High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy.

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Functional characterization of splicing regulatory elements

10.1101/2021.05.14.444228 ◽

2021 ◽

Author(s):

Scott I Adamson ◽

Lijun Zhan ◽

Brenton R Graveley

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Functional Characterization ◽

Regulatory Elements ◽

Small Subset ◽

General Sequence ◽

Splicing Regulators ◽

Splicing Regulatory Elements

Background: RNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high-throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms, and in particular, the mediating RNA binding proteins, underlying these associations. Results: Here we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins. We identify features which make splicing events more sensitive to depletion of RNA binding proteins, as well as which RNA binding proteins act as splicing regulators sensitive to depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RNA binding proteins which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied. Conclusions: This study identifies sequence and other features underlying splicing regulation mediated specific RNA binding proteins, as well as validates and identifies other potentially important regulators of splicing in other large transcriptomic datasets.

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A Deep Boosting Based Approach for Capturing the Sequence Binding Preferences of RNA-Binding Proteins from High-Throughput CLIP-Seq Data

10.1101/086421 ◽

2016 ◽

Cited By ~ 1

Author(s):

Shuya Li ◽

Fanghong Dong ◽

Yuexin Wu ◽

Sai Zhang ◽

Chen Zhang ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Binding Proteins ◽

Rna Binding ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

False Negative ◽

Functional Roles ◽

Potential Applications ◽

Validation Tests

AbstractCharacterizing the binding behaviors of RNA-binding proteins (RBPs) is important for understanding their functional roles in gene expression regulation. However, current high-throughput experimental methods for identifying RBP targets, such as CLIP-seq and RNAcompete, usually suffer from the false positive and false negative issues. Here, we develop a deep boosting based machine learning approach, called DeBooster, to accurately model the binding sequence preferences and identify the corresponding binding targets of RBPs from CLIP-seq data. Comprehensive validation tests have shown that DeBooster can outperform other state-of-the-art approaches in predicting RBP targets and recover false negatives that are common in current CLIP-seq data. In addition, we have demonstrated several new potential applications of DeBooster in understanding the regulatory functions of RBPs, including the binding effects of the RNA helicase MOV10 on mRNA degradation, the influence of different binding behaviors of the ADAR proteins on RNA editing, as well as the antagonizing effect of RBP binding on miRNA repression. Moreover, DeBooster may provide an effective index to investigate the effect of pathogenic mutations in RBP binding sites, especially those related to splicing events. We expect that DeBooster will be widely applied to analyze large-scale CLIP-seq experimental data and can provide a practically useful tool for novel biological discoveries in understanding the regulatory mechanisms of RBPs.

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Translational regulation of ANG II type 1 receptors in proliferating vascular smooth muscle cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00448.2005 ◽

2006 ◽

Vol 290 (1) ◽

pp. R50-R56 ◽

Cited By ~ 5

Author(s):

Sunghou Lee ◽

Hong Ji ◽

Zheng Wu ◽

Wei Zheng ◽

Ali Hassan ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Muscle Cells ◽

Distribution Analysis ◽

Exon 2

The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT1aR) densities (Bmax) increased by 30% between 5 and 7 days in culture [Bmax (fmol/mg protein): day 5, 379 ± 8.4 vs. day 7, 481 ± 12, n = 3, P < 0.05] under conditions in which no significant changes in AT1aR mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 ± 0.01 vs. day 7, 0.24 ± 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 ± 0.14 vs. day 7, 0.33 ± 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT1aR mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT1aR mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 ± 9.9 vs. day 7, 56.8 ± 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5′ leader sequence (5′LS) of the AT1aR [5′LS RPC (AU): day 5, 0.62 ± 0.15 vs. day 7, 0.23 ± 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 ± 5.7 vs. day 7, 17.2 ± 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT1aR expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5′LS of the AT1aR mRNA.

