scholarly journals Increased hnRNP K Expression Impacts Myelopoiesis By Altering RUNX1 Splicing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3317-3317
Author(s):  
Sean M Post ◽  
Marisa J Aitken ◽  
Prerna Malaney ◽  
Xiaorui Zhang ◽  
Todd Link ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Fetal Liver ◽  
Full Length ◽  
Clinical Samples ◽  
Differentiation Potential ◽  
Hnrnp K ◽  
Mutational Status

Abstract Mutations in RNA binding proteins have been identified as pathogenic drivers in many hematological malignancies. However, in addition to mutational status, expression changes in RNA binding proteins likely impact disease processes. Through our studies, we identified that overexpression of hnRNP K (heterogeneous ribonucleoprotein K) -a poly(C)-RNA binding protein that governs the expression of numerous genes and transcripts- plays a pivotal role in myeloid malignancies. Using clinical samples, we determined that hnRNP K overexpression is a recurrent abnormality, occurring in nearly 30% of AML cases. Importantly, elevated hnRNP K levels associate with decreased overall survival (24.3 months versus 48.7 months; HR 1.9; 95% CI 1.3-2.7). However, the role of hnRNP K overexpression in AML remains unclear. To evaluate its putative oncogenic potential, we overexpressed hnRNP K in murine fetal liver cells (FLCs). Using colony formation assays (CFAs), we demonstrated that hnRNP K-overexpressing FLCs have an altered differentiation potential (increased number of immature (c-kit +Sca-1 +) and decreased number of mature myeloid (Gr1 +CD11b +) cells) and an increase in self-renewal capacity (increased number of colonies) (p=0.008). Mice transplanted with hnRNP K overexpressing FLCs had markedly shortened survival compared to empty vector controls, despite similar engraftment (median survival 8.1 weeks versus median not reached (HR 3.0, 95% CI 1.2 - 7.3, p=0.02). Significantly, extramedullary hematopoiesis was observed in the spleens and the hepatic parenchyma of mice transplanted with FLCs that overexpress hnRNP K. This resulted in disrupted splenic architecture and the presence of immature hematopoietic cells and cells of myeloid origin (CD117, CD14, and myeloperoxidase). Furthermore, analyses of the bone marrow revealed an increase in myeloid cells in hnRNP K transplanted mice. We next used unbiased and biochemical approaches to discover a direct interaction between hnRNP K and the RUNX1 transcript-a critical transcriptional factor often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1 (delExon6) , resulting in the generation of a functionally distinct isoform that is more stable than full-length RUNX1. RNA-Seq and reporter assays demonstrated that delExon6 has a unique transcriptional profile compared to full-length RUNX1, suggesting this spliced transcript may have a pathogenic role. To examine the functionality of delExon6, we performed CFAs. Here, we observed that delExon6 expression results in an increased proliferation potential that is mediated by hnRNP K's RNA binding activity. Together, these data establish hnRNP K as an oncogene in myeloid leukemia through its ability to directly bind the RUNX1 transcript, modify RUNX1 splicing, and subsequently alter its transcriptional activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

2013 ◽  
Vol 9 (6) ◽  
pp. e1003460 ◽  
Author(s):  
Pei-Ling Tsai ◽  
Ni-Ting Chiou ◽  
Sharon Kuss ◽  
Adolfo García-Sastre ◽  
Kristen W. Lynch ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Rna Splicing ◽  
Influenza A ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Hnrnp K ◽  
Virus Rna

scholarly journals Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1

2020 ◽  
Vol 21 (3) ◽  
pp. 1166 ◽  
Author(s):  
Marian Scherer ◽  
Michal Levin ◽  
Falk Butter ◽  
Marion Scheibe
Keyword(s):  
Quantitative Proteomics ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Full Length ◽  
Interacting Proteins ◽  
Non Coding Rna ◽  
Nuclear Rna ◽  
Long Non Coding Rna ◽  
Shed Light

The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function together with RNA-binding proteins (RBPs), the composition of their RBP complex can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5′-region and one in the 3′-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.

The BCR-ABL1-Regulated hnRNP A1, hnRNP E2, and hnRNP K Are Differentially Expressed Between CD34+ and CD34+/CD38- Ph+ Cells, and After Blastic Transformation of CML.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1222-1222
Author(s):  
Jason G Harb ◽  
Paolo Neviani ◽  
Joshua J Oaks ◽  
Guido Marcucci ◽  
Peter Hokland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Disease Progression ◽  
Cell Line ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Hnrnp A1 ◽  
Hnrnp K

