Atypical mandibulofacial dysostosis with microcephaly diagnosed through the identification of a novel pathogenic mutation in EFTUD2.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536) is a rare monogenic disease that is caused by a mutation in the elongation factor Tu GTP binding domain containing 2 gene (EFTUD2, OMIM* 603892). It is characterized by mandibulofacial dysplasia, microcephaly, malformed ears, cleft palate, growth and intellectual disability. MFDM can be easily misdiagnosed due to its phenotypic overlap with other craniofacial dysostosis syndromes. The clinical presentation of MFDM is highly variable among patients. METHODS: A patient with craniofacial anomalies was enrolled and evaluated by a multidisciplinary team. To make a definitive diagnosis, whole-exome sequencing was performed, followed by validation by Sanger sequencing. RESULTS: The patient presented with extensive facial bone dysostosis, upward slanting palpebral fissures, outer and middle ear malformation, a previously unreported orbit anomaly, and spina bifida occulta. A novel, pathogenic insertion mutation (c.215_216insT: p.Tyr73Valfs*4) in EFTUD2 was identified as the likely cause of the disease. CONCLUSIONS: We diagnosed this atypical case of MFDM by the detection of a novel pathogenetic mutation in EFTUD2. We also observed previously unreported features. These findings enrich both the genotypic and phenotypic spectrum of MFDM.

Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

Environmentally relevant concentrations of 2,3,7,8-TCDD induced inhibition of multicellular alternative splicing and transcriptional dysregulation.

Alternative splicing (AS), found in approximately 95 % of human genes, significantly amplifies protein diversity and is implicated in disease pathogenesis when dysregulated. However, the precise involvement of AS in the toxic mechanisms induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains incompletely elucidated. This study conducted a thorough global AS analysis in six human cell lines following TCDD exposure. Our findings revealed that environmentally relevant concentration (0.1 nM) of TCDD significantly suppressed AS events in all cell types, notably inhibiting diverse splicing events and reducing transcript diversity, potentially attributed to modifications in the splicing patterns of the inhibitory factor family, particularly hnRNP. And we identified 151 genes with substantial AS alterations shared among these cell types, particularly enriched in immune and metabolic pathways. Moreover, TCDD induced cell-specific changes in splicing patterns and transcript levels, with increased sensitivity notably in THP-1 monocyte, potentially linked to aberrant expression of pivotal genes within the spliceosome pathway (DDX5, EFTUD2, PUF60, RBM25, SRSF1, and CRNKL1). This study extends our understanding of disrupted alternative splicing and its relation to the multisystem toxicity of TCDD. It sheds light on how environmental toxins affect post-transcriptional regulatory processes, offering a fresh perspective for toxicology and disease etiology investigations.

The feedback loop of EFTUD2/c-MYC impedes chemotherapeutic efficacy by enhancing EFTUD2 transcription and stabilizing c-MYC protein in colorectal cancer.