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Functional reconstruction of NANOS3 expression in the germ cell lineage by a novel transgenic reporter reveals distinct subcellular localizations of NANOS3

Reproduction ◽

10.1530/rep-09-0373 ◽

2010 ◽

Vol 139 (2) ◽

pp. 381-393 ◽

Cited By ~ 10

Author(s):

Masashi Yamaji ◽

Takashi Tanaka ◽

Mayo Shigeta ◽

Shinichiro Chuma ◽

Yumiko Saga ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Primordial Germ Cells ◽

Cell Lineage ◽

Regulatory Elements ◽

Initiation Factor ◽

Processing Bodies

Mutations of RNA-binding proteins such as NANOS3, TIAL1, and DND1 in mice have been known to result in the failure of survival and/or proliferation of primordial germ cells (PGCs) soon after their fate is specified (around embryonic day (E) 8.0), leading to the infertility of these animals. However, the mechanisms of actions of these RNA-binding proteins remain largely unresolved. As a foundation to explore the role of these RNA-binding proteins in germ cells, we established a novel transgenic reporter strain that expresses NANOS3 fused with EGFP under the control of Nanos3 regulatory elements. NANOS3–EGFP exhibited exclusive expression in PGCs as early as E7.25, and continued to be expressed in female germ cells until around E14.5 and in male germ cells throughout the fetal period with declining expression levels after E16.5. NANOS3–EGFP resumed strong expression in postnatal spermatogonia and continued to be expressed in undifferentiated spermatogonial cells in adults. Importantly, the Nanos3–EGFP transgene rescued the sterile phenotype of Nanos3 homozygous mutants, demonstrating the functional equivalency of NANOS3–EGFP with endogenous NANOS3. We found that throughout germ cell development, a predominant amount of NANOS3–EGFP co-localized with TIAL1 (also known as TIAR) and phosphorylated eukaryotic initiation factor 2α, markers for the stress granules, whereas a fraction of it showed co-localization with DCP1A, a marker for the processing bodies. On the other hand, NANOS3–EGFP did not co-localize with Tudor domain-containing protein 1, a marker for the intermitochondrial cements, in spermatogenic cells. These findings unveil the presence of distinct posttranscriptional regulations in PGCs soon after their specification, for which RNA-binding proteins such as NANOS3 and TIAL1 would play critical functions.

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Transcriptome-wide high-throughput mapping of protein–RNA occupancy profiles using POP-seq

Scientific Reports ◽

10.1038/s41598-020-80846-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mansi Srivastava ◽

Rajneesh Srivastava ◽

Sarath Chandra Janga

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Interaction Network ◽

K562 Cells ◽

Genomic Variation ◽

Interaction Sites ◽

Protein Occupancy ◽

Rna Interaction

AbstractInteraction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein–RNA complexes and poly-A pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non poly-A RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of poly-A pulldown. Our study demonstrates that ~ 68% of the total POP-seq peaks exhibited an overlap with publicly available protein–RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein–RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment (p value < 2.2e−16) for GWAS SNPs, phenotypic, clinically relevant germline as well as somatic variants reported in cancer genomes, suggesting the prevalence of uncharacterized genomic variation in protein occupied sites on RNA. We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein–RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.

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StoatyDive: Evaluation and classification of peak profiles for sequencing data

GigaScience ◽

10.1093/gigascience/giab045 ◽

2021 ◽

Vol 10 (6) ◽

Author(s):

Florian Heyl ◽

Rolf Backofen

Keyword(s):

Quality Control ◽

High Throughput ◽

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Typical Result ◽

Sequence Motif ◽

Peak Shape ◽

Sequencing Data

Abstract Background The prediction of binding sites (peak-calling) is a common task in the data analysis of methods such as cross-linking immunoprecipitation in combination with high-throughput sequencing (CLIP-Seq). The predicted binding sites are often further analyzed to predict sequence motifs or structure patterns. When looking at a typical result of such high-throughput experiments, the obtained peak profiles differ largely on a genomic level. Thus, a tool is missing that evaluates and classifies the predicted peaks on the basis of their shapes. We hereby present StoatyDive, a tool that can be used to filter for specific peak profile shapes of sequencing data such as CLIP. Findings With StoatyDive we are able to classify peak profile shapes from CLIP-seq data of the histone stem-loop-binding protein (SLBP). We compare the results to existing tools and show that StoatyDive finds more distinct peak shape clusters for CLIP data. Furthermore, we present StoatyDive’s capabilities as a quality control tool and as a filter to pick different shapes based on biological or technical questions for other CLIP data from different RNA binding proteins with different biological functions and numbers of RNA recognition motifs. We finally show that proteins involved in splicing, such as RBM22 and U2AF1, have potentially sharper-shaped peaks than other RNA binding proteins. Conclusion StoatyDive finally fills the demand for a peak shape clustering tool for CLIP-Seq data that fine-tunes downstream analysis steps such as structure or sequence motif predictions and that acts as a quality control.

