Post-transcriptional Mechanisms Research Articles

Pooled CRISPR Screening Identifies P-Bodies as Repressors of Cancer Epithelial-Mesenchymal Transition.

Systematic investigation of the influence of posttranscriptional regulation on cancer cell motility established a connection between P-body-mediated translational control and EMT, which could be therapeutically exploited to attenuate metastasis formation.

RNA Interference-Screening of Potentially Lethal Gene Targets in the White-Backed Planthopper Sogatella furcifera via a Spray-Induced and Nanocarrier-Delivered Gene Silencing System.

RNA interference (RNAi) is a widespread post-transcriptional silencing mechanism that targets homologous mRNA sequences for specific degradation. An RNAi-based pest management strategy is target-specific and considered a sustainable biopesticide. However, the specific genes targeted and the efficiency of the delivery methods can vary widely across species. In this study, a spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system that incorporated gene-specific dsRNAs targeting conserved genes was used to evaluate phenotypic effects in white-backed planthopper (WBPH). At 2 days postspraying, transcript levels for all target genes were significantly reduced and knockdown of two gene orthologs, hsc70-3 and PP-α, resulted in an elevated mortality (>60%) and impaired ecdysis. These results highlight the utility of the SI-NDGS system for identifying genes involved in WBPH growth and development that could be potentially exploitable as high mortality target genes to develop an alternative method for WBPH control.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Trypanosoma cruzi RNA-binding protein DRBD3: perinuclear foci formation during benznidazole exposure.

Benznidazole (BZ) is the trypanocidal compound of choice for Chagas disease, a neglected tropical disease in the Americas. However, this drug often fails to cure the infection. The regulation of gene expression in Trypanosoma cruzi, the causative agent of Chagas disease, is based on post-transcriptional mechanisms. When environmental changes cause translational arrest, RNA-binding proteins, and their target mRNAs assemble into cytoplasmic bodies, known as RNA granules, which act as RNA sorting centers. We have characterized the T. cruzi RNA-binding protein DRBD3, which has two RRMs domains, and a C-terminal low-complexity sequence rich in proline and glutamines. Using a tagged form of TcDRBD3 (rTcDRBD3), we showed that this protein resides in the cytoplasm, but localizes into perinuclear cytoplasmic foci after BZ exposure. RNA staining after BZ also showed that this molecule accumulates into perinuclear cytoplasmic foci. Moreover, BZ and puromycin treatment enhanced the colocalization of rTcDRBD3 and RNA, suggesting that TcDRBD3 granules repertoire harbors RNAs released from polysomes. Under starvation, rTcDRBD3 granules localized throughout the cytoplasm and also increased in number in the presence of puromycin. Our results suggest that TcDRBD3 accumulates into perinuclear granules that harbor RNA and also that its localization varies according to the type of stress.

Haploinsufficiency of Cnot3 Aggravates Acid-Induced Acute Lung Injury Likely Through Transcriptional and Post-Transcriptional Upregulation of Pro-Inflammatory Genes.

Acute lung injury (ALI) is caused by a variety of illnesses, including aspiration pneumonia and sepsis. The CCR4-NOT complex is a large multimeric protein complex that degrades mRNA through poly(A) tail shortening, whereas it also contributes to regulation of transcription and translation. Cnot3 is a scaffold component of the CCR4-NOT complex and is essential for the integrity of the complex; loss of Cnot3 leads to depletion of whole complex. While the significance of cytokine mRNA degradation in limiting inflammation has been established, the roles of CCR4-NOT complex-mediated in ALI remain elusive. The effects of Cnot3 haploinsufficiency in the pathology and cytokine expression were analyzed in the mouse lungs of acid aspiration-induced acute lung injury. The decay rate and transcription activity of cytokine mRNAs under Cnot3 heterozygous deletion were analyzed in lipopolysaccharide (LPS) -stimulated mouse embryonic fibroblasts (MEFs). Tamoxifen-induced heterozygous deletion of Cnot3 in adult mice (Cnot3 Hetz) did not show body weight loss or any apparent abnormality. Under acid aspiration-induced acute lung injury, Cnot3 Hetz mice exhibited increased pulmonary edema, worse lung pathologies and more severe inflammation compared with wild type mice. mRNA expression of pro-inflammatory genes Il1b and Nos2 were significantly upregulated in the lungs of Cnot3 Hetz mice. Consistently, mRNA expression of Il1b and Nos2 was upregulated in LPS-stimulated Cnot3 Hetz MEFs. Mechanistically, while heterozygous depletion of Cnot3 stabilized both Il1b and Nos2 mRNAs, the nascent pre-mRNA level of Il1b was upregulated in Cnot3 Hetz MEFs, implicating Cnot3-mediated transcriptional repression of Il1b expression in addition to destabilization of Il1b and Nos2 mRNAs. PU.1 (Spi1) was identified as a causative transcription factor to promote Il1b expression under Cnot3 haploinsufficient conditions. CNOT3 plays a protective role in ALI by suppressing expression of pro-inflammatory genes Il1b and Nos2 through both post-transcriptional and transcriptional mechanisms, including mRNA stability control of Spi1.

