Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

Enhancer release and retargeting activates disease-susceptibility genes.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

Cross-modal coherent registration of whole mouse brains.

Recent whole-brain mapping projects are collecting large-scale three-dimensional images using modalities such as serial two-photon tomography, fluorescence micro-optical sectioning tomography, light-sheet fluorescence microscopy, volumetric imaging with synchronous on-the-fly scan and readout or magnetic resonance imaging. Registration of these multi-dimensional whole-brain images onto a standard atlas is essential for characterizing neuron types and constructing brain wiring diagrams. However, cross-modal image registration is challenging due to intrinsic variations of brain anatomy and artifacts resulting from different sample preparation methods and imaging modalities. We introduce a cross-modal registration method, mBrainAligner, which uses coherent landmark mapping and deep neural networks to align whole mouse brain images to the standard Allen Common Coordinate Framework atlas. We build a brain atlas for the fluorescence micro-optical sectioning tomography modality to facilitate single-cell mapping, and used our method to generate a whole-brain map of three-dimensional single-neuron morphology and neuron cell types.

KCNH6 is essential for insulin secretion by regulating intracellular ER Ca2+ store.

Appropriate Ca2+ concentration in the endoplasmic reticulum (ER), modulating cytosolic Ca2+ signal, serves significant roles in physiological function of pancreatic ß cells. To maintaining ER homeostasis, Ca2+ movement across the ER membrane is always accompanied by a simultaneous K+ flux in the opposite direction. KCNH6 was proven to modulate insulin secretion by controlling plasma membrane action potential duration and intracellular Ca2+ influx. Meanwhile, the specific function of KCNH6 in pancreatic ß-cells remains unclear. In this study, we found that KCNH6 exhibited mainly ER localization and Kcnh6 ß-cell-specific knockout (ßKO) mice suffered from abnormal glucose tolerance and impaired insulin secretion in adulthood. ER Ca2+ store was overloaded in islets of ßKO mice, which contributed to ER stress and ER stress-induced apoptosis in ß cells. Next, we verified that ethanol treatment induced increases in ER Ca2+ store and apoptosis in pancreatic ß cells, whereas adenovirus-mediated KCNH6 overexpression in islets attenuated ethanol-induced ER stress and apoptosis. In addition, tail-vein injections of KCNH6 lentivirus rescued KCNH6 expression in ßKO mice, restored ER Ca2+ overload and attenuated ER stress in ß cells, which further confirms that KCNH6 protects islets from ER stress and apoptosis. These data suggest that KCNH6 on the ER membrane may help to stabilize intracellular ER Ca2+ stores and protect ß cells from ER stress and apoptosis. In conclusion, our study reveals the protective potential of KCNH6-targeting drugs in ER stress-induced diabetes.

Bach2 in CD4+ T cells from SLE patients modulates B-cell differentiation and IgG production.

T and B cells participate in the development of systemic lupus erythematosus (SLE). BTB and CNC homology 2 (Bach2) is an irreplaceable regulator in the T and B lineages that helps to maintain immune homeostasis. However, the function of Bach2 in the pathogenesis of SLE has not been studied in depth. Flow cytometry and qRT-PCR were used to assess Bach2 levels, bisulfite sequencing PCR was used to measure the methylation level, and silencing by electroporation and stimulation with a cytokine concentration gradient were used to investigate the effect of Bach2 on T cells. Bach2 expression was elevated in the helper T-cell subsets (T follicular helper, Th1, Th2, Th17, and Treg cells) of SLE patients and negatively correlated with disease severity and autoantibody levels. CD4+ T cells from SLE patients had decreased methylation levels in the Bach2 promoter region. Silencing Bach2 in CD4+ T cells induced increases in the CD19+ B-cell count, plasmablasts, and secretion of IgG by prompting the secretion of cytokines. The activation signals CD3/CD28, IL-6, and IL-21 upregulated Bach2 expression in CD4+ T cells. The regulation of Bach2 by cytokines and T-cell activation signals in CD4+ T cells was shown to act on B cells and play a protective role against SLE.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

Semi-automatic proximal humeral trabecular bone density assessment tool: technique application and clinical validation.

