scholarly journals Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

2019 ◽  
Author(s):  
Deanna M Tiek ◽  
Roham Razaghi ◽  
Lu Jin ◽  
Norah Sadowski ◽  
Carla Alamillo-Ferrer ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Exon Skipping ◽  
Standard Of Care ◽  
Secondary Structures ◽  
Preliminary Evidence ◽  
Clinical Samples ◽  
Sr Protein ◽  
Resistant Cells

AbstractTemozolomide (TMZ) is a chemotherapy agent that adds mutagenic adducts to guanine, and is first-line standard of care for the aggressive brain cancer glioblastoma (GBM). Methyl guanine methyl transferase (MGMT) is a DNA repair enzyme that can remove O6-methyl guanine adducts prior to the development of catastrophic mutations, and is associated with TMZ resistance. However, inhibition of MGMT fails to reverse TMZ resistance. Guanines are essential nucleotides in many DNA and RNA secondary structures. In several neurodegenerative diseases (NDs), disruption of these secondary structures is pathogenic. We therefore took a structural view of TMZ resistance, seeking to establish the role of guanine mutations in disrupting critical nucleotide secondary structures. To test whether these have functional impacts on TMZ-resistant GBM, we focused on two specific guanine-rich regions: G-quadruplexes (G4s) and splice sites. Here we report broad sequence- and conformation-based changes in G4s in acquired or intrinsic TMZ resistant vs. sensitive GBM cells, accompanied by nucleolar stress and enrichment of nucleolar RNA:DNA hybrids (r-loops). We further show widespread splice-altering mutations, exon skipping, and deregulation of splicing-regulatory serine/arginine rich (SR) protein phosphorylation in TMZ-resistant GBM cells. The G4-stabilizing ligand TMPyP4 and a novel inhibitor of cdc2-like kinases (CLKs) partially normalize G4 structure and SR protein phosphorylation, respectively, and are preferentially growth-inhibitory in TMZ-resistant cells. Lastly, we report that the G4- and RNA-binding protein EWSR1 forms aberrant cytoplasmic aggregates in response to acute TMZ treatment, and these aggregates are abundant in TMZ resistant cells. Preliminary evidence suggests these cytoplasmic EWSR1 aggregates are also present in GBM clinical samples. This work supports altered nucleotide secondary structure and splicing deregulation as pathogenic features of TMZ-resistant GBM. It further positions cytoplasmic aggregation of EWSR1 as a potential indicator for TMZ resistance, establishes the possibility of successful intervention with splicing modulatory or G4-targeting agents, and provides a new context in which to study aggregating RNA binding proteins.

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 An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

2021 ◽  
Author(s):  
Dong Cao
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Exon Skipping ◽  
Circular Rna ◽  
Circular Rnas ◽  
Rna Regulation ◽  
Fused In Sarcoma ◽  
Specific Factors ◽  
Isoform B

Circular RNAs (circRNAs) are always expressed tissue-specifically, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in thirteen conserved RBPs. Among them, loss of FUST-1, the homolog of FUS (Fused in Sarcoma), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Further, I showed that FUST-1 regulates circRNA formation without affecting the levels of the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different N-terminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation.

scholarly journals Systematic Identification of Protein Phosphorylation-Mediated Interactions

2020 ◽  
Author(s):  
Brendan M. Floyd ◽  
Kevin Drew ◽  
Edward M. Marcotte
Keyword(s):  
Mass Spectrometry ◽  
Protein Phosphorylation ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Protein ◽  
Mass Spectrometry Data ◽  
Size Exclusion ◽  
Functional Protein

ABSTRACTProtein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies.

scholarly journals LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation

2019 ◽  
Author(s):  
Kensuke Ninomiya ◽  
Shungo Adachi ◽  
Tohru Natsume ◽  
Junichi Iwakiri ◽  
Goro Terai ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intron Retention ◽  
Nuclear Bodies ◽  
Splicing Factors ◽  
Stress Recovery ◽  
Stress Exposure ◽  
Sr Protein ◽  
Retained Introns