Hidden regulators: the emerging roles of lncRNAs in brain development and disease.

Long non-coding RNAs (lncRNAs) have emerged as critical players in brain development and disease. These non-coding transcripts, which once considered as "transcriptional junk," are now known for their regulatory roles in gene expression. In brain development, lncRNAs participate in many processes, including neurogenesis, neuronal differentiation, and synaptogenesis. They employ their effect through a wide variety of transcriptional and post-transcriptional regulatory mechanisms through interactions with chromatin modifiers, transcription factors, and other regulatory molecules. Dysregulation of lncRNAs has been associated with certain brain diseases, including Alzheimer's disease, Parkinson's disease, cancer, and neurodevelopmental disorders. Altered expression and function of specific lncRNAs have been implicated with disrupted neuronal connectivity, impaired synaptic plasticity, and aberrant gene expression pattern, highlighting the functional importance of this subclass of brain-enriched RNAs. Moreover, lncRNAs have been identified as potential biomarkers and therapeutic targets for neurological diseases. Here, we give a comprehensive review of the existing knowledge of lncRNAs. Our aim is to provide a better understanding of the diversity of lncRNA structure and functions in brain development and disease. This holds promise for unravelling the complexity of neurodevelopmental and neurodegenerative disorders, paving the way for the development of novel biomarkers and therapeutic targets for improved diagnosis and treatment.

Dynamics of alternative polyadenylation in single root cells of Arabidopsis thaliana.

Single-cell RNA-seq (scRNA-seq) technologies have been widely used to reveal the diversity and complexity of cells, and pioneering studies on scRNA-seq in plants began to emerge since 2019. However, existing studies on plants utilized scRNA-seq focused only on the gene expression regulation. As an essential post-transcriptional mechanism for regulating gene expression, alternative polyadenylation (APA) generates diverse mRNA isoforms with distinct 3' ends through the selective use of different polyadenylation sites in a gene. APA plays important roles in regulating multiple developmental processes in plants, such as flowering time and stress response. In this study, we developed a pipeline to identify and integrate APA sites from different scRNA-seq data and analyze APA dynamics in single cells. First, high-confidence poly(A) sites in single root cells were identified and quantified. Second, three kinds of APA markers were identified for exploring APA dynamics in single cells, including differentially expressed poly(A) sites based on APA site expression, APA markers based on APA usages, and APA switching genes based on 3' UTR (untranslated region) length change. Moreover, cell type annotations of single root cells were refined by integrating both the APA information and the gene expression profile. We comprehensively compiled a single-cell APA atlas from five scRNA-seq studies, covering over 150,000 cells spanning four major tissue branches, twelve cell types, and three developmental stages. Moreover, we quantified the dynamic APA usages in single cells and identified APA markers across tissues and cell types. Further, we integrated complementary information of gene expression and APA profiles to annotate cell types and reveal subtle differences between cell types. This study reveals that APA provides an additional layer of information for determining cell identity and provides a landscape of APA dynamics during Arabidopsis root development.

Purification by STA-PUT Technique of Male Germ Cells from Single Mouse and RNA-Extraction for Transcriptomic Analysis.

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals.

Differential microRNA editing may drive target pathway switching in human temporal lobe epilepsy.

