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The spliceosome is known to be stored in nuclear speckles (NS), which are recognized as active subnuclear organelles for splicing. However, it remains poorly understood how spliceosomal protein\u2013protein interactions are functionally coupled to NS organization to maintain splicing robustness in plants. Here, we report the functional significance of a specific interaction between 2 U4\/U6\u00b7U5 tri-snRNP components of the spliceosome, STA1 and DOT2, in regulating NS organization, pre-mRNA splicing, and heat stress responses in Arabidopsis. We identified a missense mutation in DOT2 (a Snu66\/SART1 homolog) from a genetic suppressor of the PRP6 homolog mutant sta1-1 (named S307). This mutation restored the weakened interaction between STA1 and DOT2 in the sta1-1 mutant background. Genetic, biochemical, and cell biological analyses showed that variation in the strength of the STA1\u2013DOT2 interaction was closely associated with changes in NS formation, splicing efficiency, transcript abundance, as well as growth and heat tolerance. Pharmacological inhibition of STA1-associated NS formation by tubercidin recapitulated sta1-1-like phenotypes and splicing defects, supporting a functional link between NS organization and splicing outcomes. In addition, heat-induced weakening of the STA1\u2013DOT2 interaction was accompanied by reduced NS formation and increased intron retention at the transcriptome-wide level, including key heat-responsive transcripts. Based on these observations, we propose that the STA1\u2013DOT2 interaction, likely reflecting the assembly state of the U4\/U6\u00b7U5 tri-snRNP, may represent one of the heat-sensitive interaction nodes that couple spliceosome assembly to NS organization and splicing robustness under stress conditions.<\/jats:p>","DOI":"10.1093\/plcell\/koag207","type":"journal-article","created":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T13:21:43Z","timestamp":1783084903000},"source":"Crossref","is-referenced-by-count":1,"title":["The STA1\u2013DOT2 interaction promotes nuclear speckle formation and splicing robustness in growth and heat stress responses"],"prefix":"10.1093","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-2341-5581","authenticated-orcid":false,"given":"Heejin","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Life Science, Sogang University , Seoul 04107 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-1125-572X","authenticated-orcid":false,"given":"Kyoung-jae","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Life Science, Sogang University , Seoul 04107 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9202-0874","authenticated-orcid":false,"given":"So Young","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Life Science and Multidisciplinary Genome Institute, Hallym University , Chuncheon 24252 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1243-4466","authenticated-orcid":false,"given":"Dong Hye","family":"Seo","sequence":"additional","affiliation":[{"name":"Plant Biotechnology Research Center, Ghent University, Global Campus , Incheon 21985 ,","place":["Republic of Korea"]},{"name":"Department of Plant Biotechnology and Bioinformatics, Ghent University , Ghent 9052 ,","place":["Belgium"]},{"name":"Department of Systems Biology and Institute of Life Science and Biotechnology, Yonsei University , Seoul 03722 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5027-4622","authenticated-orcid":false,"given":"Woo Taek","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Systems Biology and Institute of Life Science and Biotechnology, Yonsei University , Seoul 03722 ,","place":["Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3638-6043","authenticated-orcid":false,"given":"Dae-Jin","family":"Yun","sequence":"additional","affiliation":[{"name":"Department of Advanced Biotechnology and Global Plant Stress Research Center, Konkuk University , Seoul 05029 ,","place":["Republic of 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