Abstract Abstract 1222 The molecular mechanism leading to disease progression of chronic myelogenous leukemia (CML) still remains to be identified although enhanced BCR/ABL expression and activity seems to play an important role in controlling genomic stability, differentiation, and self renewal of the leukemic cell clone undergoing blastic tranformation by affecting expression and function of RNA binding proteins (RBPs) like hnRNP A1, hnRNP E2, and hnRNP K (Perrotti D. et al. J. Clin Invest. 2010). We previously reported (Harb J. et al., ASH 2009) that a BCR-ABL1 dosage-dependence and hierarchical organization exists for the expression of hnRNP A1, hnRNP E2, and hnRNP K in cell line models of CML. In fact, as BCR/ABL levels increase, upregulated expression of hnRNP A1 is observed, followed by increased expression of hnRNP E2 and hnRNP K. HnRNP A1 and hnRNP K were also temporally expressed within Lineage- Sca-1+ c-kit+ (LSK), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP) in a mouse model (SCLtTA TRE-BCR/ABL) of chronic myeloid leukemia. Interestingly, hnRNP A1 and hnRNP K levels in BCR/ABL+ mouse progenitors correlated with disease severity as mice with higher levels of these RBPs presented a more progressed phenotype characterized by increased mixed lineage B220+/Mac-1+ progenitors in bone marrow and spleen when compared with mice that developed a CML-CP-like phenotype. Here we show that hnRNP A1, hnRNP E2, and hnRNP K expression levels, as well as BCR/ABL activity are different in HSC (CD34+/CD38-), CMP (CD34+/CD38+/CD45+/IL-3Ra-), and GMP (CD34+/CD38+/CD45+/IL-3Ra+) isolated from peripheral blood of patients in chronic phase (CML-CP) at diagnosis, untreated accelerated phase (CML-AP), and blast crisis (CML-BC). Interestingly, in CML-CP, the highest expression of hnRNP A1 was found in the CD34+/CD38- stem cell fraction and it gradually decreased in the more mature CMP and GMP progenitors (55% and 65% lower, respectively). By contrast, consistent with the role of hnRNP A1 as regulator of progenitor cell proliferation and survival, hnRNP A1 expression progressively increased in the HSC, CMP and GMP fractions isolated from patients in CML-AP and CML-BC. Unlike hnRNP A1, hnRNP E2 and hnRNP K were barely detected in the CD34+/CD38- from CML-CP patients but their expression was markedly pronounced in the HSC fraction of progressed CML patients. In agreement with our cell line data, expression of hnRNP A1 and hnRNP E2 in advanced CML (CML-AP and CML-BC) increases when CD34+/CD38- stem cells undergo maturation toward CMP. Conversely, it appears that hnRNP K expression is the last to increase in CML-BC, suggesting a hierarchical regulation of RBP expression during differentiation and lineage commitment. Expectedly, levels of hnRNP A1 in CMPs increase during disease progression (CP<AP<BC). Because of the highest expression of hnRNP A1 in the CD34+/CD38- fraction in CML-CP, and its previously described role in regulating survival and proliferation of Ph+ CD34+ cells (Iervolino et al., Mol Cell Biol 2002; Neviani et al. Cancer Cell 2005), we lentivirally transduced a hnRNP A1 shRNA into the CD34+/CD38- and CD34+/CD38+ cell fractions in CML-CP and assessed the role played by this RBP in survival of primitive and committed CML-CP progenitors. Western blot analysis demonstrated that hnRNP A1 was efficiently knockdown (≥ 80% reduction) in both compartments. Due to its ability to regulate BCR-ABL1 expression/function through the SET-PP2A axis, downregulation of hnRNP A1 led to reduced BCR/ABL activity in both stem and progenitor cells. However, cell survival and cytokine-dependent proliferation were severely compromised in committed progenitors transduced with the hnRNP A1 shRNA but not in the shRNA hnRNP A1-expressing CD34+/CD38- cells, consistent with the notion that progenitors but not stem cells are BCR-ABL1 oncogene addicted. Colony forming assays performed with empty vector- and hnRNP A1 shRNA-transduced CD34+/CD38- cells showed a 50% reduction in the number of CFCs upon hnRNP A1 downregulation. Accordingly, levels of the hnRNP A1-regulated anti-apoptotic Bcl-xL were substantially reduced in CD34+/CD38- cells. Taken together, these data further implicate RBPs hnRNP A1, hnRNP E2, and hnRNP K in CML disease progression and suggest their possible role in the control of survival of stem and primitive CML-CP progenitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development

2020 ◽  
Author(s):  
Jun Cao ◽  
Andrew L. Routh ◽  
Muge N. Kuyumcu-Martinez
Keyword(s):  
Heart Development ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Muscle Cells ◽  
Full Length ◽  
Actin Binding ◽  
Nanopore Sequencing ◽  
Mrna Isoforms

ABSTRACTAlternative splicing (AS) increases the variety of the proteome by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full length Tpm1 isoforms composed of distinct exons with specific linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. Nanopore sequencing combined with polyA sequencing revealed that the RNA binding protein RBFOX2 controls AS of muscle specific Tpm1 isoforms and expression of TPM1 protein via terminal exon splicing impacting its polyadenylation. Furthermore, RBFOX2 regulates Tpm1 AS antagonistically to PTBP1. In sum, we defined full length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of muscle specific isoforms of Tpm1 by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle enriched genes.

scholarly journals Regulation of Fetal Genes by Transitions among RNA-Binding Proteins during Liver Development