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin

ABSTRACT Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3’ UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

Life after transcription: control of the fate of neurons

The neocortex is a complex structure responsible for higher-order cognitive abilities in mammals. It consists of six cell layers, each comprised of various subtypes of excitatory and inhibitory neurons. These neurons project their axons to specific targets within each layer. All neocortical projection neurons are generated by neural progenitor cells located in the proliferative ventricular zone. In recent decades, significant strides have been made in comprehending the transcriptional programs governing neuronal cell fate specification. Nonetheless, posttranscriptional mechanisms involved in this process remain largely unexplored. Recently, we found that TrkC-T1, an isoform of the TrkC receptor lacking the kinase domain, determines the fate of corticofugal projection neurons (CFuPN). Our study reveals that the balance between TrkC-T1 and TrkC-TK+, a more widely recognized isoform containing the kinase domain, depends on the type of cell within the developing cortex. Additionally, we demonstrate that two RNA-binding proteins, Srsf1 and Elavl1, work in opposition to establish this balance. Additionally, our data suggests that Srsf1 stimulates the CFuPN fate while Elavl1 stimulates the callosal projection neuron (CPN) fate in vivo by regulating the different TrkC-T1 to TrkC-TK+ ratios. In this study, we identified a protein translation-dependent mechanism that governs the cell fate switch in the developing neocortex. Our results demonstrate that Ire1α, the Inositol-Requiring Enzyme 1α, regulates global translation rates in the developing neocortex through its dynamic interaction with the ribosome, as well as the regulation of expression of translation elongation factors eIF4A1 and eEF-2. Inactivation of Ire1α leads to decreased protein synthesis rates that are associated with stalled ribosomes and a reduced number of sites for translation initiation. We demonstrate the distinctive sensitivity of neurons determined for the upper layer to translation rates. While eEF-2 is necessary for cortical lamination, eIF4A1 regulates the attainment of upper layer fate in a mechanism of translational control that is dependent on structural elements embedded in the 5’UTR of genes responsible for determining fate downstream of Ire1α. Our findings reveal the developmental control of ribosome dynamics as post-transcriptional mechanisms that coordinate the establishment of neuronal diversity and the assembly of cortical layers.

Post-transcriptional mechanisms controlling neurogenesis and direct neuronal reprogramming.

Neurogenesis is a tightly regulated process in time and space both in the developing embryo and in adult neurogenic niches. A drastic change in the transcriptome and proteome of radial glial cells or neural stem cells towards the neuronal state is achieved due to sophisticated mechanisms of epigenetic, transcriptional, and post-transcriptional regulation. Understanding these neurogenic mechanisms is of major importance, not only for shedding light on very complex and crucial developmental processes, but also for the identification of putative reprogramming factors, that harbor hierarchically central regulatory roles in the course of neurogenesis and bare thus the capacity to drive direct reprogramming towards the neuronal fate. The major transcriptional programs that orchestrate the neurogenic process have been the focus of research for many years and key neurogenic transcription factors, as well as repressor complexes, have been identified and employed in direct reprogramming protocols to convert non-neuronal cells, into functional neurons. The post-transcriptional regulation of gene expression during nervous system development has emerged as another important and intricate regulatory layer, strongly contributing to the complexity of the mechanisms controlling neurogenesis and neuronal function. In particular, recent advances are highlighting the importance of specific RNA binding proteins that control major steps of mRNA life cycle during neurogenesis, such as alternative splicing, polyadenylation, stability, and translation. Apart from the RNA binding proteins, microRNAs, a class of small non-coding RNAs that block the translation of their target mRNAs, have also been shown to play crucial roles in all the stages of the neurogenic process, from neural stem/progenitor cell proliferation, neuronal differentiation and migration, to functional maturation. Here, we provide an overview of the most prominent post-transcriptional mechanisms mediated by RNA binding proteins and microRNAs during the neurogenic process, giving particular emphasis on the interplay of specific RNA binding proteins with neurogenic microRNAs. Taking under consideration that the molecular mechanisms of neurogenesis exert high similarity to the ones driving direct neuronal reprogramming, we also discuss the current advances in in vitro and in vivo direct neuronal reprogramming approaches that have employed microRNAs or RNA binding proteins as reprogramming factors, highlighting the so far known mechanisms of their reprogramming action.

Integrated multi-omics analysis for lung adenocarcinoma in Xuanwei, China.

Xuanwei lung cancer (XWLC) is well-known for its high incidence and mortality. However, the molecular mechanism is still unclear. We performed a comprehensive transcriptomic, proteomic, and phosphoproteomic characterization of tumors and matched normal adjacent tissues from three XWLC patients with lung adenocarcinoma (LUAD). Integrated transcriptome and proteome analysis revealed dysregulated molecules and pathways in tumors and identified enhanced metabolic-disease coupling. Non-coding RNAs were widely involved in post-transcriptional regulatory mechanisms to coordinate the progress of LUAD and partially explained the molecular differences between RNA and protein expression patterns. Phosphoproteome provided evidence support for new phosphate sites, reporting the potential roles of core kinase family members and key kinase pathways involved in metabolism, immunity, and homeostasis. In addition, by comparing with the previous LUAD researches, we emphasized the higher degree of oxidative phosphorylation in Xuanwei LUAD and pointed that VIPR1 deficiency aggravated metabolic dysfunction. Our integrated multi-omics analysis provided a powerful resource for a systematic understanding of the molecular structure of XWLC and proposed therapeutic opportunities based on redox metabolism.

