Effects Of RNA-binding Proteins Research Articles

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Effect of RNA-binding proteins on osteogenic differentiation of bone marrow mesenchymal stem cells.

Tissue regeneration mediated by mesenchymal stem cells (MSCs) is an ideal way to repair bone defects. RNA-binding proteins (RBPs) can affect cell function through post-transcriptional regulation. Exploring the role of RBPs in the process of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is helpful to find a key method to promote the osteogenic efficiency of BMSCs. By reviewing the literature, we obtained a differentially expressed mRNA dataset during the osteogenic differentiation of BMSCs and a human RBP dataset. A total of 82 differentially expressed RBPs in the osteogenic differentiation of BMSCs were screened by intersection of the two datasets. Functional analysis showed that the differentially expressed RBPs were mainly involved in RNA transcription, translation and degradation through the formation of spliceosomes and ribonucleoprotein complexes. The top 15 RBPs determined by degree score were FBL, NOP58, DDX10, RPL9, SNRPD3, NCL, IFIH1, RPL18A, NAT10, EXOSC5, ALYREF, PA2G4, EIF5B, SNRPD1 and EIF6. The results of this study demonstrate that the expression of many RBPs changed during osteogenic differentiation of BMSCs.

Enhanced gene regulation by cooperation between mRNA decay and gene transcription

It has become increasingly clear in the last few years that gene expression in eukaryotes is not a linear process from mRNA synthesis in the nucleus to translation and degradation in the cytoplasm, but works as a circular one where the mRNA level is controlled by crosstalk between nuclear transcription and cytoplasmic decay pathways. One of the consequences of this crosstalk is the approximately constant level of mRNA. This is called mRNA buffering and happens when transcription and mRNA degradation act at compensatory rates. However, if transcription and mRNA degradation act additively, enhanced gene expression regulation occurs. In this work, we analyzed new and previously published genomic datasets obtained for several yeast mutants related to either transcription or mRNA decay that are not known to play any role in the other process. We show that some, which were presumed only transcription factors (Sfp1) or only decay factors (Puf3, Upf2/3), may represent examples of RNA-binding proteins (RBPs) that make specific crosstalk to enhance the control of the mRNA levels of their target genes by combining additive effects on transcription and mRNA stability. These results were mathematically modeled to see the effects of RBPs when they have positive or negative effects on mRNA synthesis and decay rates. We found that RBPs can be an efficient way to buffer or enhance gene expression responses depending on their respective effects on transcription and mRNA stability.

The predictive value of RNA binding proteins in colon adenocarcinoma

RNA binding proteins (RBPs) play an important role in regulating post-transcriptional gene expression and have been reported to be closely associated with the occurrence and development of tumors. However, the effect of RBPs in colon cancer remains unclear. We downloaded clinical information and transcriptome data of colon adenocarcinoma (COAD) from The Cancer Genome Atlas database (TCGA) database. After combining this data, we identified differentially expressed RBPs in normal and cancer tissues and subsequently performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Prognosis-related RBPs were identified via Cox regression analysis. The samples were randomly divided into two groups; an experimental group and a control group. A predictive model was constructed by dividing the experimental group into high- and low-risk subgroups based on the scores of the prognostic-related RBPs, and the prognosis of samples in these two subgroups was compared. Then, this model was applied to the control group. Finally, the model results were verified based on an online survival database and the Human Protein Atlas (HPA) database. A total of 469 differentially expressed RBPs were identified in normal and cancer tissues. Ten prognosis-related RBPs were determined by Cox regression analysis. In the prognostic prediction model, the prognosis of high-risk patients in the experimental group was worse than that in the low-risk group, and the same result was obtained in the control group. In addition, the risk score in the Cox regression analysis showed that the model could be used as an independent prognostic factor (P<0.001). The results of the online survival analysis tool, HPA database, and the model were consistent. Some specific RBPs are significantly associated with the prognosis of patients with COAD, and this finding may provide important information for the future diagnosis and treatment of patients with COAD.

Amyotrophic Lateral Sclerosis: Molecular Mechanisms, Biomarkers, and Therapeutic Strategies.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with the progressive loss of motor neurons, leading to a fatal paralysis. According to whether there is a family history of ALS, ALS can be roughly divided into two types: familial and sporadic. Despite decades of research, the pathogenesis of ALS is still unelucidated. To this end, we review the recent progress of ALS pathogenesis, biomarkers, and treatment strategies, mainly discuss the roles of immune disorders, redox imbalance, autophagy dysfunction, and disordered iron homeostasis in the pathogenesis of ALS, and introduce the effects of RNA binding proteins, ALS-related genes, and non-coding RNA as biomarkers on ALS. In addition, we also mention other ALS biomarkers such as serum uric acid (UA), cardiolipin (CL), chitotriosidase (CHIT1), and neurofilament light chain (NFL). Finally, we discuss the drug therapy, gene therapy, immunotherapy, and stem cell-exosomal therapy for ALS, attempting to find new therapeutic targets and strategies. A challenge is to study the various mechanisms of ALS as a syndrome. Biomarkers that have been widely explored are indispensable for the diagnosis, treatment, and prevention of ALS. Moreover, the development of new genes and targets is an urgent task in this field.

Effects of RNA Binding Proteins on the Prognosis and Malignant Progression in Prostate Cancer.

