Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.

BACKGROUND: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. RESULTS: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. CONCLUSIONS: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

SF3A2 promotes progression and cisplatin resistance in triple-negative breast cancer via alternative splicing of MKRN1.

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer owing to the lack of effective therapeutic targets. Splicing factor 3a subunit 2 (SF3A2), a poorly defined splicing factor, was notably elevated in TNBC tissues and promoted TNBC progression, as confirmed by cell proliferation, colony formation, transwell migration, and invasion assays. Mechanistic investigations revealed that E3 ubiquitin-protein ligase UBR5 promoted the ubiquitination-dependent degradation of SF3A2, which in turn regulated UBR5, thus forming a feedback loop to balance these two oncoproteins. Moreover, SF3A2 accelerated TNBC progression by, at least in part, specifically regulating the alternative splicing of makorin ring finger protein 1 (MKRN1) and promoting the expression of the dominant and oncogenic isoform, MKRN1-T1. Furthermore, SF3A2 participated in the regulation of both extrinsic and intrinsic apoptosis, leading to cisplatin resistance in TNBC cells. Collectively, these findings reveal a previously unknown role of SF3A2 in TNBC progression and cisplatin resistance, highlighting SF3A2 as a potential therapeutic target for patients with TNBC.

YTHDC1 promotes the malignant progression of gastric cancer by promoting ROD1 translocation to the nucleus.

RNA-binding proteins (RBPs) make vital impacts on tumor progression and are important potential targets for tumor treatment. Previous studies have shown that RBP regulator of differentiation 1 (ROD1), enriched in the nucleus, is abnormally expressed and functions as a splicing factor in tumors; however, the mechanism underlying its involvement in gastric cancer (GC) is unknown. In this study, ROD1 is found to stimulate GC cell proliferation and metastasis and is related to poor patient prognosis. In vitro experiments showed that ROD1 influences GC proliferation and metastasis through modulating the imbalance of the level of the oncogenic gene OIP5 and the tumor suppressor gene GPD1L. Further studies showed that the N6-methyladenosine (m6A) "reader" protein YTHDC1 can interact with ROD1 and regulate the balance of the expression of the downstream molecules OIP5/GPD1L by promoting the nuclear enrichment of ROD1. Therefore, YTHDC1 stimulates GC development and progression through modulating nuclear enrichment of the splicing factor ROD1.

[Methyltransferase WTAP aggravates hypoxia/reoxygenation-induced myocardial cell injury by regulating the expression of activator of transcription 4].

OBJECTIVE: To investigate the regulatory role of Wilms tumor 1-associating protein (WTAP) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its molecular mechanism. METHODS: (1) Experiment I: H9C2 cardiomyocytes were divided into blank control group and H/R model group. H/R was used to induce myocardial ischemia/reperfusion (I/R) injury model in H9C2 cells. The blank control group was not treated. N6-methyladenosine (m6A) RNA methylation assay kit was used to detect the level of m6A. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases [WTAP, methyltransferase-like proteins (METTL3, METTL14)], respectively. (2) Experiment II: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, and H/R+sh-WTAP group. sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group, and the model establishment method in the other groups was the same as experiment I. At 48 hours after transfection, the apoptosis rate of cells was detected by flow cytometry. The protein expressions of WTAP, activated caspase-3, activated poly (ADP-ribose) polymerase (PARP), activating transcription factor 4 (ATF4), proline-rich receptor-like protein kinase (PERK), phosphorylated PERK (p-PERK) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by Western blotting. The positive expression of ATF4 was observed by immunofluorescence staining. (3) Experiment III: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group. The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes, and the modeling method of the other groups were modeled the same as experiment II. The apoptosis rate was detected by flow cytometry. Western blotting was used to detect the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP. RESULTS: (1) Experiment I: the methylation level of m6A in the H/R group was significantly higher than that in the blank control group. RT-qPCR results showed that the gene expressions of METTL3, METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group, and WTAP was the most significantly up-regulated. Western blotting results showed the same trend. These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes. (2) Experiment II: the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group [(14.16±1.58)% vs. (24.51±2.38)%, P < 0.05]. Western blotting results showed that the protein expressions of WTAP, activated caspase-3, activated PARP, p-PERK, ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group. Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group [(19.36±1.81)% vs. (32.83±2.69)%, P < 0.01]. The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress. (3) Experiment III: the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group [(26.61±2.76)% vs. (17.14±0.87)%, P < 0.05]. Western blotting results showed that the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group. These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes. CONCLUSIONS: Methyltransferase WTAP could regulate ATF4 expression, mediate cell apoptosis and endoplasmic reticulum stress, and promote H/R-induced myocardial cell injury.