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HuR and other turnover- and translation-regulatory RNA-binding proteins: implications for the kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00270.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. F569-F576 ◽

Cited By ~ 15

Author(s):

Rudolf Pullmann ◽

Hamid Rabb

Keyword(s):

Posttranscriptional Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Current Knowledge ◽

Regulation Of Gene Expression ◽

Regulatory Elements ◽

Matrix Remodeling ◽

Acid Base Balance ◽

Renal Metabolism

The posttranscriptional regulation of gene expression occurs through cis RNA regulatory elements by the action of trans factors, which are represented by noncoding RNAs (especially microRNAs) and turnover- and translation-regulatory (TTR) RNA-binding proteins (RBPs). These multifactorial proteins are a group of heterogeneous RBPs primarily implicated in controlling the decay and translation rates of target mRNAs. TTR-RBPs usually shuttle between cellular compartments (the nucleus and cytoplasm) in response to various stimuli and undergo posttranslational modifications such as phosphorylation or methylation to ensure their proper subcellular localization and function. TTR-RBPs are emerging as key regulators of a wide variety of genes influencing kidney physiology and pathology. This review summarizes the current knowledge of TTR-RBPs that influence renal metabolism. We will discuss the role of TTR-RBPs as regulators of kidney ischemia, fibrosis and matrix remodeling, angiogenesis, membrane transport, immunity, vascular tone, hypertension, and acid-base balance as well as anemia, bone mineral disease, and vascular calcification.

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FLASH: ultra-fast protocol to identify RNA–protein interactions in cells

Nucleic Acids Research ◽

10.1093/nar/gkz1141 ◽

2019 ◽

Vol 48 (3) ◽

pp. e15-e15 ◽

Cited By ~ 1

Author(s):

Ibrahim Avsar Ilik ◽

Tugce Aktas ◽

Daniel Maticzka ◽

Rolf Backofen ◽

Asifa Akhtar

Keyword(s):

High Throughput ◽

Protein Interactions ◽

Binding Sites ◽

Binding Proteins ◽

High Throughput Sequencing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification ◽

Sequencing Library

Abstract Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein–RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.

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RNA-Binding Proteins at the Host-Pathogen Interface Targeting Viral Regulatory Elements

Viruses ◽

10.3390/v13060952 ◽

2021 ◽

Vol 13 (6) ◽

pp. 952

Author(s):

Azman Embarc-Buh ◽

Rosario Francisco-Velilla ◽

Encarnacion Martinez-Salas

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Viruses ◽

Regulatory Elements ◽

Host Cells ◽

Viral Rnas ◽

Positive Strand ◽

Multiplication Cycle ◽

Positive Strand Rna

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.

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RBPTD: a database of cancer-related RNA-binding proteins in humans

Database ◽

10.1093/database/baz156 ◽

2020 ◽

Vol 2020 ◽

Cited By ~ 1

Author(s):

Kun Li ◽

Zhi-Wei Guo ◽

Xiang-Ming Zhai ◽

Xue-Xi Yang ◽

Ying-Song Wu ◽

...

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Sequencing Data ◽

Dna Copy Number Variation ◽

Expression Of Genes ◽

Number Variation

Abstract RNA-binding proteins (RBPs) play important roles in regulating the expression of genes involved in human physiological and pathological processes, especially in cancers. Many RBPs have been found to be dysregulated in cancers; however, there was no tool to incorporate high-throughput data from different dimensions to systematically identify cancer-related RBPs and to explore their causes of abnormality and their potential functions. Therefore, we developed a database named RBPTD to identify cancer-related RBPs in humans and systematically explore their functions and abnormalities by integrating different types of data, including gene expression profiles, prognosis data and DNA copy number variation (CNV), among 28 cancers. We found a total of 454 significantly differentially expressed RBPs, 1970 RBPs with significant prognostic value, and 53 dysregulated RBPs correlated with CNV abnormality. Functions of 26 cancer-related RBPs were explored by analysing high-throughput RNA sequencing data obtained by crosslinking immunoprecipitation, and the remaining RBP functions were predicted by calculating their correlation coefficient with other genes. Finally, we developed the RBPTD for users to explore functions and abnormalities of cancer-related RBPs to improve our understanding of their roles in tumorigenesis. Database URL: http: //www.rbptd.com

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