Myoparr-Associated and -Independent Multiple Roles of Heterogeneous Nuclear Ribonucleoprotein K during Skeletal Muscle Cell Differentiation

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2α/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and -independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

The mechanical regulation of RNA binding protein hnRNPC in the failing heart

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXβ2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXβ2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXβ2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXβ2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.

The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying reduced methamphetamine behavioral sensitivity. Mice with a heterozygous frameshift deletion in the first coding exon of Hnrnph1 showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H dysfunction with reduced methamphetamine-induced dopamine release and behavior, we surveyed the RNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine (2 mg/kg, i.p.). Methamphetamine induced opposite changes in RNA-binding targets of hnRNP H in Hnrnph1 mutants versus wild-types, including 3′UTR targets in mRNAs enriched for synaptic proteins involved in dopamine release and excitatory synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on a methamphetamine-induced upregulation of Cacna2d2 transcript and decreased 3′UTR usage in hyposensitive Hnrnph1 mutants. Our study identifies a dynamic methamphetamine-induced RNA targetome of hnRNP H that has the potential to rapidly regulate gene expression, synaptic transmission, plasticity, and behavior.

Heterogeneous Nuclear Ribonucleoprotein A1 Loads Batched Tumor-Promoting MicroRNAs Into Small Extracellular Vesicles With the Assist of Caveolin-1 in A549 Cells

MicroRNAs in small extracellular vesicle (sEV-miRNAs) have been widely investigated as crucial regulated molecules secreted by tumor cells to communicate with surroundings. It is of great significance to explore the loading mechanism of sEV-miRNAs by tumor cells. Here, we comprehensively illustrated a reasoned loading pathway of batched tumor-promoting sEV-miRNAs in non-small cell lung cancer (NSCLC) cell line A549 with the application of a multi-omics method. The protein heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was strictly selected as a powerful sEV-miRNA loading protein from miRNA-binding proteome and further verified through small RNA sequencing after hnRNPA1 silence. In terms of the mechanism, SUMOylated hnRNPA1 in sEVs was verified to control sEV-miRNA loading. Subsequently, as a scaffolding component of caveolae, caveolin-1 (CAV1) was detailedly demonstrated to assist the loading of SUMOylated hnRNPA1 and its binding miRNAs into sEVs. Inhibition of CAV1 significantly prevented SUMOylated hnRNPA1 from encapsulating into sEVs, resulting in less enrichment of sEV-miRNAs it loaded. Finally, we confirmed that hnRNPA1-loaded sEV-miRNAs could facilitate tumor proliferation and migration based on database analysis and cytological experiments. Our findings reveal a loading mechanism of batched tumor-promoting sEV-miRNAs, which may contribute to the selection of therapeutic targets for lung cancer.