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Alternative splicing genes in hnRNP A1 defect mice. (a) The top 10 affected processes from the alternative splicing genes in the embryonic hearts of hnRNP A1 defect mice determined using microarray. The numbers represent gene numbers involved in the process. (b) Various alternative splicing products of muscle-related genes in the hearts of mice analysed using qRT-PCR. The exon position, various alternative splicing products and RT-PCR primer positions are marked. The PCR products of full-length and truncated forms found using gel electrophoresis are indicated hnRNP A1+/+ (wt) and hnRNP A1+/− (A1+/−). (c) Relative expression levels in (b) presented with the ratio (full/truncated length) relative to hnRNP A1+/+ mice. (d) The alternative splicing forms quantified using real-time PCR. The exon position, alternative splicing products and RT-PCR primer positions are presented in the lower part. Solid and dotted lines represent the primers for full-length and deleted mRNA, respectively. Error bars represent standard deviation. *p < 0.05 compared with the wild-type mice. The results are summarized from observations of six mice.
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Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNP A1 in vivo. The knockout mice, hnRNP A1−/−, showed embryonic lethality because of muscle developmental defects. The...
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... Gene ontology (GO) analysis (Zhou et al., 2019) of genes differentially expressed in the MC 2 vs. MC 0 clusters showed strong enrichment of the categories ''translation factors,'' ''mRNA transport,'' and ''translation'' ( Figure 2F). One of the most significantly downregulated genes in Nkx1-2 KD cells was Hnrnpa2b1 ( Figure S1E), which encodes a nuclear ribonucleoprotein that controls pre-mRNA processing, splicing, RNA nuclear export, and mRNA translation at the ribosomes (Kwon et al., 2019;Liu et al., 2017;Vautier et al., 2001), all of which are essential functions for mammalian embryonic development (Kwon et al., 2019). Other downregulated genes included Acyp1 ( Figure S1F) and Hnrnph3 ( Figure S1G), which also encode proteins involved in controlling RNA splicing and RNA processing. ...
Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.
... 21 HnRNP A1 +/− mice exhibited disrupted blood pressure and heart rates. 22 Animal models have demonstrated that the absence of SRSF1, 23 SRSF5, 24 SRSF10, 25 hnRNP U 26 or hnRNP L 27,28 phenocopy human cardiovascular diseases such as atrial septal defects, heart failure, noncompaction of the ventricular myocardium and diabetic heart. ...
RNA splicing is an important RNA processing step required by multiexon protein-coding mRNAs and some noncoding RNAs. Precise RNA splicing is required for maintaining gene and cell function; however, mis-spliced RNA transcripts can lead to loss- or gain-of-function effects in human diseases. Mis-spliced RNAs induced by gene mutations or the dysregulation of splicing regulators may result in frameshifts, nonsense-mediated decay (NMD), or inclusion/exclusion of exons. Genetic animal models have characterised multiple splicing factors required for cardiac development or function. Moreover, sarcomeric and ion channel genes, which are closely associated with cardiovascular function and disease, are hotspots for AS. Here, we summarise splicing factors and their targets that are associated with cardiovascular diseases, introduce some therapies potentially related to pathological AS targets, and raise outstanding questions and future directions in this field.
... ficant association among hnRNP family members and their contribution to clinical and pathological features of patients with lymphoma indicates that hnRNPs are novel and promising therapeutic targets. Many of these hnRNPs are, however, essential genes therefore therapeutic windows may be limited due to toxicity at higher doses (Gallardo et al. 2016;T.-Y. Liu et al. 2017;Sapir et al. 2022). However, in tumours with loss of heterozygosity events in essential genes, monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles (Nichols et al. 2020). It should, therefore, be considered that lower concentrations of targeted therap ...
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B-cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B-cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP-RNA network in lymphoma.
... Elavl2/HuB belongs to the family of Hu proteins that have been implicated in the regulation of AS and alternative polyadenylation (Pascale et al, 2008). Cpeb4 or Elavl2 knockout mice are viable (Liu et al, 2017;Kato et al, 2019), thus suggesting a mild effect in lung development, whereas Hnrnpa1 knockout mice die perinatally with cardiac defects but analysis of lungs was not reported (Liu et al, 2017). Thus, further investigation on the biological relevance of each of these factors, and combinatorial effects, is warranted to validate their roles in lung development. ...
... Elavl2/HuB belongs to the family of Hu proteins that have been implicated in the regulation of AS and alternative polyadenylation (Pascale et al, 2008). Cpeb4 or Elavl2 knockout mice are viable (Liu et al, 2017;Kato et al, 2019), thus suggesting a mild effect in lung development, whereas Hnrnpa1 knockout mice die perinatally with cardiac defects but analysis of lungs was not reported (Liu et al, 2017). Thus, further investigation on the biological relevance of each of these factors, and combinatorial effects, is warranted to validate their roles in lung development. ...
Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found that Vegfa switches from the Vegfa 164 isoform to the longer Vegfa 188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type-specific regulation of Vegfa alternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.
... Thus, dysfunction of RBPs at specific cell states may disrupt cell fate decisions and explain broadly why mutations or dysfunction in many RBPs cause neuromuscular diseases. For example, Tardbp (TDP-43) dysfunction impairs myogenic proliferation, whereas loss of Hnrnpa1 disrupts terminal myogenic differentiation (Liu et al., 2017;Vogler et al., 2018). Focusing on these disease-associated RBPS, we found that Tardbp expression is highest in subcluster 1, and in contrast, Hnrnpa2b1 expression is highest in subcluster 3, while Tia1 expression is highest in subcluster 6 ( Figure 2B). ...
RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.
... Additional mutations in hnRNP A1 were later identified by targeted sequencing of sporadic ALS patients and in an ALS family with flail arm syndrome [114]. hnRNP A1 knockout mice are embryonic lethal, while heterozygous animals display a cardiac phenotype and show many changes in alternative splicing of muscle development-related genes [115]. ...
In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today. In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP? Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process. In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription.
... Previous studies have demonstrated that some RBPs have strong tumor-promoting functions and thus can be used as a cancer therapeutic target. However, RBPs have essential functions in non-cancerous cells, such as tissue formation and repair [42]. Thus, adverse effects are worrisome when RBPs are applied in cancer treatment. ...
MicroRNAs (miRNAs) are single-stranded short-chain RNAs that are endogenously expressed in vertebrates; they are considered the fine-tuners of cellular protein expression that act by modifying mRNA translation. miRNAs control tissue development and differentiation, cell growth, and apoptosis in cancer and non-cancer cells. Aberrant regulation of miRNAs is involved in the pathogenesis of various diseases including cancer. Numerous investigations have shown that the changes in cellular miRNA expression in cancerous tissues and extracellular miRNAs enclosed in exosomes are correlated with cancer prognosis. Therefore, miRNAs can be used as cancer biomarkers and therapeutic targets for cancer in clinical applications. In the previous decade, miRNAs have been shown to regulate cellular functions by directly binding to proteins and mRNAs, thereby controlling cancer progression. This regulatory system implies that cancer-associated miRNAs can be applied as molecular-targeted therapy. This review discusses the roles of miRNA–protein systems in cancer progression and its future applications in cancer treatment.
... Some of these terms establish an important role for hnRNP A1 in neuronal functioning, such as regulation of neuron projection, regulation of neurogenesis and regulation of neurotransmitter transport, while others, such as nervous system development, confirm previous findings. For example, previous literature has demonstrated embryonic lethality in hnRNP A1 knock-out mice, confirming the importance of hnRNP A1 in nervous system development (Liu et al., 2017). However, the effects of hnRNP A1 knock-down on axonogenesis and neuron projection have not been explored. ...
Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA binding protein (RBP) that is localized within neurons and plays crucial roles in RNA metabolism. Its importance in neuronal functioning is underscored from the study of its pathogenic features in many neurodegenerative diseases where neuronal hnRNP A1 is mislocalized from the nucleus to the cytoplasm resulting in loss of hnRNP A1 function. Here, we model hnRNP A1 loss-of-function by siRNA mediated knockdown in differentiated Neuro-2a cells. Through RNA sequencing (RNA-seq) followed by gene ontology (GO) analyses, we show that hnRNP A1 is involved in important biological processes, including RNA metabolism, neuronal function, neuronal morphology, neuronal viability, and stress granule (SG) formation. We further confirmed several of these roles by showing that hnRNP A1 knockdown results in a reduction of neurite outgrowth, increase in cell cytotoxicity and changes in SG formation. In summary, these findings indicate that hnRNP A1 loss-of-function contributes to neuronal dysfunction and cell death and implicates hnRNP A1 dysfunction in the pathogenesis of neurodegenerative diseases.Significance StatementHnRNP A1 plays a biologically important role in controlling gene expression and maintaining proper cellular functioning in neurons. Previous research has shown that many neurodegenerative diseases exhibit pathogenic features of hnRNP A1 dysfunction, whereby it is mislocalized from its homeostatic nuclear location to the cytoplasm resulting in loss of proper functioning. Here, we model hnRNP A1 loss-of-function in differentiated neuronal cells and show that it contributes to neuronal dysfunction and cell death. These data are important because it underscores the importance of loss-of-function models and implicates hnRNP A1 dysfunction in the pathogenesis of neurodegenerative diseases.
... The defects we observe in Hrb87F-IR, a Drosophila homolog of the hnRNPA family, are reminiscent of phenotypes in hnRNPA1 knockout mice. These mice are embryonic lethal due to defects in heart and skeletal muscle, most prominently aberrant splicing of both muscle structural genes and key regulators of myofiber differentiation including mef2c, notch4, Myo5a, Myo6, Pax6, and Foxo3, among others [131]. Conditional knockout of hnRNPA1 in mouse muscle promotes insulin resistance and more generally disrupts glucose metabolism [132], a parallel to GO enrichments for metabolic terms we see in DE genes in Hrb87F-IR. ...
The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.
... The defects we observe in Hrb87F-IR, a Drosophila homolog of the hnRNP-A family, are reminiscent of phenotypes in hnRNP-A1 knockout mice. These mice are embryonic lethal due to defects in heart and skeletal muscle, most prominently aberrant splicing of both muscle structural genes and key regulators of myofiber differentiation including mef2c, notch4, Myo5a, Myo6, Pax6, and Foxo3, among others [104]. Conditional knockout of hnRNP-A1 in mouse muscle promotes insulin resistance and more generally disrupts glucose metabolism [105], a parallel to GO enrichments for metabolic terms we see in DE genes in Hrb87F-IR. ...
The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability between muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a useful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.
