scholarly journals A Deep Learning approach predicts the impact of point mutations in intronic flanking regions on micro-exon splicing definition

2019 ◽  
Author(s):  
Lucas F. DaSilva ◽  
Ana C. Tahira ◽  
Vinicius Mesel ◽  
Sergio Verjovski-Almeida
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Base Point ◽  
Validation Dataset ◽  
Systematic Assessment ◽  
Exon Splicing ◽  
Flanking Sequences ◽  
The Impact

AbstractWhile mammalian exons are on average 140-nt-long, thousands of human genes harbor micro-exons (≤ 39 nt). Large numbers of micro-exons have their splicing altered in diseases such as autism and cancer, and yet there is no systematic assessment of the impact of point mutations in intronic flanking-sequences on the splicing of a neighboring micro-exon. Here, we constructed a model using the Convolutional Neural Network (CNN) to predict the impact of point mutations in intronic-flanking-sequences on the splicing of a neighboring micro-exon. The prediction model was based on both the sequence contents and conservation among species of the two 100-nt intronic regions (5’ and 3’) that flank all human micro-exons and a set with the same number of randomly selected long exons. After training our CNN model, the micro-exon splicing event prediction accuracy, using an independent validation dataset, was 0.71 with an area under the ROC curve of 0.76, showing that our model had identified sequence patterns that have been conserved in evolution in the introns that flank micro-exons. Next, we introduced in silico point mutations at each of the 200 nucleotides in the introns that flank a micro-exon and used the trained CNN algorithm to predict splicing for every mutated intronic sequence version. This analysis identified thousands of point mutations in the flanking introns that significantly decreased the power of the CNN model to correctly predict a neighboring micro-exon splicing event, thus pointing to predictive bases in intronic regions important for micro-exon splicing signaling. We found these predictive bases to locate within conserved RNA-binding-motifs for RNA-binding-proteins (RBPs) known to relate to micro-exon splicing. Experimental data of minigene splicing reporter changes upon intron-base point-mutation confirmed the effect predicted by the CNN model for some of the micro-exon splicing events. The model can be used for validating novel micro-exons de novo assembled from RNA-seq data, and for an unbiased screening of introns, identifying genomic bases that have high micro-exon-splicing predictive power, possibly revealing critical point mutations that would be related in a yet unknown manner to a given disease.

scholarly journals Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

2017 ◽  
Vol 114 (14) ◽  
pp. 3619-3624 ◽  
Author(s):  
Richard She ◽  
Anupam K. Chakravarty ◽  
Curtis J. Layton ◽  
Lauren M. Chircus ◽  
Johan O. L. Andreasson ◽  
...  
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Quantitative Description ◽  
Rna Degradation ◽  
Nutrient Sensing ◽  
Regulatory Function ◽  
Specific Sequence ◽  
The Impact

RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene “birth.” TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

scholarly journals Targeting Poison Exons to Treat Developmental and Epileptic Encephalopathy

2021 ◽  
pp. 1-6
Author(s):  
Miriam C. Aziz ◽  
Patricia N. Schneider ◽  
Gemma L. Carvill
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Autism Spectrum ◽  
Epileptic Encephalopathy ◽  
Nonsense Mediated Decay ◽  
Subsequent Degradation ◽  
Alternative Splicing Events ◽  
Genetic Epilepsies

