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

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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uvCLAP: a fast, non-radioactive method to identify in vivo targets of RNA-binding proteins

10.1101/158410 ◽

2017 ◽

Cited By ~ 1

Author(s):

Daniel Maticzka ◽

Ibrahim Avsar Ilik ◽

Tugce Aktas ◽

Rolf Backofen ◽

Asifa Akhtar

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Neurological Diseases ◽

Affinity Purification ◽

Target Selection ◽

Rna Motif

AbstractRNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation and stability of mRNAs. Understanding the exact contribution of RBPs to gene regulation is crucial as many RBPs are frequently mis-regulated in several neurological diseases and certain cancers. While recently developed techniques provide binding sites of RBPs, they are labor-intensive and generally rely on radioactive labeling of RNA. With more than 1,000 RBPs in a human cell, it is imperative to develop easy, robust, reproducible and high-throughput methods to determine in vivo targets of RBPs. To address these issues we developed uvCLAP (UV crosslinking and affinity purification) as a robust, reproducible method to measure RNA-protein interactions in vivo. To test its performance and applicability we investigated binding of 15 RBPs from fly, mouse and human cells. We show that uvCLAP generates reliable and comparable data to other methods. Unexpectedly, our results show that despite their different subcellular localizations, STAR proteins (KHDRBS1-3, QKI) bind to a similar RNA motif in vivo. Consistently a point mutation (KHDRBS1Y440F) or a natural splice isoform (QKI-6) that changes the respective RBP subcellular localization, dramatically alters target selection without changing the targeted RNA motif. Combined with the knowledge that RBPs can compete and cooperate for binding sites, our data shows that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.

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SSMART: Sequence-structure motif identification for RNA-binding proteins

10.1101/287953 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alina Munteanu ◽

Neelanjan Mukherjee ◽

Uwe Ohler

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Motif ◽

Primary Sequence ◽

Sequence Structure ◽

Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

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Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

10.1101/2020.08.24.265819 ◽

2020 ◽

Author(s):

Kotaro Chihara ◽

Lars Barquist ◽

Kenichi Takasugi ◽

Naohiro Noda ◽

Satoshi Tsuneda

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Chronic Infections ◽

Uv Crosslinking ◽

Rna Interaction

ABSTRACTPosttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5’UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa.IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.

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The kinetic landscape of an RNA binding protein in cells

10.1101/2020.05.11.089102 ◽

2020 ◽

Author(s):

Deepak Sharma ◽

Leah L. Zagore ◽

Matthew M. Brister ◽

Xuan Ye ◽

Carlos E. Crespo-Hernández ◽

...

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

High Throughput Sequencing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cumulative Probability ◽

Mrna Levels ◽

Dissociation Kinetics ◽

The Impact ◽

In Cells

ABSTRACTGene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions 2. However, these kinetics were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how previously inaccessible, kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.

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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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rec-Y2H matrix screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

10.1101/2020.09.14.296160 ◽

2020 ◽

Author(s):

Benjamin Lang ◽

Jae-Seong Yang ◽

Mireia Garriga-Canut ◽

Silvia Speroni ◽

Maria Gili ◽

...

Keyword(s):

Protein Interactions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Interaction Networks ◽

Sequence Motifs ◽

Protein Protein Interactions ◽

Binding Interaction ◽

Transcriptional Gene Regulation

AbstractRNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory possibilities. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling > 600 K interactions. We discovered 1994 new interactions and demonstrate that interacting RBPs bind RNAs adjacently in vivo. We further find that the mRNA binding region and motif preferences of RBPs can deviate, depending on their adjacently binding interaction partners. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that splicing impairing RBP mutations observed in cancer rewire spliceosomal interaction networks.Graphical abstract

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Transcriptome-wide profiles of circular RNA and RNA binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

10.1101/2020.03.19.997478 ◽

2020 ◽

Author(s):

Trine Line Hauge Okholm ◽

Shashank Sathe ◽

Samuel S. Park ◽

Andreas Bjerregaard Kamstrup ◽

Asta Mannstaedt Rasmussen ◽

...

Keyword(s):

Bladder Cancer ◽

Protein Interactions ◽

Binding Sites ◽

Target Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Circular Rna ◽

Circular Rnas ◽

Cancer Genes

AbstractCircular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA binding proteins (RBPs), however, little is known about the prevalence and strength of these interactions in different biological contexts. Here, we comprehensively evaluate the interplay between circRNAs and RBPs in the ENCODE cell lines, HepG2 and K562, by profiling the expression of circRNAs in fractionated total RNA-sequencing samples and analyzing binding sites of 150 RBPs in large eCLIP data sets. We show that KHSRP binding sites are enriched in flanking introns of circRNAs in both HepG2 and K562 cells, and that KHSRP depletion affects circRNA biogenesis. Additionally, we show that exons forming circRNAs are generally enriched with RBP binding sites compared to non-circularizing exons. To detect individual circRNAs with regulatory potency, we computationally identify circRNAs that are highly covered by RBP binding sites and experimentally validate circRNA-RBP interactions by RNA immunoprecipitations. We characterize circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, which is covered with GRWD1 binding sites. We confirm that circCDYL binds GRWD1 in vivo and functionally characterizes the effect of circCDYL-GRWD1 interactions on target genes in HepG2. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g. TP53 and MYC. Finally, we show that elevated levels of highly RBP-covered circRNAs, including circCDYL, are associated with overall survival of bladder cancer patients. Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

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Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Journal of Bacteriology ◽

10.1128/jb.00343-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2458-2469 ◽

Cited By ~ 14

Author(s):

Kayley H. Schulmeyer ◽

Manisha R. Diaz ◽

Thomas B. Bair ◽

Wes Sanders ◽

Cindy J. Gode ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Posttranscriptional Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Consensus Sequence ◽

Stem Loop ◽

Content Type

ABSTRACTCsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level.Pseudomonas aeruginosahas a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed withtssA1, anin vivoregulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent.IMPORTANCEThe CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis.P. aeruginosahas two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution toP. aeruginosaphysiology and virulence.

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Predicting in vivo binding sites of RNA-binding proteins using mRNA secondary structure

RNA ◽

10.1261/rna.2017210 ◽

2010 ◽

Vol 16 (6) ◽

pp. 1096-1107 ◽

Cited By ~ 113

Author(s):

X. Li ◽

G. Quon ◽

H. D. Lipshitz ◽

Q. Morris

Keyword(s):

Secondary Structure ◽

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Secondary Structure ◽

In Vivo Binding

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Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.5898 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5898-5909 ◽

Cited By ~ 102

Author(s):

R Stripecke ◽

C C Oliveira ◽

J E McCarthy ◽

M W Hentze

Keyword(s):

Binding Sites ◽

Untranslated Region ◽

Translational Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Yeast Cells ◽

General Mechanism ◽

Translational Repressor

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

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