["PFSMD1 (TUDOR DOMAIN, METHYLATION-DEPENDENT)","HISTONE H2A","SPT2 TRANSCRIPTIONAL REGULATOR","COLD-SHOCK DNA-BINDING PROTEIN (SN DOMAIN)","PLASMODIUM N-METHYL-D-ASPARTATE RECEPTOR ASSOCIATED PROTEIN (SN DOMAIN)","PFUBIQUITIN CONJUGATING ENZYME (SN DOMAIN)","PFMCA-2 (METACASPASE-2, CLEAVES TSN VIA TYR224/CYS157)"],"other_free_text":["Nuclease and RNA-binding activities inhibited by the micrococcal nuclease inhibitor pdTp [#0]","Essential at the trophozoite stage; silencing or pdTp inhibition blocks development [#1]","Proteolyzed by PfMCA-2 at multiple sites through a non-canonical mechanism during apoptosis-like cell death, contributing to impaired mRNA splicing [#4, #6]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"26136076","id":"PMC_26136076","title":"Disruption of cellular homeostasis induces organelle stress and triggers apoptosis like cell-death pathways in malaria parasite.","date":"2015","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/26136076","citation_count":52,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["TSN","tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18035357","id":"PMC_18035357","title":"Tudor domain proteins in protozoan parasites and characterization of Plasmodium falciparum tudor staphylococcal nuclease.","date":"2007","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18035357","citation_count":27,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["TSN","tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20713134","id":"PMC_20713134","title":"Plasmodium falciparum Tudor Staphylococcal Nuclease interacting proteins suggest its role in nuclear as well as splicing processes.","date":"2010","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/20713134","citation_count":15,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29317266","id":"PMC_29317266","title":"Biochemical characterization of unusual cysteine protease of P. falciparum, metacaspase-2 (MCA-2).","date":"2018","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/29317266","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["TSN","tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24047207","id":"PMC_24047207","title":"Organization of Plasmodium falciparum spliceosomal core complex and role of arginine methylation in its assembly.","date":"2013","source":"Malaria journal","url":"https://pubmed.ncbi.nlm.nih.gov/24047207","citation_count":12,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["TSN","tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18486605","id":"PMC_18486605","title":"Transient silencing of Plasmodium falciparum Tudor Staphylococcal Nuclease suggests an essential role for the protein.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18486605","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["tudor staphylococcal nuclease"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35670460","id":"PMC_35670460","title":"Plasmodium falciparum metacaspase-2 capture its natural substrate in a non-canonical way.","date":"2021","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35670460","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["TSN"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6591,"output_tokens":2242,"usd":0.026701,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7590,"output_tokens":3385,"usd":0.061288,"stage2_stop_reason":"end_turn"},"total_usd":0.087989,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"P. falciparum TSN (PF3D7_1136300) possesses nuclease activity that preferentially cleaves single-stranded RNAs, and its Tudor domain functions as the RNA-binding domain. The nuclease and RNA-binding activities are inhibited by 3',5'-deoxythymidine bisphosphate (pdTp), a specific inhibitor of micrococcal nucleases. TSN shows predominantly nuclear localization in the parasite.\",\n      \"method\": \"In vitro nuclease/RNA-binding activity assays; pdTp inhibition; immunolocalization\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct biochemical activity assay with domain mapping, inhibitor validation, and subcellular localization, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"18035357\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Transient silencing of TSN (PF3D7_1136300) by siRNA caused significant reduction in TSN mRNA and protein, inhibited parasite growth, and blocked parasite development at the trophozoite stage. Pharmacological inhibition with pdTp produced the same developmental block, consistent with an essential role for TSN at the trophozoite stage.\",\n      \"method\": \"siRNA knockdown; pdTp chemical inhibition; parasite growth assay; morphological examination\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function (siRNA + chemical inhibitor) with specific phenotypic readout (trophozoite-stage block), two orthogonal methods, single lab\",\n      \"pmids\": [\"18486605\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TSN (PF3D7_1136300) interacts with multiple P. falciparum nuclear proteins identified in a bacterial two-hybrid screen, including Histone H2A, SPT2 (a transcriptional regulator), and a Cold-shock DNA-binding protein. Yeast two-hybrid domain mapping showed that the SN domains of TSN mediate interactions with Plasmodium N-methyl-D-aspartate receptor associated protein, PfUbiquitin conjugating enzyme, and Cold-shock DNA-binding protein. The Tudor domain of TSN interacts with the spliceosomal Sm protein PfSmD1, implicating TSN in spliceosome assembly.\",\n      \"method\": \"Bacterial two-hybrid screen; yeast two-hybrid domain mapping; immunolocalization of interacting partners\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two-hybrid screens with domain mapping, single lab, multiple interaction partners identified but limited in vitro validation\",\n      \"pmids\": [\"20713134\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The Tudor domain of TSN (PF3D7_1136300; referred to as PfTu-TSN in this study) interacts with the spliceosomal Sm protein PfSmD1, and this interaction is methylation-dependent, as demonstrated by pull-down assays.