BACKGROUND: Chemoresistance presents a significant obstacle in the treatment of colorectal cancer (CRC), yet the molecular basis underlying CRC chemoresistance remains poorly understood, impeding the development of new therapeutic interventions. Elongation factor Tu GTP binding domain containing 2 (EFTUD2) has emerged as a potential oncogenic factor implicated in various cancer types, where it fosters tumor growth and survival. However, its specific role in modulating the sensitivity of CRC cells to chemotherapy is still unclear. METHODS: Public dataset analysis and in-house sample validation were conducted to assess the expression of EFTUD2 in 5-fluorouracil (5-FU) chemotherapy-resistant CRC cells and the potential of EFTUD2 as a prognostic indicator for CRC. Experiments both in vitro, including MTT assay, EdU cell proliferation assay, TUNEL assay, and clone formation assay and in vivo, using cell-derived xenograft models, were performed to elucidate the function of EFTUD2 in sensitivity of CRC cells to 5-FU treatment. The molecular mechanism on the reciprocal regulation between EFTUD2 and the oncogenic transcription factor c-MYC was investigated through molecular docking, ubiquitination assay, chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP). RESULTS: We found that EFTUD2 expression was positively correlated with 5-FU resistance, higher pathological grade, and poor prognosis in CRC patients. We also demonstrated both in vitro and in vivo that knockdown of EFTUD2 sensitized CRC cells to 5-FU treatment, whereas overexpression of EFTUD2 impaired such sensitivity. Mechanistically, we uncovered that EFTUD2 physically interacted with and stabilized c-MYC protein by preventing its ubiquitin-mediated proteasomal degradation. Intriguingly, we found that c-MYC directly bound to the promoter region of EFTUD2 gene, activating its transcription. Leveraging rescue experiments, we further confirmed that the effect of EFTUD2 on 5-FU resistance was dependent on c-MYC stabilization. CONCLUSION: Our findings revealed a positive feedback loop involving an EFTUD2/c-MYC axis that hampers the efficacy of 5-FU chemotherapy in CRC cells by increasing EFTUD2 transcription and stabilizing c-MYC oncoprotein. This study highlights the potential of EFTUD2 as a promising therapeutic target to surmount chemotherapy resistance in CRC patients.

Prenatal Diagnosis of Fetal Micrognathia at 11-20 Weeks of Gestation: A Prospective Observation Study.

OBJECTIVE: To prospectively evaluate the prognosis of fetuses diagnosed with micrognathia using prenatal ultrasound screening. METHODS: Between January 2019 and December 2022, a normal range of IFA to evaluate the facial profile in fetuses with micrognathia in a Chinese population between 11 and 20 gestational weeks was established, and the pregnancy outcomes of fetal micrognathia were described. The medical records of these pregnancies were collected, including family history, maternal demographics, sonographic findings, genetic testing results, and pregnancy outcomes. RESULTS: Ultrasound identified 25 patients with fetal micrognathia, with a mean IFA value of 43.6°. All cases of isolated fetal micrognathia in the initial scans were non-isolated in the following scans. A total of 78.9% (15/19) cases had a genetic cause confirmed, including 12 with chromosomal abnormalities and 3 with monogenic disorders. Monogenic disorders were all known causes of micrognathia, including two cases of campomelic dysplasia affected by SOX9 mutations and one case of mandibulofacial dysostosis with an EFTUD2 mutation. In the end, 19 cases were terminated, 1 live birth was diagnosed as Pierre Robin syndrome, and 5 cases were lost to follow-up. CONCLUSION: IFA is a useful indicator and three-dimensional ultrasound is a significant support technique for fetal micrognathia prenatal diagnosis. Repeat ultrasound monitoring and genetic testing are crucial, with CMA recommended and Whole exome sequencing performed when normal arrays are reported. Isolated fetal micrognathia may be an early manifestation of monogenic disorders.

First and second branchial arch involvement in mandibulofacial dysostosis Guion-Almeida type.

BACKGROUND: Mandibulofacial dysostosis Guion-Almeida Type (MFDGA; OMIM#610536) is a rare autosomal dominant genetic disorder caused by heterozygous pathogenic variants in the EFTUD2 gene. Mandibulofacial dysostoses are characterised by the core triad malar hypoplasia, maxillary hypoplasia and dysplastic ears, all derived by the impaired development of the first and second branchial arches. Differential diagnosis is often challenging. The early genetic diagnosis is extremely useful, not only for the correct management of cranial malformations, but also for the early diagnosis and treatment of the comorbidities associated to the disease, which greatly benefit from early treatment.

Deciphering the RNA-binding protein interaction with the mRNAs encoded from human chromosome 15q11.2 BP1-BP2 microdeletion region.

