Characterizing the Function of an RNA Binding Protein, IGF2BP3, in Hematopoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3664-3664
Author(s):  
Tiffany M Tran ◽  
Jayanth Kumar Palanichamy ◽  
Jonathan Howard ◽  
Jorge R. Contreras ◽  
Thilini Fernando ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Lymphoblastic Leukemia ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Post-transcriptional control of gene expression plays important roles in defining normal and pathological cellular phenotypes. Amongst mechanisms of post-transcriptional regulation, RNA binding proteins (RBPs) have recently been shown to play important roles. However, in vivo roles for RBPs are not well understood. Here, we identified the RBP IGF2BP3 to be specifically overexpressed in MLL-rearranged B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and a risk of high relapse. IGF2BP3 was required for the survival of B-ALL cell lines, and knockdown led to decreased proliferation and increase apoptosis. In addition, enforced expression of IGF2BP3 in murine bone marrow transplant assays caused a proliferation of hematopoietic stem and progenitor cells and a skewing of hematopoietic development to the B-cell/myeloid lineage. Using cross-link immunoprecipitation and high-throughput sequencing, we uncovered the transcriptome regulated by IGF2BP3; including novel direct targets, MYC and CDK6. These were regulated following experimental alteration of IGF2BP3 expression in vivo, and are regulated via elements within their 3'untranslated regions. Hence, IGF2BP3 mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism operant in MLL-rearranged B-ALL, highlighting IGF2BP3 and its cognate RNA binding partners as potential therapeutic targets in this disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enforcing Post-Transcriptional Circuitries to Achieve Human Hematopoietic Stem Cell Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-46-SCI-46
Author(s):  
Kristin Hope
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Self Renewal ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem

Abstract The balance between hematopoietic stem cell (HSC) differentiation and self-renewal is central to clinical regenerative paradigms. Unravelling the precise molecular mechanisms that govern HSC fate choices will thus have far reaching consequences for the development of effective therapies for hematopoietic and immunological disorders. There is an emerging recognition that beyond transcription, HSC homeostasis is subject to post-transcriptional control by RNA-binding proteins (RBPs) that ensure precise control of gene expression by modulating mRNA splicing, polyadenylation, localization, degradation or translation. RBPs can synchronously regulate the fates of operationally similar RNAs, in what have been termed RNA regulons. We have used a variety of functional approaches, in combination with unbiased genome- and proteome-scale, methods to define the tenets that govern this regulation and to determine key downstream circuitries of stem cell-regulating RBPs whose targeting could provide the basis for novel regenerative treatments. Through loss-of-function efforts, we have identified the RBP, MSI2, as a required factor for human HSC maintenance. By contrast, at supraphysiological levels, MSI2 has a profound positive effect on human HSC self-renewal decisions. Using a combination of global profiling, including mapping MSI2's targets through cross-linking immunoprecipitation (CLIP)-seq, we show that MSI2 achieves its ex vivo self-renewal-promoting effects by directing a co-ordinated post-transcriptional repression of key targets within the aryl hydrocarbon receptor (AHR) pathway. We are currently exploring the "rules" by which MSI2 influences its downstream effects on target RNAs and how it functions, in combination with other protein interactors, to instill a putative RBP regulatory code in HSCs. HSCs exhibit highly unique epigenomes, transcriptomes and proteomes and it is this distinctive molecular landscape that provides the canvas upon which MSI2, and indeed any other HSC-specific RBP exert their post-transcriptional influence over stem cell function. As such, to decipher the bona fide RNA networks that RBPs function upon in HSCs and to understand how they influence this network to enforce self-renewal, we are working towards performing systematic studies of RBP regulons in these cells specifically. In turn these approaches are elucidating a host of RBPs and post-transcriptional control mechanisms previously unappreciated for their role in HSC control. When modulated appropriately, we can successfully tailor these post-transcriptional regulons to enforce desired HSC outputs ex vivo. In summary, approaches to elucidate key HSC-regulatory RBPs and their protein and RNA interactomes provide valuable insights into a layer of HSC control previously not well understood, and one that can be capitalized on to achieve successful HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control

