Cross-linking Immunoprecipitation Research Articles

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.

LINC01432 binds to CELF2 in newly diagnosed multiple myeloma promoting short progression-free survival to standard therapy.

Multiple Myeloma (MM) is a highly prevalent and incurable form of cancer that arises from malignant plasma cells, with over 35,000 new cases diagnosed annually in the United States. While there are a growing number of approved therapies, MM remains incurable and nearly all patients will relapse and exhaust all available treatment options. Mechanisms for disease progression are unclear and in particular, little is known regarding the role of long non-coding RNAs (lncRNA) in mediating disease progression and response to treatment. In this study, we used transcriptome sequencing to compare newly diagnosed MM patients who had short progression-free survival (PFS) to standard first-line treatment (PFS < 24 months) to patients who had prolonged PFS (PFS > 24 months). We identified 157 differentially upregulated lncRNAs with short PFS and focused our efforts on characterizing the most upregulated lncRNA, LINC01432. We investigated LINC01432 overexpression and CRISPR/Cas9 knockdown in MM cell lines to show that LINC01432 overexpression significantly increases cell viability and reduces apoptosis, while knockdown significantly reduces viability and increases apoptosis, supporting the clinical relevance of this lncRNA. Next, we used individual-nucleotide resolution cross-linking immunoprecipitation with RT-qPCR to show that LINC01432 directly interacts with the RNA binding protein, CELF2. Lastly, we showed that LINC01432-targeted locked nucleic acid antisense oligonucleotides reduce viability and increases apoptosis. In summary, this fundamental study identified lncRNAs associated with short PFS to standard NDMM treatment and further characterized LINC01432, which inhibits apoptosis.

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Massively parallel dissection of RNA in RNA-protein interactions in vivo.

Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo.

Abstract LB209: YBX1 is a negative regulator of post-transcriptional program associated to EMT, invasion and metastasis in cSCC

Abstract Y-box binding protein 1 (YB-1), which is encoded by YBX1 gene, is a multifunctional cold-shock protein that binds both DNA and RNA to orchestrate transcription as well as RNA processing and translation. Recently, we demonstrated that YBX1 is a post-transcriptional effector required for the maintenance of epidermal homeostasis. Although it was well known that YBX1 plays prominent pro-oncogenic roles in malignant transformation and cell invasion in a wide variety of cancers, the role of YBX1 in cutaneous squamous cell carcinoma (cSCC) is poorly understood. Here, we report that YBX1 protein level is higher in cSCC tumor compared with non-neoplastic skin, but it goes down in metastatic states. According to this finding, YBX1-KD cSCC primary cell lines show an increase of cell migration and invasion in vitro assay and in a context that mimics the natural three-dimensional environment of tumor with organotypic skin equivalent cultures. Furthermore, we found that YBX1-KD promotes an epithelial-to-mesenchymal transition and leads to the detection of more metastases in vivo after tail vein injection of YBX1-KD cSCC cells when compared with the injection of control cells. YBX1 is predominantly found in the cytoplasm of SCC cells. Therefore, we focused on the role of YBX1 as a post-transcriptional regulatorand performed an eCLIP-seq (crosslinking immunoprecipitation sequencing) to identify its direct RNA targets. We found that YBX1 binds novel pro-invasive RNA regulons associated to cell-cell and cell-matrix adhesions. Through the interpolation of the analysis of RNA-seq and SILAC, we found that YBX1 KD up-modulates at post-transcriptional level several targets, involved into invasion and metastasis. Our study reveals that, in human cutaneous squamous cell carcinoma, loss of function of YBX1 promotes migration, invasiveness and metastasis, through the induction of an epithelial-to-mesenchymal transition regulated at post-transcriptional level. Citation Format: Stefano Sol, Fabiana Boncimino, Kristina Todorova, Anna Mandinova. YBX1 is a negative regulator of post-transcriptional program associated to EMT, invasion and metastasis in cSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB209.

Bioinformatics-Based Screening of Key LncRNAs for Modulating the Transcriptome Associated with Glaucoma in Human Trabecular Meshwork Cells.

