Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

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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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A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

10.20944/preprints202108.0137.v1 ◽

2021 ◽

Author(s):

Shao-Yen Kao ◽

Elena Nikonova ◽

Sabrina Chaabane ◽

Albiona Sabani ◽

Alexandra Martitz ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Processing ◽

Muscle Development ◽

Rna Binding ◽

Flight Muscle ◽

Rna Binding Proteins ◽

Fiber Type ◽

Expression Patterns ◽

Fine Tuning ◽

Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability between muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy, and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Interestingly, knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of Kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a useful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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A Candidate RNAi Screen Reveals Diverse RNA-Binding Protein Phenotypes in Drosophila Flight Muscle

Cells ◽

10.3390/cells10102505 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2505

Author(s):

Shao-Yen Kao ◽

Elena Nikonova ◽

Sabrina Chaabane ◽

Albiona Sabani ◽

Alexandra Martitz ◽

...

Keyword(s):

Alternative Splicing ◽

Rna Processing ◽

Muscle Development ◽

Rna Binding ◽

Flight Muscle ◽

Rna Binding Proteins ◽

Fiber Type ◽

Expression Patterns ◽

Fine Tuning ◽

Fiber Types

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa, Rm62, mub, mbl, sbr, and clu. Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

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

10.1101/2020.12.28.424570 ◽

2020 ◽

Author(s):

Mansi Srivastava ◽

Rajneesh Srivastava ◽

Sarath Chandra Janga

Keyword(s):

High Throughput ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Interaction Network ◽

Genomic Variation ◽

P Value ◽

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 polyA pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non polyA 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 polyA 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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The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.894-905.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 894-905

Author(s):

R A Voelker ◽

W Gibson ◽

J P Graves ◽

J F Sterling ◽

M T Eisenberg

Keyword(s):

Rna Processing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Open Reading Frame ◽

In Vitro Translation ◽

Rna Recognition Motif ◽

Reading Frame ◽

Cdna Sequences ◽

Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

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FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-04054-8 ◽

2021 ◽

Author(s):

Teresa Chioccarelli ◽

Geppino Falco ◽

Donato Cappetta ◽

Antonella De Angelis ◽

Luca Roberto ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Rna Binding ◽

Cannabinoid Receptor ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Circular Rna ◽

Physical Interaction ◽

Knock Out ◽

Maternal Contribution ◽

Zygote Development

AbstractCircular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1−/−) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

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Discovery of allele-specific protein-RNA interactions in human transcriptomes

10.1101/389205 ◽

2018 ◽

Author(s):

Emad Bahrami-Samani ◽

Yi Xing

Keyword(s):

Genetic Variants ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Protein ◽

Computational Method ◽

Joint Analysis ◽

Transcriptional Level ◽

Rna Seq ◽

Allele Specific ◽

Rna Interaction

AbstractGene expression is tightly regulated at the post-transcriptional level through splicing, transport, translation, and decay. RNA-binding proteins (RBPs) play key roles in post-transcriptional gene regulation, and genetic variants that alter RBP-RNA interactions can affect gene products and functions. We developed a computational method ASPRIN (Allele-Specific Protein-RNA Interaction), that uses a joint analysis of CLIP-seq (cross-linking and immunoprecipitation followed by high-throughput sequencing) and RNA-seq data to identify genetic variants that alter RBP-RNA interactions by directly observing the allelic preference of RBP from CLIP-seq experiments as compared to RNA-seq. We used ASPRIN to systematically analyze CLIP-seq and RNA-seq data for 166 RBPs in two ENCODE (Encyclopedia of DNA Elements) cell lines. ASPRIN identified genetic variants that alter RBP-RNA interactions by modifying RBP binding motifs within RNA. Moreover, through an integrative ASPRIN analysis with population-scale RNA-seq data, we showed that ASPRIN can help reveal potential causal variants that affect alternative splicing via allele-specific protein-RNA interactions.

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Coordination of RNA Processing Regulation by Signal Transduction Pathways

Biomolecules ◽

10.3390/biom11101475 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1475

Author(s):

Veronica Ruta ◽

Vittoria Pagliarini ◽

Claudio Sette

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Rna Processing ◽

Translational Regulation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulation Of Gene Expression ◽

Signal Transduction Pathways ◽

Challenges And Opportunities ◽

Common Signal

Signal transduction pathways transmit the information received from external and internal cues and generate a response that allows the cell to adapt to changes in the surrounding environment. Signaling pathways trigger rapid responses by changing the activity or localization of existing molecules, as well as long-term responses that require the activation of gene expression programs. All steps involved in the regulation of gene expression, from transcription to processing and utilization of new transcripts, are modulated by multiple signal transduction pathways. This review provides a broad overview of the post-translational regulation of factors involved in RNA processing events by signal transduction pathways, with particular focus on the regulation of pre-mRNA splicing, cleavage and polyadenylation. The effects of several post-translational modifications (i.e., sumoylation, ubiquitination, methylation, acetylation and phosphorylation) on the expression, subcellular localization, stability and affinity for RNA and protein partners of many RNA-binding proteins are highlighted. Moreover, examples of how some of the most common signal transduction pathways can modulate biological processes through changes in RNA processing regulation are illustrated. Lastly, we discuss challenges and opportunities of therapeutic approaches that correct RNA processing defects and target signaling molecules.

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Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6241-6252.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6241-6252 ◽

Cited By ~ 106

Author(s):

Kristina L. Carroll ◽

Dennis A. Pradhan ◽

Josh A. Granek ◽

Neil D. Clarke ◽

Jeffry L. Corden

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Rna Binding ◽

Rna Binding Proteins ◽

Large Degree ◽

Sequence Motifs ◽

Mobility Shift ◽

Nascent Transcript ◽

Pol Ii ◽

Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

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Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells

RNA ◽

10.1261/rna.078896.121 ◽

2021 ◽

pp. rna.078896.121

Author(s):

Yan Han ◽

Xuzhen Guo ◽

Tiancai Zhang ◽

Jiangyun Wang ◽

Keqiong Ye

Keyword(s):

Protein Interactions ◽

Rna Structure ◽

Rna Binding ◽

Rna Binding Proteins ◽

High Specificity ◽

Ester Group ◽

Yeast Cells ◽

Primary Amines ◽

Low Efficiency ◽

In Cells

Characterization of RNA-protein interaction is fundamental for understanding metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

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