Gene expression and alternative splicing contribute to adaptive divergence of ecotypes.

Regulation of gene expression is a critical link between genotype and phenotype explaining substantial heritable variation within species. However, we are only beginning to understand the ways that specific gene regulatory mechanisms contribute to adaptive divergence of populations. In plants, the post-transcriptional regulatory mechanism of alternative splicing (AS) plays an important role in both development and abiotic stress response, making it a compelling potential target of natural selection. AS allows organisms to generate multiple different transcripts/proteins from a single gene and thus may provide a source of evolutionary novelty. Here, we examine whether variation in alternative splicing and gene expression levels might contribute to adaptation and incipient speciation of dune-adapted prairie sunflowers in Great Sand Dunes National Park, Colorado, USA. We conducted a common garden experiment to assess transcriptomic variation among ecotypes and analyzed differential expression, differential splicing, and gene coexpression. We show that individual genes are strongly differentiated for both transcript level and alternative isoform proportions, even when grown in a common environment, and that gene coexpression networks are disrupted between ecotypes. Furthermore, we examined how genome-wide patterns of sequence divergence correspond to divergence in transcript levels and isoform proportions and find evidence for both cis and trans-regulation. Together, our results emphasize that alternative splicing has been an underappreciated mechanism providing source material for natural selection at short evolutionary time scales.

Exploring transcriptional and post-transcriptional epigenetic regulation of crf and 11βhsd2 in rainbow trout brain during chronic social stress

Using dominance hierarchies in juvenile rainbow trout (Oncorhynchus mykiss) as a model of chronic social stress in fish, we explored whether epigenetic transcriptional and post-transcriptional mechanisms are involved in the gene expression of corticotropin-releasing factor (crf) and 11β-hydroxysteroid dehydrogenase (11βhsd2), key factors involved in the regulation of the endocrine stress axis response. In juvenile rainbow trout pairs, subordinate individuals display sustained elevation of circulating cortisol concentrations. Cortisol production is controlled by the hypothalamic-pituitary-interrenal (HPI) axis in fish and initiated by CRF release from the preoptic area (POA). Given that crf is modulated during chronic social stress, and that such stress has been implicated in the epigenetic regulation of crf in other taxa, we probed a role for epigenetic regulation of crf transcript abundance in chronically stressed rainbow trout. We also investigated the regulation of the cortisol-metabolising enzyme 11βhsd2 in the POA, which is upregulated in subordinates. The potential involvement of DNA methylation and microRNAs (miRNAs) in the regulation of crf transcript abundance was investigated during social stress in the POA of fish, as was the potential involvement of miRNAs in 11βhsd2 regulation. Although transcript abundances of crf were elevated in subordinate fish after 4 days, DNA methylation profiles within putative promoter sequences upstream of the crf gene were not significantly affected by chronic stress. An inverse relationship between crf and its predicted posttranscriptional regulator miR-103a-3p in the POA suggests that miRNAs may be involved in mediating the effects of chronic social stress on key components of the endocrine stress axis.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Interconnections between DDX41-Dependent Post-Transcriptional and GATA Factor-Instigated Transcriptional Mechanisms That Govern Hematopoiesis