PURPOSE: This study aimed to apply a newly developed semi-automatic phantom-less QCT (PL-QCT) to measure proximal humerus trabecular bone density based on chest CT and verify its accuracy and precision. METHODS: Subcutaneous fat of the shoulder joint and trapezius muscle were used as calibration references for PL-QCT BMD measurement. A self-developed algorithm based on a convolution map was utilized in PL-QCT for semi-automatic BMD measurements. CT values of ROIs used in PL-QCT measurements were directly used for phantom-based quantitative computed tomography (PB-QCT) BMD assessment. The study included 376 proximal humerus for comparison between PB-QCT and PL-QCT. Two sports medicine doctors measured the proximal humerus with PB-QCT and PL-QCT without knowing each other's results. Among them, 100 proximal humerus were included in the inter-operative and intra-operative BMD measurements for evaluating the repeatability and reproducibility of PL-QCT and PB-QCT. RESULTS: A total of 188 patients with 376 shoulders were involved in this study. The consistency analysis indicated that the average bias between proximal humerus BMDs measured by PB-QCT and PL-QCT was 1.0 mg/cc (agreement range - 9.4 to 11.4; P > 0.05, no significant difference). Regression analysis between PB-QCT and PL-QCT indicated a good correlation (R-square is 0.9723). Short-term repeatability and reproducibility of proximal humerus BMDs measured by PB-QCT (CV: 5.10% and 3.41%) were slightly better than those of PL-QCT (CV: 6.17% and 5.64%). CONCLUSIONS: We evaluated the bone quality of the proximal humeral using chest CT through the semi-automatic PL-QCT system for the first time. Comparison between it and PB-QCT indicated that it could be a reliable shoulder BMD assessment tool with acceptable accuracy and precision. This study developed and verify a semi-automatic PL-QCT for assessment of proximal humeral bone density based on CT to assist in the assessment of proximal humeral osteoporosis and development of individualized treatment plans for shoulders.

LncRNA PVT1 promotes strong stemness and endothelial progenitor cell characteristics in renal carcinoma stem cells.

Renal cancer stem cells (RCSCs) derived from clear cell renal cell carcinoma (ccRCC) tissues with higher microvessel density (MVD) have strong stemness and endothelial progenitor cells-like (EPCs-like) characteristics. A high level of lncRNA PVT1 expression is essential for simultaneously retaining strong RCSC stemness and EPCs-like characteristics. PVT1 binds with TAZ protein and prevents its phosphorylation, which promotes RCSC stemness. Moreover, RCSCs support endothelial differentiation and angiogenesis, which are mediated via the PVT1/miR-15b/KDR axis. This report provides insight into the determinants of RCSC impact on stemness and highlights the critical role of RCSC in angiogenesis. The presented findings suggest that targeting RCSC through PVT1 expression may be a new treatment strategy for ccRCC.

Atrial cardiomyocytes contribute to the inflammatory status associated with atrial fibrillation in right heart disease.

AIMS: Right heart disease (RHD), characterized by right ventricular (RV) and atrial (RA) hypertrophy, and cardiomyocytes' (CM) dysfunctions have been described to be associated with the incidence of atrial fibrillation (AF). Right heart disease and AF have in common, an inflammatory status, but the mechanisms relating RHD, inflammation, and AF remain unclear. We hypothesized that right heart disease generates electrophysiological and morphological remodelling affecting the CM, leading to atrial inflammation and increased AF susceptibility. METHODS AND RESULTS: Pulmonary artery banding (PAB) was surgically performed (except for sham) on male Wistar rats (225-275 g) to provoke an RHD. Twenty-one days (D21) post-surgery, all rats underwent echocardiography and electrophysiological studies (EPS). Optical mapping was performed in situ, on Langendorff-perfused hearts. The contractility of freshly isolated CM was evaluated and recorded during 1 Hz pacing in vitro. Histological analyses were performed on formalin-fixed RA to assess myocardial fibrosis, connexin-43 levels, and CM morphology. Right atrial levels of selected genes and proteins were obtained by qPCR and Western blot, respectively. Pulmonary artery banding induced severe RHD identified by RV and RA hypertrophy. Pulmonary artery banding rats were significantly more susceptible to AF than sham. Compared to sham RA CM from PAB rats were significantly elongated and hypercontractile. Right atrial CM from PAB animals showed significant augmentation of mRNA and protein levels of pro-inflammatory interleukin (IL)-6 and IL1ß. Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) and junctophilin-2 were decreased in RA CM from PAB compared to sham rats. CONCLUSIONS: Right heart disease-induced arrhythmogenicity may occur due to dysfunctional SERCA2a and inflammatory signalling generated from injured RA CM, which leads to an increased risk of AF.