2019 ◽  
Vol 97 (1) ◽  
pp. 10-20 ◽  
Author(s):  
Laura P.M.H. de Rooij ◽  
Derek C.H. Chan ◽  
Ava Keyvani Chahi ◽  
Kristin J. Hope
Keyword(s):  
Transcriptional Regulation ◽  
Stem Cell ◽  
Cell Fate ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Post Transcriptional Regulation

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.

scholarly journals PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites

2020 ◽  
Vol 48 (10) ◽  
pp. 5511-5526
Author(s):  
Tiago R Ferreira ◽  
Adam A Dowle ◽  
Ewan Parry ◽  
Eliza V C Alves-Ferreira ◽  
Karen Hogg ◽  
...  
Keyword(s):  
Protein Modification ◽  
Transcriptional Control ◽  
Leishmania Major ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Capacity ◽  
Arginine Methylation ◽  
Mrna Metabolism

Abstract RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

scholarly journals Novel autoregulatory cases of alternative splicing coupled with nonsense-mediated mRNA decay

2018 ◽  
Author(s):  
Dmitri Pervouchine ◽  
Yaroslav Popov ◽  
Andy Berry ◽  
Beatrice Borsari ◽  
Adam Frankish ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Negative Feedback Loop ◽  
Control Of Gene Expression ◽  
Nonsense Mediated Decay ◽  
Premature Termination Codons

AbstractNonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic framework to identify novel such autoregulatory feedback loops by combining eCLIP assays for a large panel of RBPs with the data on shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq. We show that RBPs frequently bind their own pre-mRNAs and respond prominently to NMD pathway disruption. Poison and essential exons, i.e., exons that trigger NMD when included in the mRNA or skipped, respectively, respond oppositely to the inactivation of NMD pathway and to the depletion of their host genes, which allows identification of novel autoregulatory mechanisms for a number of human RBPs. For example, SRSF7 binds its own pre-mRNA and facilitates the inclusion of two poison exons; SFPQ binding promotes switching to an alternative distal 3’-UTR that is targeted by NMD; RPS3 activates a poison 5’-splice site in its pre-mRNA that leads to a frame shift; U2AF1 binding activates one of its two mutually exclusive exons, leading to NMD; TBRG4 is regulated by cluster splicing of its two essential exons. Our results indicate that autoregulatory negative feedback loop of alternative splicing and NMD is a generic form of post-transcriptional control of gene expression.

scholarly journals Palmitoyl Acyltransferase DHHC9 Is Required for Efficient Induction of Leukemia By Oncogenic NRAS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 893-893
Author(s):  
Ping Liu ◽  
Bo Jiao ◽  
Ruihong Zhang ◽  
Huanbin Zhao ◽  
Chun Zhang ◽  
...  
Keyword(s):  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Myeloproliferative Disease ◽  
Ras Proteins ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Palmitoyl Acyltransferase

Abstract Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high-affinity plasma membrane binding of NRAS, HRAS and KRAS4A. NRAS mutations are common in human hematological malignancies. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for RAS related cancers. In previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate the modification reaction. Protein S-palmitoylation is catalyzed by the DHHC family of palmitoyl acyltransferases (PATs). Thus far 24 mammalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9) is the ortholog of yeast Ras2 PAT and constitutes a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancers. In this study we investigated the role of DHHC9 in normal hematopoiesis and NRAS leukemogenesis in vivo. We found that DHHC9 is not the only PATs for RAS palmitoylation in vivo and the Zdhhc9 knockout mice were born and grew similarly as the wild type mice. The frequency of lineage-specific populations and hematopoietic stem cell phenotypes were also similar in mice with knockout alleles of Zdhhc9 as that of wild type mice. However, loss of DHHC9 prolonged the survival of mice with myeloproliferative disease or T-cell acute lymphoblastic leukemia evoked by oncogenic NRAS expressed either from the endogenous locus or from retroviral promoter. These results demonstrate that DHHC9 is dispensable for normal hematopoiesis, but plays an important role in the pathogenesis of NRAS-induced leukemias. The findings suggest that palmitoyl acyltransferase DHHC9 may serve as a safe and effective target for developing therapies against NRAS-related cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Long Non-Coding RNA-Ribonucleoprotein Networks in the Post-Transcriptional Control of Gene Expression

