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Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates.

ABSTRACT: The study of pre-mRNA splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which enables the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning alignments corresponding to retained introns, as well as exon-exon junction spanning alignments corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing alignments that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative alignments are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify many splice isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses pools of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach enables a dramatic enrichment in the fraction of splicing informative alignments generated per splicing event, yielding an increase in both the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of the shortcomings associated with RNA-seq that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.

SUBMITTER: Gildea MA

PROVIDER: S-EPMC6868320 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates

Project description:The study of splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which can enable the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning reads corresponding to retained introns, as well as exon-exon junction spanning reads corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing reads that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative reads are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify less abundant isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses complex pools of thousands of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach can enable a dramatic enrichment in the fraction of splicing informative reads generated in an experiment, enabling an increase both in the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of methods for calculating splicing efficiency with existing RNA-seq data, including the shortcomings associated with these approaches that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.

2019-03-12 | GSE126583 | GEO

Detection of splice isoforms and rare intermediates using multiplexed primer extension sequencing.

Project description:Targeted RNA sequencing (RNA-seq) aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-seq experiments. Here we present multiplexed primer extension sequencing (MPE-seq), an approach for targeted RNA-seq that uses complex pools of reverse-transcription primers to enable sequencing enrichment at user-selected locations across the genome. We targeted hundreds to thousands of pre-mRNA splice junctions and obtained high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

| S-EPMC6414223 | biostudies-literature

Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates

Project description:Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates

| PRJNA522443 | ENA

Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Project description:New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2â²-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems. We sequenced in-vitro transcribed RNaseP in wild type and barcoded variants and probed them with SHAPE-Seq. Comparison to existing crystalography data and previous SHAPE experiments verified the accuracy of the technique and the absence of bias due to our multiplexiing strategy.

2011-08-26 | E-GEOD-31573 | biostudies-arrayexpress

AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Project description:Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

| S-EPMC10337260 | biostudies-literature

T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.

Project description:The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.

| S-EPMC359481 | biostudies-other

Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

2011-08-26 | GSE31573 | GEO

Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms.

Project description:The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5' end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3' of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.

| S-EPMC8691633 | biostudies-literature

Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).

Project description:New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

| S-EPMC3131332 | biostudies-literature

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

Project description:Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

| S-EPMC4390264 | biostudies-literature

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