Zebrafish CERKL Enhances Host TBK1 Stability and Simultaneously Degrades Viral Protein via Ubiquitination Modulation.

In the viral infection process, host gene function is usually reported as either defending the host or assaulting the virus. In this study, we demonstrated that zebrafish ceramide kinase-like (CERKL) mediates protection against viral infection via two distinct mechanisms: stabilization of TANK-binding kinase 1 (TBK1) through impairing K48-linked ubiquitination and degradation of spring viremia of carp virus (SVCV) P protein by dampening K63-linked ubiquitination, resulting in an improvement of the host immune response and a decline in viral activity in epithelioma papulosum cyprini (EPC) cells. On SVCV infection, ifnφ1 expression was increased or blunted by CERKL overexpression or knockdown, respectively. Subsequently, we found that CERKL localized in the cytoplasm, where it interacted with TBK1 and enhanced its stability by impeding the K48-linked polyubiquitination; meanwhile, the antiviral capacity of TBK1 was significantly potentiated by CERKL. In contrast, CERKL also interacted with and degraded SVCV P protein to disrupt its function in viral proliferation. Further mechanism analysis revealed K63-linked deubiquitination is the primary means of CERKL-mediated SVCV P protein degradation. Taken together, our study reveals a novel mechanism of fish defense against viral infection: the single gene cerkl is both a shield for the host and a spear against the virus, which strengthens resistance.

Computational determination of the S1(Ã1A″) absorption spectra of HONO and DONO using full-dimensional neural network potential energy surfaces.

The Ã1A″ ← X̃1A' absorption spectra of HONO and DONO were simulated by a full six-dimensional quantum mechanical method based on the newly constructed potential energy surfaces for the ground and excited electronic states, which were represented by the neural network method utilizing over 36 000 ab initio energy points calculated at the multireference configuration interaction level with Davidson correction. The absorption spectrum of HONO/DONO comprises a superposition of the spectra from two isomers, namely, trans- and cis-HONO/DONO, due to their coexistence in the ground X̃1A' state. Our calculated spectra of both HONO and DONO were found to be in fairly good agreement with the experiment, including the energy positions and widths of the peaks. The dominant progression was assigned to the N=O stretch mode (20n) associated with trans-HONO/DONO, which can be attributed to the promotion of an electron to the π* orbital of N=O. Specifically, the resonances with higher vibrational quanta were found to be in the domain of the Feshbach-type resonances. The assignments of the spectra and mode specificity therein are discussed.

Down-conversion and frequency spectrum convolution of the broadband signal based on active mode-locking technology.

A multifunction processor for a broadband signal based on the active mode-locking optoelectronic oscillator (OEO) is proposed and experimentally demonstrated. The central frequency down-conversion and frequency spectrum convolution of the target broadband signal (TBS) are realized by just tuning the wavelength of the optical carrier or by the time domain product, respectively. To achieve the central frequency down-conversion of the TBS, an optical tunable delay line (OTDL) is adopted to match the delay time of the OEO loop with the repetition period of the TBS. Then the spectrum convolution of the TBS is produced by just injecting a lower frequency signal consistent with the free spectral range (FSR) of the OEO loop. Moreover, the frequency convolution repetition is also greatly increased by harmonic mode-locking injection. The equivalent bandwidth of the TBS is enlarged by ∼50 times, benefiting from the frequency convolution. The central frequency conversion flexibility and the bandwidth compatibility are also discussed in detail. This work provides a multifunction processor system and may have potential usage in multifunctional integrated radar systems.

Human-multimodal deep learning collaboration in 'precise' diagnosis of lupus erythematosus subtypes and similar skin diseases.

BACKGROUND: Lupus erythematosus (LE) is a spectrum of autoimmune diseases. Due to the complexity of cutaneous LE (CLE), clinical skin image-based artificial intelligence is still experiencing difficulties in distinguishing subtypes of LE. OBJECTIVES: We aim to develop a multimodal deep learning system (MMDLS) for human-AI collaboration in diagnosis of LE subtypes. METHODS: This is a multi-centre study based on 25 institutions across China to assist in diagnosis of LE subtypes, other eight similar skin diseases and healthy subjects. In total, 446 cases with 800 clinical skin images, 3786 multicolor-immunohistochemistry (multi-IHC) images and clinical data were collected, and EfficientNet-B3 and ResNet-18 were utilized in this study. RESULTS: In the multi-classification task, the overall performance of MMDLS on 13 skin conditions is much higher than single or dual modals (Sen = 0.8288, Spe = 0.9852, Pre = 0.8518, AUC = 0.9844). Further, the MMDLS-based diagnostic-support help improves the accuracy of dermatologists from 66.88% ± 6.94% to 81.25% ± 4.23% (p = 0.0004). CONCLUSIONS: These results highlight the benefit of human-MMDLS collaborated framework in telemedicine by assisting dermatologists and rheumatologists in the differential diagnosis of LE subtypes and similar skin diseases.