2020 ◽  
Vol 6 (3) ◽  
pp. 40
Author(s):  
Paola Briata ◽  
Roberto Gherzi
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Huge Number ◽  
Protein Coding ◽  
Control Of Gene Expression ◽  
Non Coding Rna ◽  
Non Coding Rnas ◽  
And Function ◽  
Long Non Coding Rna

Although mammals possess roughly the same number of protein-coding genes as worms, it is evident that the non-coding transcriptome content has become far broader and more sophisticated during evolution. Indeed, the vital regulatory importance of both short and long non-coding RNAs (lncRNAs) has been demonstrated during the last two decades. RNA binding proteins (RBPs) represent approximately 7.5% of all proteins and regulate the fate and function of a huge number of transcripts thus contributing to ensure cellular homeostasis. Transcriptomic and proteomic studies revealed that RBP-based complexes often include lncRNAs. This review will describe examples of how lncRNA-RBP networks can virtually control all the post-transcriptional events in the cell.

scholarly journals Expression, Regulation and Function of microRNA as Important Players in the Transition of MDS to Secondary AML and Their Cross Talk to RNA-Binding Proteins

2020 ◽  
Vol 21 (19) ◽  
pp. 7140 ◽  
Author(s):  
Marcus Bauer ◽  
Christoforos Vaxevanis ◽  
Nadine Heimer ◽  
Haifa Kathrin Al-Ali ◽  
Nadja Jaekel ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Significant Risk ◽  
Control Of Gene Expression ◽  
Hematopoietic Stem ◽  
Molecular Abnormalities ◽  
Acute Myeloid

Myelodysplastic syndromes (MDS), heterogeneous diseases of hematopoietic stem cells, exhibit a significant risk of progression to secondary acute myeloid leukemia (sAML) that are typically accompanied by MDS-related changes and therefore significantly differ to de novo acute myeloid leukemia (AML). Within these disorders, the spectrum of cytogenetic alterations and oncogenic mutations, the extent of a predisposing defective osteohematopoietic niche, and the irregularity of the tumor microenvironment is highly diverse. However, the exact underlying pathophysiological mechanisms resulting in hematopoietic failure in patients with MDS and sAML remain elusive. There is recent evidence that the post-transcriptional control of gene expression mediated by microRNAs (miRNAs), long noncoding RNAs, and/or RNA-binding proteins (RBPs) are key components in the pathogenic events of both diseases. In addition, an interplay between RBPs and miRNAs has been postulated in MDS and sAML. Although a plethora of miRNAs is aberrantly expressed in MDS and sAML, their expression pattern significantly depends on the cell type and on the molecular make-up of the sample, including chromosomal alterations and single nucleotide polymorphisms, which also reflects their role in disease progression and prediction. Decreased expression levels of miRNAs or RBPs preventing the maturation or inhibiting translation of genes involved in pathogenesis of both diseases were found. Therefore, this review will summarize the current knowledge regarding the heterogeneity of expression, function, and clinical relevance of miRNAs, its link to molecular abnormalities in MDS and sAML with specific focus on the interplay with RBPs, and the current treatment options. This information might improve the use of miRNAs and/or RBPs as prognostic markers and therapeutic targets for both malignancies.

41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2293-2293
Author(s):  
Ekta Kapadia ◽  
Elad Jacoby ◽  
Mark Kohler ◽  
Waleed Haso ◽  
Christopher Daniel Chien ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

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