Changes In Alternative Splicing Research Articles

Mutations in hnRNP A1 drive neurodegeneration and alternative RNA splicing of neuronal gene targets.

RNA binding protein dysfunction is a pathogenic feature of multiple neurological diseases, including multiple sclerosis (MS). Neurodegeneration (the loss of, or damage to neurons and axons) is the primary driver of disease progression in MS. Herein, we utilized a novel, neuron-specific model of neurodegeneration by transducing primary mouse neurons with mutant forms of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) identified from MS patients, including one within the M9-nuclear localization sequence of hnRNP A1 (A1(P275S)) and a second in the prion-like domain of hnRNP A1 (A1(F263S)) to test the hypothesis that neuronal hnRNP A1 dysfunction drives neurodegeneration in MS. Examination of hnRNP A1 localization in neurons revealed an increase in nucleocytoplasmic mislocalization in neurons transduced with A1(P275S), but not A1(F263S). Yet, both A1(F263S) and A1(P275S) induced neurodegeneration evidenced by significant reductions in total neurite length and complexity and an increase in FluoroJade-C neuronal cell body staining. RNA sequencing and differential alternative splicing analysis of mutant-expressing neurons revealed dramatic changes in alternative RNA splicing of transcripts critical to neuronal function. Further, amyloid precursor protein (APP), a marker for neurodegeneration in MS, showed differential splicing in mutant-expressing neurons, which was confirmed in MS brains with hnRNP A1 dysfunction. Overall, we have identified that hnRNP A1 plays a complex role in neuronal function and regulation by mediating the alternative splicing of neuron-specific transcripts. When neuronal hnRNP A1 function is impaired, as in disease, resultant dysfunction propagates through multiple pathways that may influence the progression of neurodegeneration in MS.

Persistent large-scale changes in alternative splicing in prefrontal cortical neuron types following psychedelic exposure.

Psychedelics engage the serotonergic system as potent neuromodulators, increasing neuroplasticity in humans and rodents. Persistent changes in cognitive flexibility, emotional regulation, and social cognition are thought to underlie the therapeutic effects of psychedelics. However, the underlying molecular and cellular basis of psychedelic-induced plasticity remains unclear. Here, we identify persistent, cell type-specific alternative splicing changes in the mouse medial prefrontal cortex (mPFC) induced by a single dose of psychedelics. Combining deep RiboTag sequencing and bioinformatics, we find that a single dose of psychedelics modestly alters gene expression while dramatically shifting patterns of alternative splicing lasting at least a month. We connect our functional enrichment and alternative splicing analysis with changes in the extracellular matrix, synaptic physiology, and intrinsic physiology in parvalbumin interneurons days to a week after psychedelic treatment. Our dataset is an essential resource for understanding the persistent, cell type-specific effects of psychedelics on cortical cell types and functions.

Alternative Splicing at the Crossroad of Inflammatory Bowel Diseases and Colitis-Associated Colon Cancer.

The risk of developing colorectal cancer (CRC) is increased in ulcerative colitis patients compared to the general population. This increased risk results from the state of chronic inflammation, a well-known tumour-promoting condition. This review explores the pathologic and molecular characteristics of colitis-associated colon cancer (CAC), emphasizing the distinct features from sporadic CRC. We focus on the key signalling pathways involved in the transition to CAC, highlighting the emerging role of alternative splicing in these processes, namely on how inflammation-induced alternative splicing can significantly contribute to the increased CRC risk observed among UC patients. This review calls for more transcriptomic studies to elucidate the molecular mechanisms through which inflammation-induced alternative splicing drives CAC pathogenesis. A better understanding of these splicing events is crucial as they may reveal novel biomarkers for disease progression and have the potential to target changes in alternative splicing as a therapeutic strategy.

