Nuclear OsFKBP20-1b maintains SR34 stability and promotes the splicing of retained introns upon ABA exposure in rice.

Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome-associated protein OsFKBP20-1b during AS. We determined the AS landscape of wild-type and osfkbp20-1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron-containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20-1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays. We show that OsFKBP20-1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone-like protein in the spliceosome. OsFKBP20-1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress-responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20-1b after ABA treatment. Our findings reveal that spliceosome-associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis.

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 BACT 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 BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

Their C-termini divide Brassica rapa FT-like proteins into FD-interacting and FD-independent proteins that have different effects on the floral transition.

Members of the FLOWERING LOCUS T (FT)-like clade of phosphatidylethanolamine-binding proteins (PEBPs) induce flowering by associating with the basic leucine zipper (bZIP) transcription factor FD and forming regulatory complexes in angiosperm species. However, the molecular mechanism of the FT-FD heterocomplex in Chinese cabbage (Brassica rapa ssp. pekinensis) is unknown. In this study, we identified 12 BrPEBP genes and focused our functional analysis on four BrFT-like genes by overexpressing them individually in an FT loss-of-function mutant in Arabidopsis thaliana. We determined that BrFT1 and BrFT2 promote flowering by upregulating the expression of floral meristem identity genes, whereas BrTSF and BrBFT, although close in sequence to their Arabidopsis counterparts, had no clear effect on flowering in either long- or short-day photoperiods. We also simultaneously genetically inactivated BrFT1 and BrFT2 in Chinese cabbage using CRISPR/Cas9-mediated genome editing, which revealed that BrFT1 and BrFT2 may play key roles in inflorescence organogenesis as well as in the transition to flowering. We show that BrFT-like proteins, except for BrTSF, are functionally divided into FD interactors and non-interactors based on the presence of three specific amino acids in their C termini, as evidenced by the observed interconversion when these amino acids are mutated. Overall, this study reveals that although BrFT-like homologs are conserved, they may have evolved to exert functionally diverse functions in flowering via their potential to be associated with FD or independently from FD in Brassica rapa.

SUMO Modification of OsFKBP20-1b Is Integral to Proper Pre-mRNA Splicing upon Heat Stress in Rice.

OsFKBP20-1b, a plant-specific cyclophilin protein, has been implicated to regulate pre-mRNA splicing under stress conditions in rice. Here, we demonstrated that OsFKBP20-1b is SUMOylated in a reconstituted SUMOylation system in E.coli and in planta, and that the SUMOylation-coupled regulation was associated with enhanced protein stability using a less SUMOylated OsFKBP20-1b mutant (5KR_OsFKBP20-1b). Furthermore, OsFKBP20-1b directly interacted with OsSUMO1 and OsSUMO2 in the nucleus and cytoplasm, whereas the less SUMOylated 5KR_OsFKBP20-1b mutant had an impaired interaction with OsSUMO1 and 2 in the cytoplasm but not in the nucleus. Under heat stress, the abundance of an OsFKBP20-1b-GFP fusion protein was substantially increased in the nuclear speckles and cytoplasmic foci, whereas the heat-responsiveness was remarkably diminished in the presence of the less SUMOylated 5KR_OsFKBP20-1b-GFP mutant. The accumulation of endogenous SUMOylated OsFKBP20-1b was enhanced by heat stress in planta. Moreover, 5KR_OsFKBP20-1b was not sufficiently associated with the U snRNAs in the nucleus as a spliceosome component. A protoplast transfection assay indicated that the low SUMOylation level of 5KR_OsFKBP20-1b led to inaccurate alternative splicing and transcription under heat stress. Thus, our results suggest that OsFKBP20-1b is post-translationally regulated by SUMOylation, and the modification is crucial for proper RNA processing in response to heat stress in rice.

Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs.

Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line "50", whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.

Gibberellin Promotes Bolting and Flowering via the Floral Integrators RsFT and RsSOC1-1 under Marginal Vernalization in Radish.

