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Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

ABSTRACT: Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC10484994 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

Zhang Zhiping Z Bae Bongmin B Cuddleston Winston H WH Miura Pedro P

Nature communications 20230907 1

Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA in Drosophila was investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing com ...[more]

PMID: 37679364

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Project description:Alternative splicing (AS) is an important mechanism in the development of many cancers, as novel or aberrant AS patterns play an important role as an independent onco-driver. In addition, cancer-specific AS is potentially an effective target of personalized cancer therapeutics. However, detecting AS events remains a challenging task, especially if these AS events are novel. This is exacerbated by the fact that existing transcriptome annotation databases are far from being comprehensive, especially with regard to cancer-specific AS. Additionally, traditional sequencing technologies are severely limited by the short length of the generated reads, which rarely spans more than a single splice junction site. Given these challenges, transcriptomic long-read (LR) sequencing presents a promising potential for the detection and discovery of AS. We present Freddie, a computational annotation-independent isoform discovery and detection tool. Freddie takes as input transcriptomic LR sequencing of a sample alongside its genomic split alignment and computes a set of isoforms for the given sample. It then partitions the input reads into sets that can be processed independently and in parallel. For each partition, Freddie segments the genomic alignment of the reads into canonical exon segments. The goal of this segmentation is to be able to represent any potential isoform as a subset of these canonical exons. This segmentation is formulated as an optimization problem and is solved with a dynamic programming algorithm. Then, Freddie reconstructs the isoforms by jointly clustering and error-correcting the reads using the canonical segmentation as a succinct representation. The clustering and error-correcting step is formulated as an optimization problem-the Minimum Error Clustering into Isoforms (MErCi) problem-and is solved using integer linear programming (ILP). We compare the performance of Freddie on simulated datasets with other isoform detection tools with varying dependence on annotation databases. We show that Freddie outperforms the other tools in its accuracy, including those given the complete ground truth annotation. We also run Freddie on a transcriptomic LR dataset generated in-house from a prostate cancer cell line with a matched short-read RNA-seq dataset. Freddie results in isoforms with a higher short-read cross-validation rate than the other tested tools. Freddie is open source and available at https://github.com/vpc-ccg/freddie/.

Project description:BackgroundAlternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC).MethodsHere, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4-5 exclusion was introduced to inhibit neoplasm growth.ResultsA total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients' prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4-5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4-5 exclusion have the potential to restrain the CRC growth.ConclusionsThis data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Dataset Information

Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

Publications

Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing.

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