The morphology and functions of the human trabecular meshwork (HTM) are dysregulated in glaucoma, and the molecular mechanisms of this dysregulation remain unknown. According to an established in vitro model, whose function was to study the regulatory networks sustaining the response of HTM cells to the increased substrate stiffness, we systematically analyzed the expression pattern of long noncoding RNAs (lncRNAs), the important regulatory RNAs in cells. Bioinformatics analysis was performed to identify the dysregulated lncRNAs in response to increased substrate stiffness using transcriptome sequencing data (RNA-seq). Then we interfered with the expression of several dysregulated lncRNAs in HTM cells to explore their molecular targets. The cross-linking immunoprecipitation and sequencing method (CLIP-seq) was used to identify enhancer of zeste homolog 2 (EZH2)-targeted RNAs in HTM cells. The chromatin IP and sequencing method (ChIP-seq) was used to identify the targets of EZH2 and histone H3 at lysine 27 (H3K27me3). The response of thousands of dysregulated lncRNAs to increased substrate stiffness was identified through RNA-seq. Functional prediction of these lncRNAs revealed that they potentially regulated key biological processes, including extracellular matrix (ECM) organization. By interfering with the expression of lncRNA SHNG8, ZFHX4-AS1, and RP11-552M11.4, the results demonstrated that those lncRNAs extensively regulated the expression levels of ECM-associated genes. Moreover, we found that EZH2 expression was significantly decreased at high substrate stiffness. Using CLIP-seq to identify EZH2-targeted RNAs in HTM cells, we found that SNHG8 was bound by EZH2. According to the CLIP-seq data of EZH2, we found that EZH2 binding sites were observed in the transcripts of SNHG8-regulated genes, but not in the ChIP-seq results of EZH2 and H3K27me3. Our results suggest that SNHG8 and EZH2 may cooperate to regulate the expression of a subset of genes by influencing their RNA abundance, explaining how they support HTM cell morphology and high density. This study contributes to the understanding of the alteration of HTM during the progression of glaucoma by identifying functional lncRNAs, especially SNHG8, and suggests novel therapeutic targets to treat glaucoma.

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice.

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Splicing factor SRSF1 is essential for homing of precursor spermatogonial stem cells in mice

Spermatogonial stem cells (SSCs) are essential for continuous spermatogenesis and male fertility. The underlying mechanisms of alternative splicing (AS) in mouse SSCs are still largely unclear. We demonstrated that SRSF1 is essential for gene expression and splicing in mouse SSCs. Crosslinking immunoprecipitation and sequencing data revealed that spermatogonia-related genes (e.g. Plzf, Id4, Setdb1, Stra8, Tial1/Tiar, Bcas2, Ddx5, Srsf10, Uhrf1, and Bud31) were bound by SRSF1 in the mouse testes. Specific deletion of Srsf1 in mouse germ cells impairs homing of precursor SSCs leading to male infertility. Whole-mount staining data showed the absence of germ cells in the testes of adult conditional knockout (cKO) mice, which indicates Sertoli cell-only syndrome in cKO mice. The expression of spermatogonia-related genes (e.g. Gfra1, Pou5f1, Plzf, Dnd1, Stra8, and Taf4b) was significantly reduced in the testes of cKO mice. Moreover, multiomics analysis suggests that SRSF1 may affect survival of spermatogonia by directly binding and regulating Tial1/Tiar expression through AS. In addition, immunoprecipitation mass spectrometry and co-immunoprecipitation data showed that SRSF1 interacts with RNA splicing-related proteins (e.g. SART1, RBM15, and SRSF10). Collectively, our data reveal the critical role of SRSF1 in spermatogonia survival, which may provide a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying homing of precursor SSCs.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking

ABSTRACT Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3’ untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

GCLiPP: global crosslinking and protein purification method for constructing high-resolution occupancy maps for RNA binding proteins

GCLiPP is a global RNA interactome capture method that detects RNA-binding protein (RBP) occupancy transcriptome-wide. GCLiPP maps RBP-occupied sites at a higher resolution than phase separation-based techniques. GCLiPP sequence tags correspond with known RBP binding sites and are enriched for sites detected by RBP-specific crosslinking immunoprecipitation (CLIP) for abundant cytosolic RBPs. Comparison of human Jurkat T cells and mouse primary T cells uncovers shared peaks of GCLiPP signal across homologous regions of human and mouse 3′ UTRs, including a conserved mRNA-destabilizing cis-regulatory element. GCLiPP signal overlapping with immune-related SNPs uncovers stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1.

Hnrnpu mutations Are Haploinsufficient and Alter the Transcriptome of MYC-Driven Lymphomas