Microdeletion of the 15q11.2 BP1-BP2 region, also known as Burnside-Butler susceptibility region, is associated with phenotypes like delayed developmental language abilities along with motor skill disabilities, combined with behavioral and emotional problems. The 15q11.2 microdeletion region harbors four evolutionarily conserved and non-imprinted protein-coding genes: NIPA1, NIPA2, CYFIP1, and TUBGCP5. This microdeletion is a rare copy number variation frequently associated with several pathogenic conditions in humans. The aim of this study is to investigate the RNA-binding proteins binding with the four genes present in 15q11.2 BP1-BP2 microdeletion region. The results of this study will help to better understand the molecular intricacies of the Burnside-Butler Syndrome and also the possible involvement of these interactions in the disease aetiology. Our results of enhanced crosslinking and immunoprecipitation data analysis indicate that most of the RBPs interacting with the 15q11.2 region are involved in the post-transcriptional regulation of the concerned genes. The RBPs binding to this region are found from the in silico analysis, and the interaction of RBPs like FASTKD2 and EFTUD2 with exon-intron junction sequence of CYFIP1 and TUBGCP5 has also been validated by combined EMSA and western blotting experiment. The exon-intron junction binding nature of these proteins suggests their potential involvement in splicing process. This study may help to understand the intricate relationship of RBPs with mRNAs within this region, along with their functional significance in normal development, and lack thereof, in neurodevelopmental disorders. This understanding will help in the formulation of better therapeutic approaches.

Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2.

Background: An increase in the demand for a functional cure has accelerated research on new methods of therapy for chronic hepatitis B, which is mainly focused on restoring antiviral immunity for controlling viral infections. Previously, we had described elongation factor Tu GTP-binding domain containing 2 (EFTUD2) as an innate immune regulator and suggested that it might be an antiviral target. Methods: In this study, we generated the Epro-LUC-HepG2 cell model for screening compounds that target EFTUD2. Plerixafor and resatorvid were screened from 261 immunity and inflammation-related compounds due to their ability to highly upregulate EFTUD2. The effects of plerixafor and resatorvid on hepatitis B virus (HBV) were examined in HepAD38 cells and HBV-infected HepG2-NTCP cells. Results: The dual-luciferase reporter assays showed that the EFTUD2 promoter hEFTUD2pro-0.5 kb had the strongest activity. In Epro-LUC-HepG2 cells, plerixafor and resatorvid significantly upregulated the activity of the EFTUD2 promoter and the expression of the gene and protein. In HepAD38 cells and HBV-infected HepG2-NTCP cells, treatment with plerixafor and resatorvid strongly inhibited HBsAg, HBV DNA, HBV RNAs, and cccDNA in a dose-dependent manner. Furthermore, the anti-HBV effect was enhanced when entecavir was administered along with either of the previous two compounds, and the effect could be blocked by knocking down EFTUD2. Conclusion: We established a convenient model for screening compounds that target EFTUD2 and further identified plerixafor and resatorvid as novel HBV inhibitors in vitro. Our findings provided information on the development of a new class of anti-HBV agents that act on host factors rather than viral enzymes.

Weighted gene co-expression network analysis reveals the hub genes associated with pulmonary hypertension.

Pulmonary hypertension (PH) is a cardiopulmonary vascular disease that acutely endangers human health and can be fatal. It progresses rapidly and has a high mortality rate. Its pathophysiology is complicated and still not completely elucidated; therefore, achieving treatment breakthroughs are difficult. In this study, data from 58 normal controls and 135 patients with PH were extracted from the GSE24988, GSE113439, and GSE117261 datasets in the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (DEGs). In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Weighted gene co-expression network analysis (WGCNA) was used to identify the key modules and hub genes associated with PH. Eight PH-associated hub genes were identified. Furthermore, correlation analysis between immune cell infiltration and hub genes was performed, and the receiver operating characteristic (ROC) curves showed that TARDBP had the best diagnostic efficacy. Moreover, a rat hypoxic pulmonary hypertension (HPH) model was generated, and the expression of hub genes in the lungs and pulmonary arteries of HPH rats was verified using western blotting assays. Our results showed that mTOR, PSMD2, RBM8A, SMARCA4, TARDBP, and UBXN7 were highly expressed in the lungs. In addition, EFTUD2, mTOR, RBM8A, SMARCA4, TARDBP, and UBXN7 were significantly upregulated, whereas DDB1 was significantly downregulated in the pulmonary arteries of HPH rats compared with those of controls. In conclusion, we identified PH hub genes with diagnostic and predictive value by performing WGCNA on data from the GEO database. Furthermore, we provided novel insights of PH that might be utilized to evaluate potential biomarker genes and therapeutic targets.