Endoloop pre-ligation assisted resection of a giant Brunner's gland adenoma of the duodenal bulb.

Brunner's gland adenoma (BGA), also known as Brunneroma or polypoid hamartoma, is a rare benign duodenal tumor that proliferates from Brunner's glands of the duodenum. They are usually asymptomatic and discovered by chance during endoscopy. Some giant lesions can sometimes present with chronic abdominal pain, nausea, vomiting, and anemia, including gastrointestinal bleeding and obstructive symptoms, and need to be resected by surgery or endoscopy. Here we report a giant BGA that was easily and safely removed by Endoloop pre-ligation assisted resection.

[Correlations between tanshinones and soil factors of Salviae Miltiorrhizae Radix et Rhizoma from different producing areas].

Soil nutrients and inorganic elements affect not only the growth and development of medicinal plants but also the formation and accumulation of active ingredients in traditional Chinese medicines. The content of tanshinones and 28 inorganic elements in Salviae Miltiorrhizae Radix et Rhizoma samples from 18 producing areas in 6 provinces was determined, and 35 physical and chemical properties of the corresponding soil samples were determined. The enrichment characteristics of inorganic elements in Salviae Miltiorrhizae Radix et Rhizoma were analyzed. The correlation analysis and stepwise regression analysis were performed to screen out the main soil factors affecting the content of tanshinones in Salviae Miltiorrhizae Radix et Rhizoma. The results showed that the content of tanshinones in the samples from different areas varied significantly, being the highest in the samples from Shandong, the second in the samples from Henan, and low in the samples from Shanxi and Sichuan. K, Mg, Ca, and Na were rich in Salviae Miltiorrhizae Radix et Rhizoma samples, among which Na and K had the highest enrichment coefficients. The results of correlation and regression analyses showed that soil K, Na, Ti, and total nitrogen were the main soil factors affecting the tanshinones in Salviae Miltiorrhizae Radix et Rhizoma. Specifically, the content of tanshinones was positively correlated with Ti and negatively correlated with Na, K, and total nitrogen in the soil. Therefore, during the planting of Salvia miltiorrhiza, the land should be selected with full consideration to the salinity and saline land should be avoided. Secondly, the application of nitrogen and potassium fertilizers can be appropriately reduced, and water-soluble elemental fertilizers for S. miltiorrhiza should be developed.

Physiological and Biochemical Properties of Cotton Seedlings in Response to Cu2+ Stress.

Copper(II) (Cu2+) is essential for plant growth and development. However, high concentrations are extremely toxic to plants. We investigated the tolerance mechanism of cotton under Cu2+ stress in a hybrid cotton variety (Zhongmian 63) and two parent lines with different Cu2+ concentrations (0, 0.2, 50, and 100 µM). The stem height, root length, and leaf area of cotton seedlings had decreased growth rates in response to increasing Cu2+ concentrations. Increasing Cu2+ concentration promoted Cu2+ accumulation in all three cotton genotypes' roots, stems, and leaves. However, compared with the parent lines, the roots of Zhongmian 63 were richer in Cu2+ and had the least amount of Cu2+ transported to the shoots. Moreover, excess Cu2+ also induced changes in cellular redox homeostasis, causing accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Conversely, antioxidant enzyme activity increased, while photosynthetic pigment content decreased. Our findings indicated that the hybrid cotton variety fared well under Cu2+ stress. This creates a theoretical foundation for the further analysis of the molecular mechanism of cotton resistance to copper and suggests the potential of the large-scale planting of Zhongmian 63 in copper-contaminated soils.