Heterozygous DDX41 germline mutations occur in familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and acute erythroid leukemia (AEL). DDX41 encodes an RNA helicase that regulates RNA splicing, cGAS-Sting signaling in innate immunity, and maintains genome stability in erythropoiesis. We described the innovation of a facile genetic rescue system to compare RNA-regulatory activities of wild type and disease-associated DDX41 variants in engineered HoxB8-immortalized (hi) murine myeloid progenitor cells (hi-Ddx41+/+ and Ddx41+/- cells) (Kim et al., Leukemia 2023). We identified DDX41-regulated transcripts and membrane proteins as quantitative metrics of DDX41 activity that discriminate benign from pathogenic DDX41 alleles. As these transcripts and the cognate proteins were not known to be components of GATA2 genetic networks that govern hematopoietic stem/progenitor cell (HSPC) transitions, we hypothesized that DDX41 mechanisms in progenitors operate in parallel with or independent of GATA2 mechanisms. Considering the biological and pathological importance of DDX41 and many unanswered questions on how clinical variants impact DDX41 activity, we instituted studies to discover DDX41-regulated transcript isoforms. The datasets are being used to unveil how DDX41 controls cellular functions, how clinical variants derail mechanisms, and to establish interconnections or independence with GATA factor mechanisms. To develop a broader perspective, we extended the myeloid data by innovating a rescue system to analyze DDX41 structure/function in G1E-ER-GATA1 erythroblasts that express a conditionally active ER-GATA1 allele. We used CRISPR/Cas9 to generate Ddx41 +/- clonal lines that expressed ~50% lower levels of DDX41 protein. A comparable system is being generated in human HUDEP2 erythroid precursor cells. We compared DDX41 with two disease-associated germline variants: G173R and K331deletion. G1E-ER-GATA1- Ddx41 +/-cells were infected with retrovirus expressing GFP, or GFP and DDX41 (WT, G173R, or K331del), GFP + cells were sorted, and total RNA was analyzed by RNA-seq. Differential expression analysis revealed 138 Ddx41-regulated transcripts (P < 0.05), with 63 elevated and 75 decreased. Disease-associated variants failed to regulate these transcripts. To establish relationships between DDX41-regulated and GATA1-regulated transcripts in erythroblasts, we compared DDX41-regulated transcripts with our GATA1 transcriptomic data (Tanimura et al., EMBO Rep. 2016; Dev. Cell 2018; Liao et al., Cell Rep. 2020), which revealed 78 transcripts co-regulated by DDX41 and GATA1. The RNA analysis is being extended to the proteome. To investigate similarities between DDX41-dependent post-transcriptional and GATA1-dependent transcriptional mechanisms, we analyzed the genes encoding the 78 co-regulated transcripts using gene ontology and pathway analyses. The gene signatures included downregulation of apoptosis (p=0.0006). We are testing the hypothesis that DDX41 and GATA1 operate additively or synergistically to control levels of transcripts encoding apoptotic regulators in developing erythroblasts, thus establishing a balance between pro-apoptotic and anti-apoptotic factors. We compared myeloid and erythroid DDX41-regulated transcript datasets to identify common and cell type-specific DDX41-regulated transcripts. This analysis unveiled 11 transcripts that are DDX41-regulated in both systems. One transcript encodes CLK3, a member of Cdc2-like kinase family that phosphorylates splicing factors. DDX41 increased intron 3 retention in Clk3 mRNA in both systems. By quantifying exon-intron junctions, we demonstrated that DDX41 promoted intron 3 retention in hi- Ddx41 +/- cells 1.7-fold (P = 0.0002) and in G1E-ER-GATA1- Ddx41 +/- cells 3.3-fold (P = 0.0002). We are testing models to explain the functional significance of DDX41-regulated Clk3 intron retention and discovered DDX41 regulates intron retention at other loci. We are assessing how DDX41 controls alternative splicing globally in the erythroid and myeloid systems and elucidating the biological and pathological consequences of corrupting these mechanisms. In aggregate, these studies established a system to discriminate pathogenic from benign DDX41 variants, and mechanistic insights that provide the basis for new myeloid and erythroid paradigms.

MiR-277 regulates the phase of circadian activity-rest rhythm in Drosophila melanogaster.

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12h light: 12h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila.

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.

The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells

Lipid metabolism is the key to ferroptosis susceptibility. However, little is known about the underlying mechanisms in osteosarcoma cells. Functional restriction of bromodomain-containing protein 4 (BRD4) reduced the susceptibility to erastin-induced ferroptosis of osteosarcoma cells both in vitro and in vivo. Mechanically, BRD4 controls the splicing efficiency of the RNA precursor (pre-mACSL3) of ACSL3 (ACSL3) by recruiting serinerich/threonine protein kinase 2 (SRPK2) to assemble the splicing catalytic platform. Moreover, the AMP-binding domain of ACSL3 significantly influences arachidonic acid synthesis and thus determines the susceptibility to erastin-induced ferroptosis. Overall, we found a BRD4-mediated pre-mACSL3 splicing influences erastin-induced ferroptosis by affecting arachidonic acid synthesis in osteosarcoma cells. Data in this study fills some of the gap in understanding the post-transcriptional regulatory mechanisms of ACSL3 and provides new insights into the mechanisms of lipid metabolism regulation and its effect on susceptibility to ferroptosis in osteosarcoma cells.