2020 ◽  
Vol 21 (23) ◽  
pp. 9319
Author(s):  
Toru Suzuki ◽  
Shungo Adachi ◽  
Chisato Kikuguchi ◽  
Shinsuke Shibata ◽  
Saori Nishijima ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fetal Liver ◽  
Regulatory Protein ◽  
Rna Decay ◽  
Translation Efficiency ◽  
Mrna Binding ◽  
Post Transcriptional Regulation

Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3′ regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.

scholarly journals Assembly of the α-Globin mRNA Stability Complex Reflects Binary Interaction between the Pyrimidine-Rich 3′ Untranslated Region Determinant and Poly(C) Binding Protein αCP

1999 ◽  
Vol 19 (7) ◽  
pp. 4572-4581 ◽  
Author(s):  
Alexander N. Chkheidze ◽  
Dmitry L. Lyakhov ◽  
Alexander V. Makeyev ◽  
Julia Morales ◽  
Jian Kong ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Direct Interaction ◽  
Binary Interaction ◽  
Globin Mrna ◽  
Hnrnp K

ABSTRACT Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human α-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3′ untranslated region (3′UTR), the α-complex. The proteins of the α-complex are widely expressed. The α-complex or a closely related complex also assembles at pyrimidine-rich 3′UTR segments of other stable mRNAs. These data suggest that the α-complex may constitute a general determinant of mRNA stability. One or more αCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the α-complex. The ability of αCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the α-complex is a multisubunit structure. In the present study, we have addressed the composition of the α-complex. An RNA titration recruitment assay revealed that αCPs were quantitatively incorporated into the α-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major αCP isoforms and the α-globin 3′UTR was detected, suggesting that each of these proteins might be sufficient for α-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant αCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the α-complex could accommodate only a single αCP. These data established that a single αCP molecule binds directly to the α-globin 3′UTR, resulting in a simple binary structure for the α-complex.

scholarly journals hnRNP K Overexpression Drives Myeloid Malignancy Via Interaction with RUNX1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2622-2622
Author(s):  
Marisa J Hornbaker ◽  
Prerna Malaney ◽  
Xiaorui Zhang ◽  
Ruizhi Duan ◽  
Vrutant Shah ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Marker Chromosome ◽  
Similar Proportion ◽  
Extra Copy ◽  
Differentiation Potential ◽  
Hnrnp K ◽  
Myeloid Malignancy ◽  
Protein Levels

Abstract Acute myeloid leukemia (AML) is a conglomerate of hematologic malignancies characterized by recurrent genetic and/or chromosomal aberrations. Recent development of targeted agents has bolstered our armamentarium of therapeutic options and improved outcomes for many patients. Acquiring deeper mechanistic understanding of myeloid leukemogenesis will provide a basis for development of even more therapeutic strategies and further improve patient outcomes. Our clinical data have revealed that overexpression of HNRNPK is a recurrent abnormality that occurs in upwards of 20% of AML cases at both the RNA and protein levels. Using bone marrow samples from a similar proportion of patients, we have recently discovered a supernumerary marker chromosome containing an extra copy of the HNRNPK locus that is not detectable with routine cytogenetic testing. We have further associated high hnRNP K protein levels with decreased overall survival in de novo AML, emphasizing the need to understand the role of hnRNP K in myeloid malignancy. To directly evaluate the oncogenic capacity of hnRNP K, we have overexpressed hnRNP K in murine fetal liver cells (FLCs). Using CyTOF and colony formation assays, we demonstrated that hnRNP K-overexpressing FLCs have altered differentiation potential and self-renewal capacity compared to empty vector controls in vitro. These findings are recapitulated in vivo, as murine recipients of hnRNP K-overexpressing FLCs develop myeloid lineage disease, often manifesting as fatal megakaryocytic leukemia. To elucidate a mechanism by which hnRNP K causes myeloid disease, we performed hnRNP K immunoprecipitation followed by mass spectrometry in an AML cell line and identified that hnRNP K preferentially interacts with translational machinery, ribosomal subunits, and proteins involved in RNA processing. In conjunction with data from our hnRNP K overexpression models that indicate overexpression of hnRNP K occurs primarily in the cytoplasm, we then performed hnRNP K-RNA immunoprecipitation followed by sequencing (RIP-Seq). We determined that hnRNP K interacts with the transcript of RUNX1-a master regulator of hematopoiesis and a critical player in a myriad of leukemias-including megakaryocytic leukemias like those observed in our mouse models. Using biochemical assays, we have demonstrated that hnRNP K directly binds to consensus sequences in the RUNX1 transcript, and ultimately alters RUNX1 translation. Indeed, mice exhibiting hnRNP K overexpression have increased protein levels of Runx1 in hematopoietic tissues. Our data demonstrate that hnRNP K overexpression drives myeloid malignancy. Currently, we are screening compounds that will disrupt the interaction between hnRNP K and RUNX1 in our efforts to further understand myeloid biology and ultimately improve outcomes for patients with these diseases. Disclosures No relevant conflicts of interest to declare.

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