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during mouse skeletal development. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting and widespread alternative RNA splicing (AS) changes. Here, we demonstrated via enhanced UV-crosslinking and immunoprecipitation of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. We found that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during mouse skeletal development. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting and widespread alternative RNA splicing (AS) changes. Here, we demonstrated via enhanced UV-crosslinking and immunoprecipitation of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. We found that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

Phylogenetic analysis and pathogenesis study of a new deletion mutation causing inherited FⅩ deficiency

Objective: To analyze the F10 gene mutations in a Chinese pedigree affected with the deficiency of the hereditary coagulation factor X (FX), resulting from a new deletion mutation, and to study the associated molecular pathogenesis. Methods: Next generation sequencing (NGS) was performed to screen the genetic mutations in the proband which were then verified by Sanger sequencing. The FX activity (FX∶C) of probands and their family members was detected using the blood clotting method, and the mutation sites of the family members were analyzed using Sanger sequencing. The pathogenicity of the mutation site was predicted by using the online bioinformatics software, Mutation Taster. The SWISS-MODEL software was used for stimulating the three-dimensional models of the wild-type and mutant proteins for analyzing the influence of the mutation site on the structure and function of the proteins, and for analyzing the difference between the catalytic residues of the wild-type and the mutant proteins. The level of the F10 gene mRNA was quantitatively analyzed by qRT-PCR (quantitative reverse transcription polymerase chain reaction) method by constructing plasmids, transfecting human embryonic kidney 293T cells (HEK 293T), and analyzing the splicing of the mutated site by RT-PCR method. The levels of FⅩ∶Ag in cell lysates and cell culture media (both inside and outside the cells) were detected by the ELISA (enzyme linked immunosorbent assay) method. Results: A medium-grade factor X deficiency with a 36.42% FⅩ∶C ratio was detected in the proband by the coagulation method. NGS analysis demonstrated a heterozygous deletion mutation in exon 8:c.902_919del (p.Ala301_Glu306del) in the proband. Sanger sequencing analysis indicated that some members of the family (mother and grandfather) were also carriers of the corresponding deletion mutation. Online bioinformatics software predicted the pathogenic nature of the c.902_919del mutation, with a pathogenic score of 0.999. The 3D protein structure model analysis indicated that the c.902_919del mutation resulted in the disappearance of a segment of β-fold in the protein structure, thereby shortening the preceding segment of the β-fold and a subsequent loss of hydrogen bonds between adjacent amino acids with no significant difference in the side chain conformation of the key catalytic residues compared to the wild-type. mRNA splicing analysis indicated the absence of alternative splicing changes in the mutation, and qRT-PCR results indicated the absence of a statistically significant difference between the mRNA levels of F10 gene and wild-type mRNA in cells expressing c.902_919del mutant. The ELISA results indicated that there was no statistically significant difference in the FX∶Ag levels of the mutant cell culture medium and the lysate. Conclusions: In this pedigree, the heterozygous mutation in exon 8 of F10 gene (c.902_919del, p.Ala301_Glu306del) caused the hereditary factor Ⅹ deficiency.

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking.

Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRα signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRα signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.

Disruption of nuclear speckle integrity dysregulates RNA splicing in C9ORF72-FTD/ALS

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.

RNA splicing as a biomarker and phenotypic driver of meningioma DNA-methylation groups.

Advances in our understanding of the molecular biology of meningiomas have led to significant gains in the ability to predict patient prognosis and tumor recurrence and to identify novel targets for therapeutic design. Specifically, classification of meningiomas based on DNA methylation has greatly improved our ability to risk stratify patients, however new questions have arisen in terms of the underlying impact these DNA-methylation signatures have on meningioma biology. This study utilizes RNA-sequencing data from 486 meningioma samples corresponding to 3 meningioma DNA-methylation groups (merlin-intact, immune-enriched, and hypermitotic), followed by in vitro experiments utilizing human meningioma cell lines. We identify alterations in RNA splicing between meningioma DNA-methylation groups including individual splicing events that correlate with hypermitotic meningiomas and predict tumor recurrence and overall patient prognosis and compile a set of splicing events that can accurately predict DNA-methylation classification based on RNA-seq data. Furthermore, we validate these events using reverse transcription polymerase chain reaction (RT-PCR) in patient samples and meningioma cell lines. Additionally, we identify alterations in RNA-binding proteins and splicing factors that lie upstream of RNA splicing events, including upregulation of SRSF1 in hypermitotic meningiomas which we show drives alternative RNA splicing changes. Finally, we design splice-switching antisense oligonucleotides to target RNA splicing changes in NASP and MFF observed in hypermitotic meningiomas, providing a rationale for RNA-based therapeutic design. RNA splicing is an important driver of meningioma phenotypes that can be useful in prognosticating patients and as a potential exploit for therapeutic vulnerabilities.