Developmental and epileptic encephalopathies (DEEs) describe a subset of neurodevelopmental disorders categorized by refractory epilepsy that is often associated with intellectual disability and autism spectrum disorder. The majority of DEEs are now known to have a genetic basis with de novo coding variants accounting for the majority of cases. More recently, a small number of individuals have been identified with intronic <i>SCN1A</i> variants that result in alternative splicing events that lead to ectopic inclusion of poison exons (PEs). PEs are short highly conserved exons that contain a premature truncation codon, and when spliced into the transcript, lead to premature truncation and subsequent degradation by nonsense-mediated decay. The reason for the inclusion/exclusion of these PEs is not entirely clear, but research suggests an autoregulatory role in gene expression and protein abundance. This is seen in proteins such as RNA-binding proteins and serine/arginine-rich proteins. Recent studies have focused on targeting these PEs as a method for therapeutic intervention. Targeting PEs using antisense oligonucleotides (ASOs) has shown to be effective in modulating alternative splicing events by decreasing the amount of transcripts harboring PEs, thus increasing the abundance of full-length transcripts and thereby the amount of protein in haploinsufficient genes implicated in DEE. In the age of personalized medicine, cellular and animal models of the genetic epilepsies have become essential in developing and testing novel precision therapeutics, including PE-targeting ASOs in a subset of DEEs.

scholarly journals Current insights into the implications of m6A RNA methylation and autophagy interaction in human diseases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuechai Chen ◽  
Jianan Wang ◽  
Muhammad Tahir ◽  
Fangfang Zhang ◽  
Yuanyuan Ran ◽  
...  
Keyword(s):  
Intervertebral Disc Degeneration ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Degradation Process ◽  
Human Diseases ◽  
Rna Modification ◽  
Rna Methylation ◽  
M6a Rna Methylation ◽  
The Impact

AbstractAutophagy is a conserved degradation process crucial to maintaining the primary function of cellular and organismal metabolism. Impaired autophagy could develop numerous diseases, including cancer, cardiomyopathy, neurodegenerative disorders, and aging. N6-methyladenosine (m6A) is the most common RNA modification in eukaryotic cells, and the fate of m6A modified transcripts is controlled by m6A RNA binding proteins. m6A modification influences mRNA alternative splicing, stability, translation, and subcellular localization. Intriguingly, recent studies show that m6A RNA methylation could alter the expression of essential autophagy-related (ATG) genes and influence the autophagy function. Thus, both m6A modification and autophagy could play a crucial role in the onset and progression of various human diseases. In this review, we summarize the latest studies describing the impact of m6A modification in autophagy regulation and discuss the role of m6A modification-autophagy axis in different human diseases, including obesity, heart disease, azoospermatism or oligospermatism, intervertebral disc degeneration, and cancer. The comprehensive understanding of the m6A modification and autophagy interplay may help in interpreting their impact on human diseases and may aid in devising future therapeutic strategies.

scholarly journals De-novo protein function prediction using DNA binding and RNA binding proteins as a test case

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Sapir Peled ◽  
Olga Leiderman ◽  
Rotem Charar ◽  
Gilat Efroni ◽  
Yaron Shav-Tal ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Function ◽  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Protein Function Prediction ◽  
Function Prediction ◽  
Test Case

scholarly journals De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Kana Mitsuhashi ◽  
Daisuke Ito ◽  
Kyoko Mashima ◽  
Munenori Oyama ◽  
Shinichi Takahashi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
De Novo Design

scholarly journals Massively parallel identification of zipcodes in primary cortical neurons

2021 ◽  
Author(s):  
Nicolai von Kuegelgen ◽  
Samantha Mendonsa ◽  
Sayaka Dantsuji ◽  
Maya Ron ◽  
Marieluise Kirchner ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Primary Cortical Neurons ◽  
Subcellular Compartments ◽  
Local Functions ◽  
Massively Parallel Reporter Assay

Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

scholarly journals Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Lulu Deng ◽  
Long Li ◽  
Cheng Zou ◽  
Chengchi Fang ◽  
Changchun Li
Keyword(s):  
Single Molecule ◽  
Posttranscriptional Regulation ◽  
Muscle Development ◽  
Target Genes ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Fast And Slow Muscles ◽  
Molecular Features ◽  
Polyadenylation Sites

Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PAS-associated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs.

scholarly journals Identification of proteins and miRNAs that specifically bind an mRNA in vivo