AbstractA number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly dephosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure.

scholarly journals Dynamic Variations of 3′UTR Length Reprogram the mRNA Regulatory Landscape

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1560
Author(s):  
Estanislao Navarro ◽  
Adrián Mallén ◽  
Miguel Hueso
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Exon Skipping ◽  
Alternative Polyadenylation ◽  
Point Of View ◽  
Cleavage Sites ◽  
Dynamic Variations ◽  
Human Disorders ◽  
Functional Features ◽  
Control Stability

This paper concerns 3′-untranslated regions (3′UTRs) of mRNAs, which are non-coding regulatory platforms that control stability, fate and the correct spatiotemporal translation of mRNAs. Many mRNAs have polymorphic 3′UTR regions. Controlling 3′UTR length and sequence facilitates the regulation of the accessibility of functional effectors (RNA binding proteins, miRNAs or other ncRNAs) to 3′UTR functional boxes and motifs and the establishment of different regulatory landscapes for mRNA function. In this context, shortening of 3′UTRs would loosen miRNA or protein-based mechanisms of mRNA degradation, while 3′UTR lengthening would strengthen accessibility to these effectors. Alterations in the mechanisms regulating 3′UTR length would result in widespread deregulation of gene expression that could eventually lead to diseases likely linked to the loss (or acquisition) of specific miRNA binding sites. Here, we will review the mechanisms that control 3′UTR length dynamics and their alterations in human disorders. We will discuss, from a mechanistic point of view centered on the molecular machineries involved, the generation of 3′UTR variability by the use of alternative polyadenylation and cleavage sites, of mutually exclusive terminal alternative exons (exon skipping) as well as by the process of exonization of Alu cassettes to generate new 3′UTRs with differential functional features.

scholarly journals Computational Discovery of Evolutionary Conserved Enterovirus 71 RNA Secondary Structures

2018 ◽  
Author(s):  
Nathan Fridlyand ◽  
Alexander N Lukashev ◽  
Andrey Chursov ◽  
Franco M Venanzi ◽  
Jonathan W Yewdell ◽  
...  
Keyword(s):  
Rna Structure ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Enterovirus 71 ◽  
Secondary Structures ◽  
Rna Structures ◽  
Alternative Structures ◽  
Evolutionarily Conserved ◽  
Computational Discovery ◽  
Rna Switch

Viral RNAs store information in their sequence and structure. Little, however is known about the contribution of RNA structure to viral replication and evolution. We previously described RNA ISRAEU to computationally identify structured regions in viral RNA based on evolutionary conservation of predicted structures. Here, we apply RNA GUESS (Generator of Uniformly Evolved Secondary Structures) to better understand enterovirus 71, a highly pathogenic human picornavirus. RNA GUESS identified previously known evolutionarily conserved picornavirus RNA structures, and uniquely predicted RNA structures with significant structural conservation despite the lack of an obvious sequence conservation. One structure consists of a stretch of obligatorily unpaired nucleotides that can potentially interact with RNA-binding proteins and siRNAs. Another, forms a potential RNA switch that can form two alternative structures while the third and fourth constitute hairpin-like structures. These findings provide a launching point for physical and genetic studies regarding the function and structures of these RNA elements.

scholarly journals Deep neural networks for inferring binding sites of RNA-binding proteins by using distributed representations of RNA primary sequence and secondary structure

BMC Genomics ◽  
2020 ◽  
Vol 21 (S13) ◽  
Author(s):  
Lei Deng ◽  
Youzhi Liu ◽  
Yechuan Shi ◽  
Wenhao Zhang ◽  
Chun Yang ◽  
...  
Keyword(s):  
Neural Networks ◽  
Secondary Structure ◽  
Binding Sites ◽  
Large Scale ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Secondary Structures ◽  
Rna Sequences ◽  
Distributed Representations