\",\n      \"method\": \"Yeast two-hybrid analysis; in vitro pull-down assay; immunofluorescence co-localization\",\n      \"journal\": \"Malaria journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal pull-down and yeast two-hybrid for the TSN–SmD1 interaction, with methylation-dependence demonstrated, single lab\",\n      \"pmids\": [\"24047207\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Under persistent cellular stress inducing apoptosis-like cell death in P. falciparum, activation of VAD-FMK-binding proteases leads to proteolytic degradation of TSN (PF3D7_1136300), a phylogenetically conserved metacaspase target. Degradation of TSN and other spliceosomal components impairs mRNA splicing, causing accumulation of unprocessed RNAs and dysregulation of vital cellular functions.\",\n      \"method\": \"Quantitative global proteomics; detection of TSN degradation under stress; functional readout of mRNA splicing impairment\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification of TSN degradation linked to splicing defects under defined stress conditions, single lab, multiple readouts\",\n      \"pmids\": [\"26136076\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"P. falciparum metacaspase-2 (PfMCA-2) directly cleaves TSN (PF3D7_1136300) in vitro. Proteolysis of TSN was abolished by treatment with the Z-FA-FMK inhibitor of PfMCA-2, establishing TSN as a natural substrate of PfMCA-2 in the context of parasite cell death.\",\n      \"method\": \"In vitro cleavage assay (recombinant PfMCA-2 + TSN substrate); pharmacological inhibition with Z-FA-FMK; western blot detection of cleavage products\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro cleavage assay establishing TSN as PfMCA-2 substrate, with inhibitor confirmation, but single lab and limited mutagenesis at this stage\",\n      \"pmids\": [\"29317266\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PfMCA-2 cleaves TSN (PF3D7_1136300) at multiple sites both in vitro and in vivo. Active-site mutagenesis showed that Cys157 and His205 (catalytic dyad) of PfMCA-2 are essential for its general enzymatic activity, while Tyr224 and Cys157 are specifically required for interaction with and cleavage of TSN. The Tyr224A mutant retained activity on an artificial substrate but abolished TSN cleavage, indicating that PfMCA-2 engages TSN through a non-canonical mechanism using non-conserved residues.\",\n      \"method\": \"In vitro and in vivo cleavage assays; active-site mutagenesis of PfMCA-2 (Cys157A, His205A, Tyr224A); artificial peptide substrate (z-GRR-AMC) activity assay\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro cleavage combined with site-directed mutagenesis of catalytic residues and orthogonal in vivo confirmation, multiple mutants tested\",\n      \"pmids\": [\"35670460\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"P. falciparum TSN (PF3D7_1136300) is a nuclear, multifunctional protein with intrinsic nuclease activity (cleaving single-stranded RNA via its SN domains) and RNA-binding activity (via its Tudor domain); the Tudor domain also mediates methylation-dependent interaction with the spliceosomal Sm protein PfSmD1, implicating TSN in spliceosome assembly, while its SN domains interact with transcriptional regulators and other nuclear proteins; TSN is essential for parasite survival at the trophozoite stage, and during apoptosis-like cell death it is proteolytically cleaved by metacaspase-2 (PfMCA-2) at multiple sites through a non-canonical mechanism requiring Tyr224 and Cys157 of PfMCA-2, leading to impaired mRNA splicing and parasite death.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSN (PF3D7_1136300) is a multifunctional nuclear protein of P. falciparum that couples intrinsic nuclease activity to RNA metabolism and spliceosome assembly [#0, #2]. It preferentially cleaves single-stranded RNA and binds RNA through its Tudor domain, with both activities sensitive to the micrococcal nuclease inhibitor pdTp, indicating a staphylococcal-nuclease-type catalytic mechanism [#0]. TSN engages the parasite nuclear machinery through domain-partitioned interactions: its SN domains bind transcriptional and other nuclear regulators including SPT2, a cold-shock DNA-binding protein, and a ubiquitin-conjugating enzyme, while its Tudor domain mediates a methylation-dependent interaction with the spliceosomal Sm protein PfSmD1, linking TSN to spliceosome assembly [#2, #3]. TSN function is essential at the trophozoite stage, as both siRNA silencing and pdTp inhibition block parasite development at this point [#1]. During apoptosis-like cell death, TSN is a substrate of the parasite metacaspase PfMCA-2, which cleaves it at multiple sites through a non-canonical mechanism requiring the non-conserved Tyr224 (together with catalytic Cys157) of PfMCA-2; this proteolysis of TSN and other spliceosomal components impairs mRNA splicing and contributes to parasite death [#4, #5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing whether the parasite TSN was catalytically active resolved that it is a bona fide ssRNA nuclease whose RNA-binding function maps to the Tudor domain, defining its core biochemical identity.\",\n      \"evidence\": \"In vitro nuclease/RNA-binding assays with pdTp inhibition and immunolocalization in P. falciparum\",\n      \"pmids\": [\"18035357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological RNA substrates in the cell not identified\",\n        \"Relative contribution of the individual SN domains to catalysis not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Loss-of-function tests addressed whether TSN activity is required for the parasite, showing it is essential specifically at the trophozoite stage.