Reference-informed prediction of alternative splicing and splicing-altering mutations from sequences.

Alternative splicing plays a crucial role in protein diversity and gene expression regulation in higher eukaryotes, and mutations causing dysregulated splicing underlie a range of genetic diseases. Computational prediction of alternative splicing from genomic sequences not only provides insight into gene-regulatory mechanisms but also helps identify disease-causing mutations and drug targets. However, the current methods for the quantitative prediction of splice site usage still have limited accuracy. Here, we present DeltaSplice, a deep neural network model optimized to learn the impact of mutations on quantitative changes in alternative splicing from the comparative analysis of homologous genes. The model architecture enables DeltaSplice to perform "reference-informed prediction" by incorporating the known splice site usage of a reference gene sequence to improve its prediction on splicing-altering mutations. We benchmarked DeltaSplice and several other state-of-the-art methods on various prediction tasks, including evolutionary sequence divergence on lineage-specific splicing and splicing-altering mutations in human populations and neurodevelopmental disorders, and demonstrated that DeltaSplice outperformed consistently. DeltaSplice predicted ∼15% of splicing quantitative trait loci (sQTLs) in the human brain as causal splicing-altering variants. It also predicted splicing-altering de novo mutations outside the splice sites in a subset of patients affected by autism and other neurodevelopmental disorders (NDDs), including 19 genes with recurrent splicing-altering mutations. Integration of splicing-altering mutations with other types of de novo mutation burdens allowed the prediction of eight novel NDD-risk genes. Our work expanded the capacity of in silico splicing models with potential applications in genetic diagnosis and the development of splicing-based precision medicine.

CCAR1 promotes DNA repair via alternative splicing

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

Lung Transcriptomics Links Emphysema to Barrier Dysfunction and Macrophage Subpopulations.

While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

SRSF2 promotes glioblastoma cell proliferation by inducing alternative splicing of FSP1 and inhibiting ferroptosis

Objective: To investigate the effect of serine/arginine-rich splicing factor 2 (SRSF2) on ferroptosis and its possible mechanism in glioblastoma cells. Methods: The online database of gene expression profiling interactive analysis 2 (GEPIA 2) and Chinese Glioma Genome Atlas were used to analyze the expression of SRSF2 in glioblastoma tissue and its association with patients prognosis. To validate the findings of the online databases, the pathological sections of glioblastoma and non-tumor brain tissues from Tianjin Medical University General Hospital, Tianjin, China were collected and analyzed by using immunohistochemistry. Silencing SRSF2 gene expression in glioblastoma cells by siRNA was analyzed with Western blot. The proliferation index was detected by using CCK8 assay. The rescued experiment was conducted by using expression plasmid of pcDNA3.1(+)-SRSF2. The activity of ferroptosis was assessed by using the levels of iron ions and malondialdehyde in glioblastoma cells and the changes in the ratio of glutathione to oxidized glutathione. The changes of gene expression and differential pre-mRNA alternative splicing (PMAS) induced by SRSF2 were monitored by using the third-generation sequencing technology analysis, namely Oxford nanopore technologies (ONT) sequencing analysis. Results: SRSF2 expression was higher in glioblastoma tissues than non-tumor brain tissues. Immunohistochemistry also showed a positive rate of 88.48%±4.60% in glioblastoma tissue which was much higher than the 9.97%±4.57% in non-tumor brain tissue. The expression of SRSF2 was inversely correlated with overall and disease-free disease survivals (P<0.01). The proliferation index of glioblastoma cells was significantly reduced by silencing with SRSF2 siRNA (P<0.01) and could be reversed with transfection of exogenous SRSF2. The levels of intracellulariron ions and malondialdehyde increased (P<0.05), but the glutathione/oxidized glutathione ratio and the expression of key proteins in the glutathione pathway remained unchanged (P>0.05). ONT sequencing results showed that silencing SRSF2 in glioblastoma cells could induce a significant alternative 3' splice site change on ferroptosis suppressor protein 1 (FSP1). Conclusion: SRSF2 inhibits the ferroptosis in glioblastoma cells and promotes their proliferation, which may be achieved by regulating FSP1 PMAS.

Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

The Role of mRNA Alternative Splicing in Macrophages Infected with Mycobacterium tuberculosis: A Field Needing to Be Discovered.

Mycobacterium tuberculosis (Mtb) is one of the major causes of human death. In its battle with humans, Mtb has fully adapted to its host and developed ways to evade the immune system. At the same time, the human immune system has developed ways to respond to Mtb. The immune system responds to viral and bacterial infections through a variety of mechanisms, one of which is alternative splicing. In this study, we summarized the overall changes in alternative splicing of the transcriptome after macrophages were infected with Mtb. We found that after infection with Mtb, cells undergo changes, including (1) directly reducing the expression of splicing factors, which affects the regulation of gene expression, (2) altering the original function of proteins through splicing, which can involve gene truncation or changes in protein domains, and (3) expressing unique isoforms that may contribute to the identification and development of tuberculosis biomarkers. Moreover, alternative splicing regulation of immune-related genes, such as IL-4, IL-7, IL-7R, and IL-12R, may be an important factor affecting the activation or dormancy state of Mtb. These will help to fully understand the immune response to Mtb infection, which is crucial for the development of tuberculosis biomarkers and new drug targets.

Non-concordant epigenetic and transcriptional responses to acute thermal stress in western mosquitofish (Gambusia affinis).

Climate change is intensifying the frequency and severity of extreme temperatures. Understanding the molecular mechanisms underlying the ability to cope with acute thermal stress is key for predicting species' responses to extreme temperature events. While many studies have focused on the individual roles of gene expression, post-transcriptional processes and epigenetic modifications in response to acute thermal stress, the relative contribution of these molecular mechanisms remains unclear. The wide range of thermal limits of western mosquitofish (Gambusia affinis) provides an opportunity to explore this interplay. Here, we quantified changes in gene expression, alternative splicing, DNA methylation and microRNA (miRNA) expression in muscle tissue dissected from mosquitofish immediately after reaching high (CTmax) or low thermal limit (CTmin). Although the numbers of genes showing expression and splicing changes in response to acute temperature stress were small, we found a possibly larger and non-redundant role of splicing compared to gene expression, with more genes being differentially spliced (DSGs) than differentially expressed (DEGs), and little overlap between DSGs and DEGs. We also identified a small proportion of CpGs showing significant methylation change (i.e. differentially methylated cytosines, DMCs) in fish at thermal limits; however, there was no overlap between DEGs and genes annotated with DMCs in both CTmax and CTmin experiments. The weak interplay between epigenetic modifications and gene expression was further supported by our discoveries of no differentially expressed miRNAs. These findings provide novel insights into the relative role of different molecular mechanisms underlying immediate responses to extreme temperatures and demonstrate non-concordant responses of epigenetic and transcriptional mechanisms to acute temperature stress.

Abstract 5851: Dysregulated RNA splicing promotes AML quiescence and chemoresistance

Abstract Chemotherapy is the first-line treatment for patients with acute myeloid leukemia (AML). Although most achieve initial remission, an estimated 50% of patients will relapse with poor clinical outcome. The paucity of genetic lesions in AML patients that relapse from chemotherapy implicate non-genetic alterations that mediate chemoresistance. In this study, we conducted a longitudinal analysis of alternative splicing changes in a cohort of 19 AML patients at diagnosis and relapse post-chemotherapy treatment. We found that the long isoform of the transcription factor-encoding gene RUNX1 (also known as RUNX1C) is upregulated in AML relapse cohorts and conferred chemotherapy resistance. Mechanistically, we show that B cell translocation gene 2 (BTG2) is an RUNX1C isoform-specific transcriptional target that promote transition into a quiescent and treatment-resistant state. Direct targeting of RUNX1C mRNA augments chemotherapy response in AML patient-derived xenograft (PDX) models in vivo. These findings uncover an isoform-specific functionality that influences chemotherapy responsiveness and offers a therapeutic strategy to sensitize leukemic cells to conventional chemotherapy treatments. Citation Format: Eric Wang, Cuijuan Han, Alexander Calderon, Abimbo Lawal, Nidhi Hariramani, Edie Crosse, Robert Bradley. Dysregulated RNA splicing promotes AML quiescence and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5851.

Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.