HYPER RECOMBINATION1 of the THO/TREX Complex Plays a Role in Controlling Transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 Gene in Arabidopsis

February HYPER RECOMBINATION1 of the THO/TREX Complex Plays a Role in Controlling Transcription of the REVERSION-TO- ETHYLENE SENSITIVITY1 Gene in Arabidopsis Congyao Xu 0 1 2 Xin Zhou 0 1 2 Chi-Kuang Wen 0 1 2 0 Current address: State Key Laboratory of Genetic Engineering and Fudan Institute of Plant Biology, School of Life Sciences, Fudan University , Shanghai , China 1 National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China 2 Editor: James Giovannoni, Cornell University, UNIT- ED STATES Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) represses ethylene hormone responses by promoting ethylene receptor ETHYLENE RESPONSE1 (ETR1) signaling, which negatively regulates ethylene responses. To investigate the regulation of RTE1, we performed a genetic screening for mutations that suppress ethylene insensitivity conferred by RTE1 overexpression in Arabidopsis. We isolated HYPER RECOMBINATION1 (HPR1), which is required for RTE1 overexpressor (RTE1ox) ethylene insensitivity at the seedling but not adult stage. HPR1 is a component of the THO complex, which, with other proteins, forms the TRanscription EXport (TREX) complex. In yeast, Drosophila, and humans, the THO/TREX complex is involved in transcription elongation and nucleocytoplasmic RNA export, but its role in plants is to be fully determined. We investigated how HPR1 is involved in RTE1ox ethylene insensitivity in Arabidopsis. The hpr1-5 mutation may affect nucleocytoplasmic mRNA export, as revealed by in vivo hybridization of fluorescein-labeled oligo(dT)45 with unidentified mRNA in the nucleus. The hpr1-5 mutation reduced the total and nuclear RTE1 transcript levels to a similar extent, and RTE1 transcript reduction rate was not affected by hpr1-5 with cordycepin treatment, which prematurely terminates transcription. The defect in the THO-interacting TEX1 protein of TREX but not the mRNA export factor SAC3B also reduced the total and nuclear RTE1 levels. SERINE-ARGININE-RICH (SR) proteins are involved mRNA splicing, and we found that SR protein SR33 co-localized with HPR1 in nuclear speckles, which agreed with the association of human TREX with the splicing machinery. We reveal a role for HPR1 in RTE1 expression during transcription elongation and less likely during export. Gene expression involved in ethylene signaling suppression was not reduced by the hpr1-5 mutation, which indicates selectivity of HPR1 for - Funding: This work was supported by the the Chinese Ministry of Science and Technology (2011CB100700 and 2012AA10A302-2; http:// program.most.gov.cn/)and National Natural Sciences Foundation of China (31123006 and 31370314; http:// www.nsfc.org.cn/) to CKW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The transcription and export of RNA is a dynamic and highly coordinated process. mRNA species that are selectively mediated by the THO/TRanscription EXport (THO/TREX) complex for their transcription and export remain to be identified. As well, the specific roles of complex components in transcription-coupled export are unclear. We reveal a role for HYPER RECOMBINATION1 (HPR1) [the yeast HYPER RECOMBINATION1 (Hpr1) homolog] in REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) transcription elongation but not activation or export, which agrees with the role of yeast Hpr1 in transcription elongation. Defects in the THO/TREX component TEX1 but not the RNA-export TREX-2 component SAC3B also reduced the native RTE1 level. Our data suggest a specific role of the THO/TREX component HPR1 in RTE1 expression. Whether TEX1 is involved in RTE1 transcription or stability remains to be determined. The yeast Sub2 protein is an RNA helicase involved in unwinding the inhibitory structure in the nascent RNA, and SUB2 overexpression suppresses yeast hpr1 defects; HPR1 could be involved in expression of selected genes with higher-order structure, where RNA polymerase movement could pause. Studies of the gene structure and transcription activity could shed light on roles of these components in gene expression regulation at the transcription-export level. In Arabidopsis, the gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members that structurally resemble prokaryotic two-component histidine kinases (HKs). Ethylene receptor signaling is not associated with HK activity, and the biochemical nature of the receptor signal remains to be determined [1]. Without biochemical knowledge of the ethylene receptor signal output, ethylene receptor signaling is largely evaluated by ethylene-induced growth inhibition or altered gene expression. Through molecular genetic studies, a model for ethylene receptor signaling has been proposed. Without ethylene, the ethylene receptors mediate the receptor signal output to suppress ethylene signaling; ethylene binding to the receptors prevents receptor signaling and suppression of ethylene signaling is relieved [2, 3]. The five members of the ethylene receptor family have common and divergent functions and act cooperatively as complexes [47]. The ethylene receptor ETR1 can function largely independently of other family members to suppress the ethylene response to a great extent. In contrast, ETHYLENE RESPONSE SENSOR1 (ERS1) functions differentially depending on other family members [6, 7]. REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) was isolated from a mutation that suppresses the dominant ethylene-insensitive etr12 mutation [8]. RTE1 encodes a membrane protein that is associated with the endoplasmic reticulum (ER) and Golgi apparatus [9, 10]. RTE1 overexpression results in ethylene insensitivity in wild-type plants depending on ETR1 ethylene receptor but not on other family members possibly via the ETR1 N-terminus [8, 1012]. RTE1 homologs are prevalent in higher eukaryotes, and expression of the rice RTE1 HOMOLOG1 (OsRTH1) complements the rte12 loss-of-function mutation and promotes ETR1 receptor signaling in Arabidopsis [13]. Elevated expression of the tomato RTE1 homolog GREEN RIPE (GR), with the dominant Gr mutation or CaMV35S:GR transgene, results in reduced ethylene responsiveness in fruit tissue and thus the non-ripening phenotype [14]. RTE1 functions in ethylene signaling may be highly conserved across higher plant species. To identify components mediating RTE1 functions, we performed a suppressor screen for RTE1 overexpressor (RTE1ox) and isolated HYPER RECOMBINATION1 (HPR1). HPR1 encodes a protein homologous to yeast (Saccharomyces cerevisiae) HYPER-RECOMBINATION1 (Hpr1) protein, a component of the mRNA transcription export machinery, namely the THO complex. Of note, mutations that affect the mRNA transcription export m (...truncated)