SART3 associates with a post-splicing complex.

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.

Retrospective evaluation of clinical and molecular data of 148 cases of esophageal atresia.

Developmental abnormalities provide a unique opportunity to seek for the molecular mechanisms underlying human organogenesis. Esophageal development remains incompletely understood and elucidating causes for esophageal atresia (EA) in humans would contribute to achieve a better comprehension. Prenatal detection, syndromic classification, molecular diagnosis, and prognostic factors in EA are challenging. Some syndromes have been described to frequently include EA, such as CHARGE, EFTUD2-mandibulofacial dysostosis, Feingold syndrome, trisomy 18, and Fanconi anemia. However, no molecular diagnosis is made in most cases, including frequent associations, such as Vertebral-Anal-Cardiac-Tracheo-Esophageal-Renal-Limb defects (VACTERL). This study evaluates the clinical and genetic test results of 139 neonates and 9 fetuses followed-up at the Necker-Enfants Malades Hospital over a 10-years period. Overall, 52 cases were isolated EA (35%), and 96 were associated with other anomalies (65%). The latter group is divided into three subgroups: EA with a known genomic cause (9/148, 6%); EA with Vertebral-Anal-Cardiac-Tracheo-Esophageal-Renal-Limb defects (VACTERL) or VACTERL/Oculo-Auriculo-Vertebral Dysplasia (VACTERL/OAV) (22/148, 14%); EA with associated malformations including congenital heart defects, duodenal atresia, and diaphragmatic hernia without known associations or syndromes yet described (65/148, 44%). Altogether, the molecular diagnostic rate remains very low and may underlie frequent non-Mendelian genetic models.

MDF is a conserved splicing factor and modulates cell division and stress response in Arabidopsis.

The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants.

High RIG-I and EFTUD2 expression predicts poor survival in endometrial cancer.

PURPOSE: Endometrial cancer is the most common gynecological malignancy. The helicase RIG-I, a part of the innate immune system, and EFTUD2, a splicing factor which can upregulate RIG-I expression, are shown to influence tumor growth and disease progression in several malignancies. For endometrial cancer, an immunogenic cancer, data about RIG-I and EFTUD2 are still missing. The aim of this study was to examine the expression of RIG-I and EFTUD2 in endometrial cancer. METHODS: 225 specimen of endometrial cancer were immunohistochemically stained for RIG-I and EFTUD2. The results were correlated to clinicopathological data, overall survival (OS) and progression-free survival (PFS). RESULTS: High RIG-I expression correlated with advanced tumor stages (FIGO: p = 0.027; pT: p = 0.010) and worse survival rates (OS: p = 0.009; PFS: p = 0.022). High EFTUD2 expression correlated to worse survival rates (OS: p = 0.026; PFS: p < 0.001) and was determined to be an independent marker for progression-free survival. CONCLUSION: Our data suggest that the expression of RIG-I and EFTUD2 correlates with survival data, which makes both a possible therapeutic target in the future.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

A functional loop between YTH domain family protein YTHDF3 mediated m6A modification and phosphofructokinase PFKL in glycolysis of hepatocellular carcinoma.

