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

PROVIDER: PRJNA522443 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8579870.fastq.gz	Fastqsanger.gz
	SRR8579869.fastq.gz	Fastqsanger.gz

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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.

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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 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.

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Project description:The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3’ to 5’ degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5’ monophosphate ends on mRNAs in wild-type and dcp2∆ xrn1∆ yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low level endonucleotyic cleavage were observed suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5’ monophosphates on some mRNAs suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential new step to regulate gene expression. 5' monophosphorylated ends of poly(A) RNA from wild-type and dcp2D xrn1D strains were identified in duplicates and triplicates, respectively.

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pre-mRNA splicing is regulated by an Aurora-A-mediated phopsho-signaling network

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