WTAP regulates the production of reactive oxygen species, promotes malignant progression, and is closely related to the tumor microenvironment in glioblastoma.

RNA modifications have been substantiated to regulate the majority of physiological activities in the organism, including the metabolism of reactive oxygen species (ROS), which plays an important role in cells. As for the effect of RNA modification genes on ROS metabolism in glioblastoma (GBM), it has not been studied yet. Therefore, this study aims to screen the RNA modification genes that are most related to ROS metabolism and explore their effects on the biological behavior of GBM in vitro. Here, an association between WTAP and ROS metabolism was identified by bioinformatics analysis, and WTAP was highly expressed in GBM tissue compared with normal brain tissue, which was confirmed by western blotting and immunohistochemical staining. When using a ROS inducer to stimulate GBM cells in the WTAP overexpression group, the ROS level increased more significantly and the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) also increased. Next, colony formation assay, wound healing assay, and transwell assay were performed to investigate the proliferation, migration, and invasion of GBM cells. The results showed that WTAP, as an oncogene, promoted the malignant progression of GBM cells. Functional enrichment analysis predicted that WTAP was involved in the regulation of tumor/immune-related functional pathways. Western blotting was used to identify that WTAP had a regulatory effect on the phosphorylation of PI3K/Akt signaling. Finally, based on functional enrichment analysis, we further performed immune-related analysis on WTAP. In conclusion, this study analyzed WTAP from three aspects, which provided new ideas for the treatment of GBM.

Transcriptomic analysis reveals mitochondrial dysfunction in the pathogenesis of Nager syndrome in sf3b4-depleted zebrafish.

Nager syndrome (NS) is a rare acrofacial dysostosis caused by heterozygous loss-of-function variants in the splicing factor 3B subunit 4 (SF3B4). The main clinical features of patients with NS are characterized by facial-mandibular and preaxial limb malformations. The migration and specification of neural crest cells are crucial for craniofacial development, and mitochondrial fitness appears to play a role in such processes. Here, by analyzing our previously published transcriptome dataset, we aim to investigate the potential involvement of mitochondrial components in the pathogenesis of craniofacial malformations, especially in sf3b4 mutant zebrafish. We identified that oxidative phosphorylation (OXPHOS) defects and overproduction of reactive oxygen species (ROS) due to decreased antioxidants defense activity, which leads to oxidative damage and mitochondrial dysfunction. Furthermore, our results highlight that fish lacking sf3b4 gene, primarily display defects in mitochondrial complex I. Altogether, our findings suggest that mitochondrial dysfunction may contribute to the development of the craniofacial anomalies observed in sf3b4-depleted zebrafish.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

Unique transcriptomes of sensory and non-sensory neurons: insights from Splicing Regulatory States.

Alternative Splicing (AS) programs serve as instructive signals of cell type specificity, particularly within the brain, which comprises dozens of molecularly and functionally distinct cell types. Among them, retinal photoreceptors stand out due to their unique transcriptome, making them a particularly well-suited system for studying how AS shapes cell type-specific molecular functions. Here, we use the Splicing Regulatory State (SRS) as a novel framework to discuss the splicing factors governing the unique AS pattern of photoreceptors, and how this pattern may aid in the specification of their highly specialized sensory cilia. In addition, we discuss how other sensory cells with ciliated structures, for which data is much scarcer, also rely on specific SRSs to implement a proteome specialized in the detection of sensory stimuli. By reviewing the general rules of cell type- and tissue-specific AS programs, firstly in the brain and subsequently in specialized sensory neurons, we propose a novel paradigm on how SRSs are established and how they can diversify. Finally, we illustrate how SRSs shape the outcome of mutations in splicing factors to produce cell type-specific phenotypes that can lead to various human diseases.