BACKGROUND & AIMS: N6-methyladenosine (m6A) modification plays a critical role in progression of hepatocellular carcinoma (HCC), and aerobic glycolysis is a hallmark of cancer including HCC. However, the role of YTHDF3, one member of the core readers of the m6A pathway, in aerobic glycolysis and progression of HCC is still unclear. METHODS: Expression levels of YTHDF3 in carcinoma and surrounding tissues of HCC patients were evaluated by immunohistochemistry. Loss and gain-of-function experiments in vitro and in vivo were used to assess the effects of YTHDF3 on HCC cell proliferation, migration and invasion. The role of YTHDF3 in hepatocarcinogenesis was observed in a chemically induced HCC model with Ythdf3-/- mice. Untargeted metabolomics and glucose metabolism phenotype assays were performed to evaluate relationship between YTHDF3 and glucose metabolism. The effect of YTHDF3 on PFKL was assessed by methylated RNA immunoprecipitation assays (MeRIP). Co-immunoprecipitation and immunofluorescence assays were performed to investigate the connection between YTHDF3 and PFKL. RESULTS: We found YTHDF3 expression was greatly upregulated in carcinoma tissues and it was correlated with poor prognosis of HCC patients. Gain-of-function and loss-of-function assays demonstrated YTHDF3 promoted proliferation, migration and invasion of HCC cells in vitro, and YTHDF3 knockdown inhibited xenograft tumor growth and lung metastasis of HCC cells in vivo. YTHDF3 knockout significantly suppressed hepatocarcinogenesis in chemically induced mice model. Mechanistically, YTHDF3 promoted aerobic glycolysis by promoting phosphofructokinase PFKL expression at both mRNA and protein levels. MeRIP assays showed YTHDF3 suppressed PFKL mRNA degradation via m6A modification. Surprisingly, PFKL positively regulated YTHDF3 protein expression, not as a glycolysis rate-limited enzyme, and PFKL knockdown effectively rescued the effects of YTHDF3 overexpression on proliferation, migration and invasion ability of Sk-Hep-1 and HepG2 cells. Notably, co-immunoprecipitation assays demonstrated PFKL interacted with YTHDF3 via EFTUD2, a core subunit of spliceosome involved in pre-mRNA splicing process, and ubiquitination assays showed PFKL could positively regulate YTHDF3 protein expression via inhibiting ubiquitination of YTHDF3 protein by EFTUD2. CONCLUSIONS: our study uncovers the key role of YTHDF3 in HCC, characterizes a positive functional loop between YTHDF3 and phosphofructokinase PFKL in glucose metabolism of HCC, and suggests the connection between pre-mRNA splicing process and m6A modification.

A forward genetic screen in C. elegans identifies conserved residues of spliceosomal proteins PRP8 and SNRNP200/BRR2 with a role in maintaining 5' splice site identity.

The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5' splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5'ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5'ss identified by U1snRNA for entry into the catalytic core.

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8.

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 (PRP8F RH) is reported, revealing a significantly different interaction between the two proteins compared with that in yeast. Based on the structure of the AAR2-PRPF8 RH complex, the importance of the interacting regions and residues was probed and AAR2 variants were designed that failed to stably bind PRPF8 in vitro. Protein-interaction studies of AAR2 with U5 proteins using size-exclusion chromatography reveal similarities and marked differences in the interaction patterns compared with yeast Aar2p and imply phosphorylation-dependent regulation of AAR2 reminiscent of that in yeast. It is found that in vitro AAR2 seems to lock PRPF8 RH in a conformation that is only compatible with the first transesterification step of the splicing reaction and blocks a conformational switch to the step 2-like, Mg2+-coordinated conformation that is likely during U5 snRNP biogenesis. These findings extend the picture of AAR2 PRP8 interaction from yeast to humans and indicate a function for AAR2 in the spliceosomal assembly process beyond its role as an SNRNP200 placeholder in yeast.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion.

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc-/-) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc-/- embryos with pifithrin-α, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-α-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc-/-; Trp53ncc-/-) mutant embryos. Eftud2ncc-/- embryos that were treated with pifithrin-α or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc-/-; Trp53ncc-/- embryos compared to Eftud2ncc-/- mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc-/-; Trp53ncc-/- embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc-/- embryos.