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

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

BMC Genomics ◽

10.1186/s12864-020-07239-w ◽

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/.

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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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An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene

ACS Synthetic Biology ◽

10.1021/acssynbio.8b00378 ◽

2018 ◽

Vol 7 (12) ◽

pp. 2765-2774 ◽

Cited By ~ 8

Author(s):

Noa Katz ◽

Roni Cohen ◽

Oz Solomon ◽

Beate Kaufmann ◽

Orna Atar ◽

...

Keyword(s):

Reporter Gene ◽

Binding Proteins ◽

Rna Binding ◽

Binding Assay ◽

Rna Binding Proteins ◽

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.14.9.5898-5909.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 5898-5909

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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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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Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.23.19.7055-7067.2003 ◽

2003 ◽

Vol 23 (19) ◽

pp. 7055-7067 ◽

Cited By ~ 45

Author(s):

Shelly A. Waggoner ◽

Stephen A. Liebhaber

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Cell Function ◽

Rna Binding Proteins ◽

Translation Control ◽

Viral Mrnas ◽

Posttranscriptional Gene Regulation ◽

Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

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