Abstract Background RNA binding proteins (RBPs) play a vital role in post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA transport, and modulation of mRNA translation and decay. The identification of RBP binding sites is a crucial step in understanding the biological mechanism of post-transcriptional gene regulation. However, the determination of RBP binding sites on a large scale is a challenging task due to high cost of biochemical assays. Quite a number of studies have exploited machine learning methods to predict binding sites. Especially, deep learning is increasingly used in the bioinformatics field by virtue of its ability to learn generalized representations from DNA and protein sequences. Results In this paper, we implemented a novel deep neural network model, DeepRKE, which combines primary RNA sequence and secondary structure information to effectively predict RBP binding sites. Specifically, we used word embedding algorithm to extract features of RNA sequences and secondary structures, i.e., distributed representation of k-mers sequence rather than traditional one-hot encoding. The distributed representations are taken as input of convolutional neural networks (CNN) and bidirectional long-term short-term memory networks (BiLSTM) to identify RBP binding sites. Our results show that deepRKE outperforms existing counterpart methods on two large-scale benchmark datasets. Conclusions Our extensive experimental results show that DeepRKE is an efficacious tool for predicting RBP binding sites. The distributed representations of RNA sequences and secondary structures can effectively detect the latent relationship and similarity between k-mers, and thus improve the predictive performance. The source code of DeepRKE is available at https://github.com/youzhiliu/DeepRKE/.

scholarly journals RGS4 RNA Secondary Structure Mediates Staufen2 RNP Assembly in Neurons

2021 ◽  
Vol 22 (23) ◽  
pp. 13021
Author(s):  
Sandra M. Fernández-Moya ◽  
Janina Ehses ◽  
Karl E. Bauer ◽  
Rico Schieweck ◽  
Anob M. Chakrabarti ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Secondary Structure ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Underlying Mechanism ◽  
Secondary Structures ◽  
Rna Granules ◽  
Rna Hairpins ◽  
Sequence Elements ◽  
Rna Hairpin

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3′-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

scholarly journals Modeling RNA-binding protein specificity in vivo by precisely registering protein-RNA crosslink sites

2018 ◽  
Author(s):  
Huijuan Feng ◽  
Suying Bao ◽  
Sebastien M. Weyn-Vanhentenryck ◽  
Aziz Khan ◽  
Justin Wong ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Specificity ◽  
Computational Method ◽  
Sr Protein ◽  
Genome Wide ◽  
Sequence Elements ◽  
Nucleotide Resolution

AbstractRNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence elements in their target transcripts. Despite the expanding list of RBPs with in vivo binding sites mapped genomewide using crosslinking and immunoprecipitation (CLIP), defining precise RBP binding specificity remains challenging. We previously demonstrated that the exact protein-RNA crosslink sites can be mapped using CLIP data at single-nucleotide resolution and observed that crosslinking frequently occurs at specific positions in RBP motifs. Here we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the resulting motifs by genome-wide analysis of allelic binding sites also detected by CLIP. We found that the prototypical SR protein SRSF1 recognizes GGA clusters to regulate splicing in a much larger repertoire of transcripts than previously appreciated.

scholarly journals Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

2021 ◽  
Author(s):  
Sean R Kundinger ◽  
Eric B Dammer ◽  
Luming Yin ◽  
Cheyenne Hurst ◽  
Lingyan Ping ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Solubility ◽  
Immunocytochemical Staining ◽  
Sr Protein ◽  
Nuclear Extracts

Post-translational modifications (PTMs) within splicing factor RNA-binding proteins (RBPs), such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Notably, the arginine-/serine-rich (RS) domains in SR proteins are densely modified by phosphorylation compared with the remainder of the proteome. Previously, we showed that dephosphorylation of SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich to the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. Phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high molecular weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine-/arginine protein kinase 2 (SRPK2) in vitro prevented high molecular weight SRSF2 species formation. Furthermore, we pharmacologically inhibited SRPKs in mammalian cells and observed increased cytoplasmic granules as well as the formation of cytoplasmic SRSF2 tubular structures that associate with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

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