Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes

Abstract RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on UV-crosslinking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP- and cell type-specific RNA-protein interactions for multiple RBPs and cell types in single-pooled experiments. Pairing STAMP with long-read sequencing also yields RBP target sites for full-length isoforms. Finally, conducting STAMP using small ribosomal subunits (Ribo-STAMP) allows analysis of transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

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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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Genomic sequences and RNA binding proteins predict RNA splicing kinetics in various single-cell contexts

10.1101/2021.05.02.442314 ◽

2021 ◽

Author(s):

Ruiyan Hou ◽

Yuanhua Huang

Keyword(s):

Single Cell ◽

Rna Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Genomic Sequence ◽

Rna Binding Proteins ◽

Single Cells ◽

Regulatory Machinery ◽

Kinetic Rates ◽

Splicing Efficiency

RNA splicing is a key step of gene expression in higher organisms. Accurate quantification of the two-step splicing kinetics is of high interests not only for understanding the regulatory machinery, but also for estimating the RNA velocity in single cells. However, the kinetic rates remain poorly understood due to the intrinsic low content of unspliced RNAs and its stochasticity across contexts. Here, we estimated the relative splicing efficiency across a variety of single-cell RNA-Seq data with scVelo. We further extracted three large feature sets including 92 basic genomic sequence features, 65,536 octamers and 120 RNA binding proteins features and found they are highly predictive to RNA splicing efficiency across multiple tissues on human and mouse. A set of important features have been identified with strong regulatory potentials on splicing efficiency. This predictive power brings promise to reveal the complexity of RNA processing and to enhance the estimation of single-cell RNA velocity.

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The role of disorder in RNA binding affinity and specificity

Open Biology ◽

10.1098/rsob.200328 ◽

2020 ◽

Vol 10 (12) ◽

pp. 200328

Author(s):

Diana S. M. Ottoz ◽

Luke E. Berchowitz

Keyword(s):

Protein Interactions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Multiple Partners ◽

Cellular Environment ◽

Rna Targets

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.

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Splicing in a single neuron is coordinately controlled by RNA binding proteins and transcription factors

eLife ◽

10.7554/elife.46726 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Morgan Thompson ◽

Ryan Bixby ◽

Robert Dalton ◽

Alexa Vandenburg ◽

John A Calarco ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Single Neuron ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fluorescent Reporters ◽

Post Transcriptional Regulation ◽

Splicing Patterns ◽

Phenotypic Convergence

Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single Caenorhabditis elegans neurons, identifying complex splicing patterns in the neuronal kinase sad-1. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs and two RBPs are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, which interact with sad-1 pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron sad-1 splicing. Additionally, we find ‘phenotypic convergence,’ previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.

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Corrigendum to “The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons” [FEBS Open Bio 4 (2014) 1-10]

FEBS Open Bio ◽

10.1016/j.fob.2014.11.007 ◽

2014 ◽

Vol 4 (1) ◽

pp. 1030-1030 ◽

Cited By ~ 1

Author(s):

Daiyu Honda ◽

Shinsuke Ishigaki ◽

Yohei Iguchi ◽

Yusuke Fujioka ◽

Tsuyoshi Udagawa ◽

...

Keyword(s):

Cortical Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Targets

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The ALS/FTLD-related RNA-binding proteins TDP-43 and FUS have common downstream RNA targets in cortical neurons

FEBS Open Bio ◽

10.1016/j.fob.2013.11.001 ◽

2013 ◽

Vol 4 (1) ◽

pp. 1-10 ◽

Cited By ~ 36

Author(s):

Daiyu Honda ◽

Shinsuke Ishigaki ◽

Yohei Iguchi ◽

Yusuke Fujioka ◽

Tsuyoshi Udagawa ◽

...

Keyword(s):

Cortical Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Targets

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DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets

Journal of Molecular Biology ◽

10.1016/j.jmb.2016.09.019 ◽

2017 ◽

Vol 429 (21) ◽

pp. 3139-3145 ◽

Cited By ~ 17

Author(s):

Martin Dutertre ◽

Stéphan Vagner

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

New Class ◽

Rna Targets ◽

Damage Response

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Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method

BIO-PROTOCOL ◽

10.21769/bioprotoc.2274 ◽

2017 ◽

Vol 7 (10) ◽

Cited By ~ 9

Author(s):

Pamela Bielli ◽

Claudio Sette

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cross Linking ◽

Rna Targets

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Posttranscription Initiation Control of Gene Expression Mediated by Bacterial RNA-Binding Proteins

Annual Review of Microbiology ◽

10.1146/annurev-micro-020518-115907 ◽

2019 ◽

Vol 73 (1) ◽

pp. 43-67 ◽

Cited By ~ 16

Author(s):

Paul Babitzke ◽

Ying-Jung Lai ◽

Andrew J. Renda ◽

Tony Romeo

Keyword(s):

Gene Expression ◽

Binding Proteins ◽

Rna Binding ◽

Tertiary Structure ◽

Rna Binding Proteins ◽

True Nature ◽

Control Of Gene Expression ◽

Global Regulators ◽

Rna Targets ◽

Bacterial Rna

RNA-binding proteins play vital roles in regulating gene expression and cellular physiology in all organisms. Bacterial RNA-binding proteins can regulate transcription termination via attenuation or antitermination mechanisms, while others can repress or activate translation initiation by affecting ribosome binding. The RNA targets for these proteins include short repeated sequences, longer single-stranded sequences, RNA secondary or tertiary structure, and a combination of these features. The activity of these proteins can be influenced by binding of metabolites, small RNAs, or other proteins, as well as by phosphorylation events. Some of these proteins regulate specific genes, while others function as global regulators. As the regulatory mechanisms, components, targets, and signaling circuitry surrounding RNA-binding proteins have become better understood, in part through rapid advances provided by systems approaches, a sense of the true nature of biological complexity is becoming apparent, which we attempt to capture for the reader of this review.

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