Prostate cancer (PCa) is a common lethal malignancy in men. RNA binding proteins (RBPs) have been proven to regulate the biological processes of various tumors, but their roles in PCa remain less defined. In the present study, we used bioinformatics analysis to identify RBP genes with prognostic and diagnostic values. A total of 59 differentially expressed RBPs in PCa were obtained, comprising 28 upregulated and 31 downregulated RBP genes, which may play important roles in PCa. Functional enrichment analyses showed that these RBPs were mainly involved in mRNA processing, RNA splicing, and regulation of RNA splicing. Additionally, we identified nine RBP genes (EXO1, PABPC1L, REXO2, MBNL2, MSI1, CTU1, MAEL, YBX2, and ESRP2) and their prognostic values by a protein–protein interaction network and Cox regression analyses. The expression of these nine RBPs was validated using immunohistochemical staining between the tumor and normal samples. Further, the associations between the expression of these nine RBPs and pathological T staging, Gleason score, and lymph node metastasis were evaluated. Moreover, these nine RBP genes showed good diagnostic values and could categorize the PCa patients into two clusters with different malignant phenotypes. Finally, we constructed a prognostic model based on these nine RBP genes and validated them using three external datasets. The model showed good efficiency in predicting patient survival and was independent of other clinical factors. Therefore, our model could be used as a supplement for clinical factors to predict patient prognosis and thereby improve patient survival.

Cataloguing and Selection of mRNAs Localized to Dendrites in Neurons and Regulated by RNA-Binding Proteins in RNA Granules.

Spatiotemporal translational regulation plays a key role in determining cell fate and function. Specifically, in neurons, local translation in dendrites is essential for synaptic plasticity and long-term memory formation. To achieve local translation, RNA-binding proteins in RNA granules regulate target mRNA stability, localization, and translation. To date, mRNAs localized to dendrites have been identified by comprehensive analyses. In addition, mRNAs associated with and regulated by RNA-binding proteins have been identified using various methods in many studies. However, the results obtained from these numerous studies have not been compiled together. In this review, we have catalogued mRNAs that are localized to dendrites and are associated with and regulated by the RNA-binding proteins fragile X mental retardation protein (FMRP), RNA granule protein 105 (RNG105, also known as Caprin1), Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2) in RNA granules. This review provides comprehensive information on dendritic mRNAs, the neuronal functions of mRNA-encoded proteins, the association of dendritic mRNAs with RNA-binding proteins in RNA granules, and the effects of RNA-binding proteins on mRNA regulation. These findings provide insights into the mechanistic basis of protein-synthesis-dependent synaptic plasticity and memory formation and contribute to future efforts to understand the physiological implications of local regulation of dendritic mRNAs in neurons.

Effect of RNA binding protein 6 on proliferation, migration and apoptosis of laryngeal cancer cells

Objective To investigate the effect of RNA binding protein 6 (RBM6) on the proliferation, migration and apoptosis of laryngeal cancer cells. Methods The expression of RBM6 protein was analyzed by Western blotting in 47 cases of laryngeal cancer and adjacent tissues. RBM6 overexpression cell line and control cell line (RBM6 group and control group) were constructed by RBM6 overexpression lentivirus and control lentivirus respectively. The proliferation of two groups was analyzed by cell counting kit-8 (CCK-8). The migration was analyzed in RBM6 group and control group by scratch test. The apoptosis level in RBM6 group and control group was analyzed by flow cytometry. The growth of tumor cells in vivo in RBM6 group and control group was analyzed by allogeneic tumor transplantation. SPSS 13.0 statistical software was used to analyze the measurement data. Mean±SD was used to express the measurement data, and t-test was used to compare between groups. Results As compared with the level of RBM6 protein in adjacent tissues (1.01±0.23), the expression level of RBM6 protein (0.34±0.12) in laryngeal cancer significantly decreased (t=2.913, P<0.05). As compared with the control group, the cell proliferation ability of RBM6 group significantly decreased (F=3.019, P<0.05). As compared with the control group [(84.18±8.32)%], Scratch healing rate in RBM6 group [(30.11±6.89)%] significantly enhanced (t=4.319, P<0.05). As compared with the control group [(35.61±7.01)%], the apoptosis rate in RBM6 group [(5.23±2.12)%] significantly increased (t=2.192, P<0.05). The results showed that the proliferation rate of cells in the control group was significantly higher than that in the RBM6 group (F=2.908, P<0.05). Conclusion RBM6 can inhibit the proliferation and migration of laryngeal cancer cells, and promote the apoptosis of tumor cells. Key words: RNA binding protein 6; Laryngeal cancer; Proliferation; Migration; Apoptosis

Inferring RBP-Mediated Regulation in Lung Squamous Cell Carcinoma.

RNA-binding proteins (RBPs) play key roles in post-transcriptional regulation of mRNAs. Dysregulations in RBP-mediated mechanisms have been found to be associated with many steps of cancer initiation and progression. Despite this, previous studies of gene expression in cancer have ignored the effect of RBPs. To this end, we developed a lasso regression model that predicts gene expression in cancer by incorporating RBP-mediated regulation as well as the effects of other well-studied factors such as copy-number variation, DNA methylation, TFs and miRNAs. As a case study, we applied our model to Lung squamous cell carcinoma (LUSC) data as we found that there are several RBPs differentially expressed in LUSC. Including RBP-mediated regulatory effects in addition to the other features significantly increased the Spearman rank correlation between predicted and measured expression of held-out genes. Using a feature selection procedure that accounts for the adaptive search employed by lasso regularization, we identified the candidate regulators in LUSC. Remarkably, several of these candidate regulators are RBPs. Furthermore, majority of the candidate regulators have been previously found to be associated with lung cancer. To investigate the mechanisms that are controlled by these regulators, we predicted their target gene sets based on our model. We validated the target gene sets by comparing against experimentally verified targets. Our results suggest that the future studies of gene expression in cancer must consider the effect of RBP-mediated regulation.