Overexpressed CD44 is associated with B-cell activation via the HA-CD44-AIM2 pathway in lupus B cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by aberrant development of B cells and excess production of autoantibodies. Our team previously reported that absent in melanoma 2 (AIM2) regulates B-cell differentiation via the Bcl-6-Blimp-1 axis. Notably, in keyhole limpet hemocyanin (KLH)-immunized CD19creAim2f/f mice, the frequency of CD19+CD44+ B cells was decreased, accompanied by a weakened KLH response, indicating that AIM2 deficiency suppressed the antigen-induced B-cell immune response by downregulating the expression of CD44. CD44, a surface marker of T-cell activation and memory, was overexpressed in T cells of SLE patients, but its roles and mechanism in B cells have not been elucidated. In the current work, we revealed that CD44 expression was upregulated in the B cells of SLE patients and MRL/lpr mice, accompanied by elevated AIM2 expression in CD19+CD44+ B-cell subsets, and that its ligand hyaluronan (HA) was also abnormally increased in the serum of SLE patients. Notably, the extrafollicular (EF) region serves as an important site of B-cell activation and differentiation separate from the germinal center, while CD44 expression is concentrated in EF B cells. In addition, in vitro experiments demonstrated that the HA-CD44 interaction stimulated B-cell activation and upregulated the expression of AIM2 and the transcription factor STAT3. Either blocking CD44, knocking down AIM2 expression or suppressing the activity of STAT3 in B cells suppressed B-cell activation and proliferation. Moreover, blocking CD44 downregulated the expression of STAT3 and AIM2, while suppressing the activity of STAT3 decreased the expression of CD44 and AIM2. In summary, overexpressed CD44 in B cells might participate in B-cell activation and proliferation in the EF region via the HA-CD44-AIM2 pathway, providing potential targets for SLE therapy.

A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms.

The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process.

An Arabidopsis Retention and Splicing complex regulates root and embryo development through pre-mRNA splicing.

Pre-mRNA splicing is an important step in the posttranscriptional processing of transcripts and a key regulator of development. The heterotrimeric retention and splicing (RES) complex plays vital roles in the growth and development of yeast, zebrafish, and humans by mediating pre-mRNA splicing of multiple genes. However, whether the RES complex is conserved in plants and what specific functions it has remain unknown. In this study, we identified Arabidopsis (Arabidopsis thaliana) BUD13 (AtBUD13), GROWTH, DEVELOPMENT AND SPLICING 1 (GDS1), and DAWDLE (DDL) as the counterparts of the yeast RES complex subunits Bud site selection protein 13 (Bud13), U2 snRNP component Snu17 (Snu17), and Pre-mRNA leakage protein 1, respectively. Moreover, we showed that RES is an ancient complex evolutionarily conserved in eukaryotes. GDS1 directly interacts with both AtBUD13 and DDL in nuclear speckles. The BUD13 domain of AtBUD13 and the RNA recognition motif domain of GDS1 are necessary and sufficient for AtBUD13-GDS1 interaction. Mutants of AtBUD13, GDS1, and DDL failed to properly splice multiple genes involved in cell proliferation and showed defects in early embryogenesis and root development. In addition, we found that GDS1 and DDL interact, respectively, with the U2 small nuclear ribonucleoproteins auxiliary factor AtU2AF65B and the NineTeen Complex-related splicing factor SKIP, which are essential for early steps of spliceosome assembly and recognition of splice sites. Altogether, our work reveals that the Arabidopsis RES complex is important for root and early embryo development by modulating pre-mRNA splicing.

Arabidopsis KETCH1 Is Critical for the Nuclear Accumulation of Ribosomal Proteins and Gametogenesis.

Male and female gametophytes are generated from micro- or megaspore mother cells through consecutive meiotic and mitotic cell divisions. Defects in these divisions often result in gametophytic lethality. Gametophytic lethality was also reported when genes encoding ribosome-related proteins were mutated. Although numerous ribosomal proteins (RPs) have been identified in plants based on homology with their yeast and metazoan counterparts, how RPs are regulated, e.g., through dynamic subcellular targeting, is unknown. We report here that an Arabidopsis (Arabidopsis thaliana) importin ß, KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1), is critical for gametogenesis. Karyopherins are molecular chaperones mediating nucleocytoplasmic protein transport. However, the role of KETCH1 during gametogenesis is independent of HYPONASTIC LEAVES 1 (HYL1), a previously reported KETCH1 cargo. Instead, KETCH1 interacts with several RPs and is critical for the nuclear accumulation of RPL27a, whose mutations caused similar gametophytic defects. We further showed that knocking down KETCH1 caused reduced ribosome biogenesis and translational capacity, which may trigger the arrest of mitotic cell cycle progression and lead to gametophytic lethality.