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kathrin Theil ◽  
Koshi Imami ◽  
Nikolaus Rajewsky
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Noncoding Rnas ◽  
Shotgun Proteomics ◽  
Small Rna Sequencing ◽  
C Elegans ◽  
Specific Transcript ◽  
Proteomics Approach

Abstract Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems.

scholarly journals miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies

2012 ◽  
Vol 23 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Yu-Hong Cui ◽  
Lan Xiao ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Coding Region ◽  
Processing Bodies ◽  
Target Mrnas ◽  
Repressive Effect

microRNAs (miRNAs) and RNA-binding proteins (RBPs) jointly regulate gene expression at the posttranscriptional level and are involved in many aspects of cellular functions. The RBP CUG-binding protein 1 (CUGBP1) destabilizes and represses the translation of several target mRNAs, but the exact mechanism that regulates CUGBP1 abundance remains elusive. In this paper, we show that miR-503, computationally predicted to associate with three sites of the CUGBP1 mRNA, represses CUGBP1 expression. Overexpression of an miR-503 precursor (pre-miR-503) reduced the de novo synthesis of CUGBP1 protein, whereas inhibiting miR-503 by using an antisense RNA (antagomir) enhanced CUGBP1 biosynthesis and elevated its abundance; neither intervention changed total CUGBP1 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-503 through the CUGBP1 coding region sites than through the single CUGBP1 3′-untranslated region target site. CUGBP1 mRNA levels in processing bodies (P-bodies) increased in cells transfected with pre-miR-503, while silencing P-body resident proteins Ago2, RCK, or LSm4 decreased miR-503–mediated repression of CUGBP1 expression. Decreasing the levels of cellular polyamines reduced endogenous miR-503 levels and promoted CUGBP1 expression, an effect that was prevented by ectopic miR-503 overexpression. Repression of CUGBP1 by miR-503 in turn altered the expression of CUGBP1 target mRNAs and thus increased the sensitivity of intestinal epithelial cells to apoptosis. These findings identify miR-503 as both a novel regulator of CUGBP1 expression and a modulator of intestinal epithelial homoeostasis.

scholarly journals Molecular dynamics of FMRP and other RNA-binding proteins in MEG-01 differentiation: the role of mRNP complexes in non-neuronal development

2016 ◽  
Vol 94 (6) ◽  
pp. 597-608 ◽  
Author(s):  
M. McCoy ◽  
D. Poliquin-Duchesneau ◽  
F. Corbin
Keyword(s):  
Protein Content ◽  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Morphological Changes ◽  
Fragile X ◽  
Cellular Protein ◽  
The Body ◽  
Total Protein Content

Asymmetrically differentiating cells are formed with the aid of RNA-binding proteins (RBPs), which can bind, stabilize, regulate, and transport target mRNAs. The loss of RBPs in neurons may lead to severe neurodevelopmental diseases such as the Fragile X Syndrome with the absence of the Fragile X Mental Retardation Protein (FMRP). Because the latter is ubiquitous and shares many similarities with other RBPs involved in the development of peripheral cells, we suggest that FMRP would have a role in the differentiation of all tissues where it is expressed. A MEG-01 differentiation model was, therefore, established to study the global developmental functions of FMRP. PMA induction of MEG-01 cells causes important morphological changes driven by cytoskeletal dynamics. Cytoskeleton change and colocalization analyses were performed by confocal microscopy and sucrose gradient fractionation. Total cellular protein content and de novo synthesis were also analyzed. Microtubular transport mediates the displacement of FMRP and other RBP-containing mRNP complexes towards regions of the cell in development. De novo protein synthesis decreases significantly upon differentiation and total protein content composition is altered. Because those results are comparable with those obtained in neurons, the absence of FMRP would have significant consequences in cells everywhere in the body. The latter should be further investigated to give a better understanding of the systemic implications of imbalances of FMRP and other functionally similar RBPs.

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