Areum Lee¹, Hyun Ji Park¹, Seung Hee Jo^1,2, Haemyeong Jung^1,2, Hyun‐Soon Kim¹, Hyo‐Jun Lee^1,3, Youn‐Sung Kim⁴, Choonkyun Jung^5,6 and Hye Sun Cho^1,2*

¹ Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea
² Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST),Daejeon 34141, Korea
³ Department of Functional Genomics, KRIBB School of Bioscience, UST, Daejeon 34113, Korea
⁴ Department of Biotechnology, NongWoo Bio, Anseong 17558, Korea
⁵ Department of International Agricultural Technology and Crop Biotechnology Institute/Green Bio Science and Technology, SeoulNational University, Pyeongchang 25354, Korea
⁶ Department of Agriculture, Forestry, and Bioresources and Integrated Major in Global Smart Farm, College of Agriculture and LifeSciences, Seoul National University, Seoul 08826, Korea

^*Correspondence: Hye Sun Cho

Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex B^ACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the B^ACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.