SF3B1 mutations provide genetic vulnerability to copper ionophores in human acute myeloid leukemia.

In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase.

G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3' splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.

METTL3-mediated the m6A modification of SF3B4 facilitates the development of non-small cell lung cancer by enhancing LSM4 expression.

BACKGROUND: Splicing factor B subunit 4 (SF3B4) has been confirmed to participate in the progression of many cancers and is considered to be a potential target for non-small cell lung cancer (NSCLC). Thus, the role and molecular mechanism of SF3B4 in NSCLC progression deserves further study. METHODS: Quantitative real-time PCR and western blot were employed to detect the mRNA and protein levels of SF3B4, Sm-like protein 4 (LSM4) and methyltransferase-like 3 (METTL3). Cell proliferation, apoptosis, invasion, migration and stemness were tested by cell counting kit-8, colony formation, flow cytometry, transwell, wound healing, and sphere formation assays. The interaction between SF3B4 and METTL3 or LSM4 was confirmed by MeRIP, RIP and Co-IP assays. Mice xenograft models were constructed to assess the effects of METTL3 and SF3B4 on NSCLC tumorigenesis. RESULTS: SF3B4 had high expression in NSCLC tissues and was associated with the shorter overall survival of NSCLC patients. Knockdown of SF3B4 suppressed NSCLC cell proliferation, invasion, migration and stemness, while inducing apoptosis. METTL3 promoted SF3B4 mRNA stability by m6A modification, and its knockdown inhibited NSCLC cell growth, metastasis and stemness by downregulating SF3B4. SF3B4 could interact with LSM4, and sh-SF3B4-mediated the inhibition on NSCLC cell functions could be reversed by LSM4 overexpression. In addition, reduced METTL3 expression restrained NSCLC tumor growth, and this effect was reversed by SF3B4 overexpression. CONCLUSION: METTL3-stablized SF3B4 promoted NSCLC cell growth, metastasis and stemness via positively regulating LSM4.

PRPF19 mRNA Encodes a Small Open Reading Frame That Is Important for Viability of Human Cells.

High-throughput ribosome profiling demonstrates the translation of thousands of small open reading frames located in the 5' untranslated regions of messenger RNAs (upstream ORFs). Upstream ORF can both perform a regulatory function by influencing the translation of the downstream main ORF and encode a small functional protein or microprotein. In this work, we showed that the 5' untranslated region of the PRPF19 mRNA encodes an upstream ORF that is translated in human cells. Inactivation of this upstream ORF reduces the viability of human cells.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

Identification of splicing factors signature predicting prognosis risk and the mechanistic roles of novel oncogenes in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the most frequent subtype of head and neck cancer, generally with a poor prognosis and limited therapeutic options due to its highly heterogeneous malignancy. In this study, we screened functional splicing regulatory RNA binding proteins (RBPs) that were closely related with the prognosis of HNSCC patients and showed significant expression differences between HNSCC tumors and normal tissues. Based on this finding, we chose six candidate genes (HNRNPC, ZCRB1, RBM12B, SF3A2, SF3B3, and SRSF11) to generate a prognostic prediction model and validated the accuracy of the prognostic model for predicting patient survival outcomes. We found that the risk score predicted by our model can serve as an independent prognostic predictor. Notably, HNSCC tumors showing higher expression of SF3B3, HNRNPC, or ZCRB1 possessed higher risk scores in the discovered prediction model. The investigation of the underlying mechanism validated that knockdown of SF3B3, HNRNPC, and ZCRB1 separately induced a substantial impairment of HNSCC cell survival. Conversely, overexpression of each of the three genes promoted tumor cellular proliferation. High throughput RNA sequencing analysis revealed that changes in the expression of SF3B3 and HNRNPC remarkably affected alternative splicing of genes related to cell cycle regulation, whereas the depletion of ZCRB1 contributed to aberrant splicing events involving in DNA damage response. In addition, the prognostic prediction model's risk score was demonstrated to be related with the immune infiltration score. Particularly, SF3B3 has a negative correlation with CD8A expression. Therefore, our findings provide promising prognosis predictors and potential therapeutic targets for better treatment efficacy of HNSCC.