Introduction: Burkitt lymphoma (BL) is an aggressive B-cell cancer, characterized by translocations that juxtapose a potent immunoglobulin enhancer with the MYC oncogene. MYC translocations are also found in 10% of tumors with diffuse large B-cell lymphoma morphology. When a BCL2 translocation is also present, these high-grade B-cell lymphomas (HGBCL-DH- BCL2) have poor prognosis. While sustained MYC expression can promote cell activation and proliferation, it also has potent and acute effects on programmed cell death, which cancer cells must overcome in order to survive. Identification and functional characterization of common mutations that cooperate with MYC are thus important for understanding its role in pathobiology. Methods: Patient samples were obtained from an ongoing meta-analysis of mature B-cell neoplasms including unpublished data from the Lymphoma/Leukemia Molecular Profiling Project. Simple somatic mutations were identified from whole genome or exome sequencing data using an ensemble of variant callers. MYC rearrangements breakpoints were identified using GRIDSS and Manta. All functional studies were performed in an EBV+ BL cell line (Raji). HNRNPU mutations were introduced into cells using IDTs ALT-R CRISPR-Cas9 system, clonally isolated, and verified by sequencing. Subsequent RNA-sequencing was performed. Knockdown and overexpression experiments were performed using an HNRNPU expression vector or siRNA targeting endogenous HNRNPU. HNRNPU eCLIP in HepG2 and K562 cells were obtained from the ENCODE project consortium. Differential gene expression was analysed with DESeq2. Peak-calling and individual crosslink sites were detected using PureCLIP. Results: Within the sequencing data, we noted that several RNA binding proteins were commonly mutated in BL and HGBCL-DH- BCL2. Mutations affecting HNRNPU were identified in tumors from 11.6% of HGBL-DH- BCL2 and 5.6% of BL patients. These mutations were predicted to be inactivating but, unlike conventional tumor suppressor genes, an inactivating mutation on the other allele was never observed. HNRNPU mutations were significantly enriched in EBV+ BLs. The gene product (hnRNP U) is an RNA- and DNA-binding protein that plays a central role in gene expression regulation. To understand their biological effect in aggressive lymphomas, we introduced heterozygous inactivating mutations in the most affected region of HNRNPU. Clones with confirmed knockout exhibited reduced expression at the mRNA and protein level. Through gene expression analysis, comparing the parental to two mutant lines, we identified 615 differentially expressed genes (358 downregulated, 219 upregulated). Pathway enrichment analysis revealed reduced expression of genes in several relevant pathways such as MYC, TP53 and DNA damage response. This was consistent with our observation of reduced MYC protein in the mutant lines. A role of hnRNPU-in MYC modulation was further in experiments when HNRNPU knockdown reduced and overexpression increased MYC expression. Overexpression of hnRNP U also resulted in cellular stress and a decrease in cell proliferation. Using actinomycin D chase experiments, we found that hnRNPU loss leads to reduced MYC transcript stability. Crosslinking immunoprecipitation suggests that hnRNP U binds directly to the MYC transcript in poly G tracts in intron 1. This region is predicted to fold into G-quadruplexes, a secondary structure that can be bound by hnRNP U. HNRNPU mutations are only observed in tumors with an intact MYC locus rather than those with a rearrangement in intron 1. Taken together, these findings suggest that the region of MYC upstream of intron 1 may be relevant for HNRNPU-mediated modulation of MYC. Conclusion: HNRNPU mutations are novel recurrent driver mutations specifically within MYC translocated B-cell lymphomas. HNRNPU acts as a modulator of MYC expression, and the most common mutations are predicted to negatively impact this role. We propose a model where HNRNPU mutations moderate MYC expression, thus buffering MYC-induced apoptosis and proteotoxic stress. Further experiments to explore the role of HNRNPU binding on MYC expression and the potential role in buffering the proteotoxic stress associated with MYC translocations are ongoing. Insights into the mechanisms contributing to disease will support ongoing work to establish in vitro and in vivo models for future therapeutic investigations.

IGF2BP1 Promotes Multiple Myeloma with Chromosome 1q Gain Via Increasing CDC5L Expression in an m6A-Dependent Manner

Background: Multiple myeloma (MM) patients with chromosome 1q gain (1q+) are clinically and biologically heterogeneous. The underlying molecular mechanisms are still under investigation, while identification of targets for effective therapy of this subgroup of MM patients is urgently needed. We aimed to investigate the clinical significance and the regulatory mechanisms of insulin-like growth factor 2 messenger RNA (mRNA) binding proteins 1 (IGF2BP1), a N6-methyladenosine (m6A) reader, in MM patients with 1q+. Methods: Comprehensive in silico analyses of existed public datasets (MMRF CoMMpass study) and in-house generated high-throughput sequencing datasets (RNA-sequencing, methylated RNA immunoprecipitation sequencing, and crosslinking immunoprecipitation sequencing), along with routine molecular and cellular in vitro assays, as well as in vivo animal model studies, were conducted. Results: IGF2BP1 displays a high expression level in MM patients with 1q+ and its high expression predicts poor prognosis in these patients. IGF2BP1 overexpression promoted cell proliferation and G1-to-S phase transition of the cell cycle in H929 cells. Combinatory analyses of MeRIP-seq, RNA-seq, CLIP-seq and patients' survival data identified CDC5L as a target of IGF2BP1, which can bind the m6A sites of CDC5L mRNA to upregulate its protein abundance. Higher CDC5L expression also predicted worse prognosis of MM patients with 1q+. Moreover, both knockdown and mutation of CDC5L attenuated the pro-proliferative effect of IGF2BP1. Furthermore, IGF2BP1 inhibitor BTYNB effectively inhibited CDC5L expression in MM cells with 1q+, and suppressed the proliferation of these cells in vitro and in vivo. Conclusions: IGF2BP1 acts as a post-transcriptional enhancer of CDC5L in an m6A manner to promote the proliferation of MM cells with 1q+. Our work identified a novel IGF2BP1-CDC5L axis and provided new insight in developing targeted therapeutics for MM patients with 1q+.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

AimChemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches.MethodsGene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses.ResultsWe identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells.ConclusionsIGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

Consequences of depleting TNRC6, AGO, and DROSHA proteins on expression of microRNAs.

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.