\",\n      \"evidence\": \"siRNA knockdown plus pdTp chemical inhibition with growth and morphological readouts in P. falciparum\",\n      \"pmids\": [\"18486605\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular cause of the trophozoite-stage arrest not defined\",\n        \"pdTp is not TSN-specific, so off-target contribution to phenotype not excluded\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Interaction screening addressed which nuclear partners TSN engages and through which domains, revealing SN-domain interactions with transcriptional/nuclear regulators and a Tudor-domain link to the spliceosomal Sm protein PfSmD1.\",\n      \"evidence\": \"Bacterial and yeast two-hybrid screens with domain mapping and partner immunolocalization in P. falciparum\",\n      \"pmids\": [\"20713134\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Interactions largely rest on two-hybrid data with limited in vitro validation\",\n        \"Functional consequence of each interaction not tested\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Characterizing the Tudor domain–PfSmD1 interaction determined that it is methylation-dependent, mechanistically connecting TSN to spliceosome assembly.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pull-down, and immunofluorescence co-localization in P. falciparum\",\n      \"pmids\": [\"24047207\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The methyltransferase generating the PfSmD1 methyl mark not identified\",\n        \"Direct role of this interaction in splicing not functionally demonstrated\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Proteomic analysis of stress-induced death addressed the fate of TSN during apoptosis-like death, showing it is degraded by VAD-FMK-binding proteases with concurrent splicing impairment.\",\n      \"evidence\": \"Quantitative global proteomics with mRNA splicing readouts under apoptosis-inducing stress in P. falciparum\",\n      \"pmids\": [\"26136076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Correlative link between TSN degradation and splicing defect not causally isolated\",\n        \"Specific protease responsible not identified in this study\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Reconstitution identified the responsible protease, establishing TSN as a direct natural substrate of the parasite metacaspase PfMCA-2.\",\n      \"evidence\": \"In vitro cleavage of recombinant TSN by PfMCA-2 with Z-FA-FMK inhibition and western blot detection in P. falciparum\",\n      \"pmids\": [\"29317266\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Cleavage sites within TSN not mapped\",\n        \"Catalytic residues of PfMCA-2 required for TSN cleavage not yet defined\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Active-site mutagenesis dissected how PfMCA-2 recognizes TSN, showing a non-canonical mechanism in which Tyr224 (with catalytic Cys157) is required specifically for TSN engagement and cleavage.\",\n      \"evidence\": \"In vitro and in vivo cleavage assays with PfMCA-2 mutants (Cys157A, His205A, Tyr224A) and an artificial z-GRR-AMC substrate in P. falciparum\",\n      \"pmids\": [\"35670460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Cleaved TSN fragments' downstream fate and effect on nuclease/splicing function not resolved\",\n        \"Structural basis of the Tyr224-dependent TSN recognition not determined\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TSN's nuclease and spliceosome-assembly functions are integrated to sustain the essential trophozoite-stage role, and how their disruption by PfMCA-2 cleavage executes parasite death, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"In vivo RNA substrates of TSN nuclease activity unknown\",\n        \"Mechanistic connection between TSN loss and trophozoite arrest undefined\",\n        \"Whether PfMCA-2 cleavage inactivates the splicing versus nuclease function specifically is untested\"\n      ],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004518\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0004521\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0044877\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0000387\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0008380\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0097153\", \"supporting_discovery_ids\": [4, 5, 6]}\n    ],\n    \"complexes\": [\n      \"spliceosomal Sm complex (via Tudor-domain interaction with PfSmD1)\"\n    ],\n    \"partners\": [\n      \"PfSmD1 (Tudor domain, methylation-dependent)\",\n      \"Histone H2A\",\n      \"SPT2 transcriptional regulator\",\n      \"Cold-shock DNA-binding protein (SN domain)\",\n      \"Plasmodium N-methyl-D-aspartate receptor associated protein (SN domain)\",\n      \"PfUbiquitin conjugating enzyme (SN domain)\",\n      \"PfMCA-2 (metacaspase-2, cleaves TSN via Tyr224/Cys157)\"\n    ],\n    \"other_free_text\": [\n      \"Nuclease and RNA-binding activities inhibited by the micrococcal nuclease inhibitor pdTp [#0]\",\n      \"Essential at the trophozoite stage; silencing or pdTp inhibition blocks development [#1]\",\n      \"Proteolyzed by PfMCA-2 at multiple sites through a non-canonical mechanism during apoptosis-like cell death, contributing to impaired mRNA splicing [#4, #6]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1136300","name":null,"product":"tudor staphylococcal nuclease","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0912300","name":null,"product":"tudor staphylococcal nuclease, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0934500","name":null,"product":"tudor staphylococcal nuclease, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"TSN","evaluation":null}