The spectrum of pre-mRNA splicing in autism.

Disruptions in spatiotemporal gene expression can result in atypical brain function. Specifically, autism spectrum disorder (ASD) is characterized by abnormalities in pre-mRNA splicing. Abnormal splicing patterns have been identified in the brains of individuals with ASD, and mutations in splicing factors have been found to contribute to neurodevelopmental delays associated with ASD. Here we review studies that shed light on the importance of splicing observed in ASD and that explored the intricate relationship between splicing factors and ASD, revealing how disruptions in pre-mRNA splicing may underlie ASD pathogenesis. We provide an overview of the research regarding all splicing factors associated with ASD and place a special emphasis on five specific splicing factors-HNRNPH2, NOVA2, WBP4, SRRM2, and RBFOX1-known to impact the splicing of ASD-related genes. In the discussion of the molecular mechanisms influenced by these splicing factors, we lay the groundwork for a deeper understanding of ASD's complex etiology. Finally, we discuss the potential benefit of unraveling the connection between splicing and ASD for the development of more precise diagnostic tools and targeted therapeutic interventions. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA RNA-Based Catalysis > RNA Catalysis in Splicing and Translation.

WTAP Mediated N6-methyladenosine RNA Modification of ELF3 Drives Cellular Senescence by Upregulating IRF8.

N6-methyladenosine (m6A), the most prevalent posttranscriptional RNA modification, involved in various diseases and cellular processes. However, the underlying mechanisms of m6A regulation in skin aging are still not fully understood. In this study, proteomics analysis revealed a significant correlation between Wilms' tumor 1-associating protein (WTAP) expression and cellular senescence. Next, upregulated WTAP was detected in aging skin tissues and senescent human dermal fibroblasts (HDFs). Functionally, overexpressed WTAP induced senescence and knockdown of WTAP rescued senescence of HDFs. Mechanistically, WTAP directly targeted ELF3 and promoted its expression in an m6A-dependent manner. Exogenous-ELF3 overexpression evidently reversed shWTAP-suppressed fibroblast senescence. Furthermore, ELF3 induced IRF8-mediated senescence-associated secretory phenotype (SASP) by binding to the (-817 to -804) site of the IRF8 promoter directly. In vivo, overexpression of WTAP evidently increased senescence cells in skin and induced skin aging. In summary, these findings revealed the critical role of WTAP-mediated m6A modification in skin aging and identified ELF3 as an important target of m6A modification in HDFs senescence, providing a new idea for delaying the aging process.

Research Progress of Group II Intron Splicing Factors in Land Plant Mitochondria.

Mitochondria are important organelles that provide energy for the life of cells. Group II introns are usually found in the mitochondrial genes of land plants. Correct splicing of group II introns is critical to mitochondrial gene expression, mitochondrial biological function, and plant growth and development. Ancestral group II introns are self-splicing ribozymes that can catalyze their own removal from pre-RNAs, while group II introns in land plant mitochondria went through degenerations in RNA structures, and thus they lost the ability to self-splice. Instead, splicing of these introns in the mitochondria of land plants is promoted by nuclear- and mitochondrial-encoded proteins. Many proteins involved in mitochondrial group II intron splicing have been characterized in land plants to date. Here, we present a summary of research progress on mitochondrial group II intron splicing in land plants, with a major focus on protein splicing factors and their probable functions on the splicing of mitochondrial group II introns.

The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.