Gibberellic acid (GA) is one of the factors that promotes flowering in radish (Raphanus Sativus L.), although the mechanism mediating GA activation of flowering has not been determined. To identify this mechanism in radish, we compared the effects of GA treatment on late-flowering (NH-JS1) and early-flowering (NH-JS2) radish lines. GA treatment promoted flowering in both lines, but not without vernalization. NH-JS2 plants displayed greater bolting and flowering pathway responses to GA treatment than NH-JS1. This variation was not due to differences in GA sensitivity in the two lines. We performed RNA-seq analysis to investigate GA-mediated changes in gene expression profiles in the two radish lines. We identified 313 upregulated, differentially expressed genes (DEGs) and 207 downregulated DEGs in NH-JS2 relative to NH-JS1 in response to GA. Of these, 21 and 8 genes were identified as flowering time and GA-responsive genes, respectively. The results of RNA-seq and quantitative PCR (qPCR) analyses indicated that RsFT and RsSOC1-1 expression levels increased after GA treatment in NH-JS2 plants but not in NH-JS1. These results identified the molecular mechanism underlying differences in the flowering-time genes of NH-JS1 and NH-JS2 after GA treatment under insufficient vernalization conditions.

Golgi-localized cyclophilin 21 proteins negatively regulate ABA signalling via the peptidyl prolyl isomerase activity during early seedling development.

KEY MESSAGE: Plant possesses particular Golgi-resident cyclophilin 21 proteins (CYP21s) and the catalytic isomerase activities have a negative effect on ABA signalling gene expression during early seedling development. Cyclophilins (CYPs) are essential for diverse cellular process, as these catalyse a rate-limiting step in protein folding. Although Golgi proteomics in Arabidopsis thaliana suggests the existence of several CYPs in the Golgi apparatus, only one putative Golgi-resident CYP protein has been reported in rice (Oryza sativa L.; OsCYP21-4). Here, we identified the Golgi-resident CYP21 family genes and analysed their molecular characteristics in Arabidopsis and rice. The CYP family genes (CYP21-1, CYP21-2, CYP21-3, and CYP21-4) are plant-specific, and their appearance and copy numbers differ among plant species. CYP21-1 and CYP21-4 are common to all angiosperms, whereas CYP21-2 and CYP21-3 evolved in the Malvidae subclass. Furthermore, all CYP21 proteins localize to cis-Golgi, trans-Golgi or both cis- and trans-Golgi membranes in plant cells. Additionally, based on the structure, enzymatic function, and topological orientation in Golgi membranes, CYP21 proteins are divided into two groups. Genetic analysis revealed that Group I proteins (CYP21-1 and CYP21-2) exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity and regulate seed germination and seedling growth and development by affecting the expression levels of abscisic acid signalling genes. Thus, we identified the Golgi-resident CYPs and demonstrated that their PPIase activities are required for early seedling growth and development in higher plants.

Autophagy Controls CSL/RBPJκ Stability through a p62/SQSTM1-Dependent Mechanism.

Cancer-associated fibroblasts (CAFs) are important at all tumor stages. CSL/RBPJκ suppresses the gene expression program leading to CAF activation and associated metabolic reprogramming, as well as autophagy. Little is known about CSL protein turnover, especially in the tumor microenvironment. We report that, in human dermal fibroblasts (HDFs), conditions inducing autophagy-often found in tumor stroma-down-regulate CSL protein levels but do not affect its mRNA levels. Genetic or pharmacologic targeting of the autophagic machinery blocks CSL down-modulation. Mechanistically, endogenous CSL associates with the autophagy and signaling adaptor p62/SQSTM1, which is required for CSL down-modulation by autophagy. This is functionally significant, because both CSL and p62 levels are lower in skin cancer-derived CAFs, in which autophagy is increased. Increasing cellular CSL levels stabilizes p62 and down-modulates the autophagic process. We reveal here an autophagy-initiated mechanism for CSL down-modulation, which could be targeted for stroma-focused cancer prevention and treatment.

Notch-effector CSL promotes squamous cell carcinoma by repressing histone demethylase KDM6B.

Notch 1/2 genes play tumor-suppressing functions in squamous cell carcinoma (SCC), a very common malignancy in skin and internal organs. In contrast with Notch, we show that the transcription factor CSL (also known as RBP-Jκ), a key effector of canonical Notch signaling endowed with intrinsic transcription-repressive functions, plays a tumor-promoting function in SCC development. Expression of this gene decreased in upper epidermal layers and human keratinocytes (HKCs) undergoing differentiation, while it increased in premalignant and malignant SCC lesions from skin, head/neck, and lung. Increased CSL levels enhanced the proliferative potential of HKCs and SCC cells, while silencing of CSL induced growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels gave rise to rapidly expanding tumors, while cells with silenced CSL formed smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKCs and SCC cells with silenced CSL revealed major modulation of apoptotic, cell-cycle, and proinflammatory genes. We also show that the histone demethylase KDM6B is a direct CSL-negative target, with inverse roles of CSL in HKC and SCC proliferative capacity, tumorigenesis, and tumor-associated inflammatory reaction. CSL/KDM6B protein expression could be used as a biomarker of SCC development and indicator of cancer treatment.

Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation.

Cancer-associated fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a stress-responsive transcriptional repressor, is down-modulated in skin cancer stromal cells, and Atf3 knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of IL6 induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous cancer-stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to cancer- and stroma-focused intervention.

PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation.

The Notch/CSL pathway plays an important role in skin homeostasis and carcinogenesis. CSL, the key effector of canonical Notch signaling endowed with an intrinsic transcription repressive function, suppresses stromal fibroblast senescence and Cancer Associated Fibroblast (CAF) activation through direct down-modulation of key effector genes. Interacting proteins that participate with CSL in this context are as yet to be identified. We report here that Programmed Cell Death 4 (PDCD4), a nuclear/cytoplasmic shuttling protein with multiple functions, associates with CSL and plays a similar role in suppressing dermal fibroblast senescence and CAF activation. Like CSL, PDCD4 is down-regulated in stromal fibroblasts of premalignant skin actinic keratosis (AKs) lesions and squamous cell carcinoma (SCC). While devoid of intrinsic DNA binding capability, PDCD4 is present at CSL binding sites of CAF marker genes as well as canonical Notch/CSL targets and suppresses expression of these genes in a fibroblast-specific manner. Thus, we propose that PDCD4 is part of the CSL repressive complex involved in negative control of stromal fibroblasts conversion into CAFs.

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53.

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation.

Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.

Multifactorial ERß and NOTCH1 control of squamous differentiation and cancer.

Downmodulation or loss-of-function mutations of the gene encoding NOTCH1 are associated with dysfunctional squamous cell differentiation and development of squamous cell carcinoma (SCC) in skin and internal organs. While NOTCH1 receptor activation has been well characterized, little is known about how NOTCH1 gene transcription is regulated. Using bioinformatics and functional screening approaches, we identified several regulators of the NOTCH1 gene in keratinocytes, with the transcription factors DLX5 and EGR3 and estrogen receptor ß (ERß) directly controlling its expression in differentiation. DLX5 and ERG3 are required for RNA polymerase II (PolII) recruitment to the NOTCH1 locus, while ERß controls NOTCH1 transcription through RNA PolII pause release. Expression of several identified NOTCH1 regulators, including ERß, is frequently compromised in skin, head and neck, and lung SCCs and SCC-derived cell lines. Furthermore, a keratinocyte ERß-dependent program of gene expression is subverted in SCCs from various body sites, and there are consistent differences in mutation and gene-expression signatures of head and neck and lung SCCs in female versus male patients. Experimentally increased ERß expression or treatment with ERß agonists inhibited proliferation of SCC cells and promoted NOTCH1 expression and squamous differentiation both in vitro and in mouse xenotransplants. Our data identify a link between transcriptional control of NOTCH1 expression and the estrogen response in keratinocytes, with implications for differentiation therapy of squamous cancer.

IRF-4 suppresses BCR/ABL transformation of myeloid cells in a DNA binding-independent manner.

Interferon regulatory factor 4 (IRF-4) is essential for B and T cell development and immune response regulation, and has both nuclear and cytoplasmic functions. IRF-4 was originally identified as a proto-oncogene resulting from a t(6;14) chromosomal translocation in multiple myeloma and its expression was shown to be essential for multiple myeloma cell survival. However, we have previously shown that IRF-4 functions as a tumor suppressor in the myeloid lineage and in early stages of B cell development. In this study, we found that IRF-4 suppresses BCR/ABL transformation of myeloid cells. To gain insight into the molecular pathways that mediate IRF-4 tumor suppressor function, we performed a structure-function analysis of IRF-4 as a suppressor of BCR/ABL transformation. We found that the DNA binding domain deletion mutant of IRF-4, which is localized only in the cytoplasm, is still able to inhibit BCR/ABL transformation of myeloid cells. IRF-4 also functions as a tumor suppressor in bone marrow cells deficient in MyD88, an IRF-4-interacting protein found in the cytoplasm. However, IRF-4 tumor suppressor activity is lost in IRF association domain (IAD) deletion mutants. These results demonstrate that IRF-4 suppresses BCR/ABL transformation by a novel cytoplasmic function involving its IAD domain.