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Updated: Nov 26, 2025
Identification of Alternative Splicing and Polyadenylation in RNA-seq Data
Published on: June 24, 2021
Direct RNA sequencing dataset of SMG1 KO mutant Physcomitrella (Physcomitrium patens)
Andrey Knyazev1, Anna Glushkevich1, Igor Fesenko1
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 16/10, Ulitsa Miklukho-Maklaya, Moscow, 117997, Russian Federation.
Abstract:
Nonsense-mediated mRNA decay (NMD) is a system that controls the quality of mRNA transcripts in eukaryotes by degradation of aberrant transcripts in a pioneer round of translation. In mammals, NMD targets one-third of mutated, disease-causing mRNAs and ∼10% of unmutated mRNAs, facilitating appropriate cellular responses to environmental changes [1]. In plants, NMD plays an important role in development and regulating abiotic and biotic stress responses [2]. The transcripts with premature termination codons (PTCs), upstream ORFs or long 3'-UTRs can be targeted to NMD. It was shown that alternative splicing plays a crucial role in regulation of NMD triggering, for example, by the introduction of a PTC in transcripts. Therefore, the correct identification of mRNA isoforms is a key step in the study of the principles of regulation of the cell transcriptome by the NMD pathway. Here, we performed long-read sequencing of Physcomitrella (Physcomitrium patens) mutant smg1Δ line 2 native transcriptome by Oxford Nanopore Technology (ONT). The smg1Δ is a knockout (KO) mutant deficient in SMG1 kinase is a key component of NMD system in plants and animals [3]. RNA was isolated with Trizol from 5 day old protonemata and sequenced using kit SQK-RNA002, flow cells FLO-MIN106 and a MinION device (Oxford Nanopore Technologies Ltd., UK (ONT)) in three biological repeats. Basecalling was performed with Guppy v.4.0.15. The presented transcriptomes give advantages in the identification and functional characterization of RNA transcripts that are direct targets of the Nonsense-mediated mRNA decay system.
Insights
Nonsense-mediated mRNA decay (NMD) quality control identifies aberrant transcripts. This study used long-read sequencing in a Physcomitrella patens mutant to characterize NMD targets, aiding transcriptome regulation studies.
Area of Science:
- Molecular Biology
- Genetics
- Plant Science
Background:
- Nonsense-mediated mRNA decay (NMD) is a crucial eukaryotic post-transcriptional quality control pathway.
- NMD regulates gene expression by degrading aberrant mRNAs, including those with premature termination codons (PTCs).
- In plants, NMD is vital for development and stress responses, but identifying its direct targets remains challenging.
Purpose of the Study:
- To characterize the native transcriptome of a Physcomitrella patens mutant lacking SMG1 kinase, a key NMD component.
- To identify direct targets of the Nonsense-mediated mRNA decay pathway in plants using long-read sequencing.
- To advance the understanding of NMD-mediated transcriptome regulation in plants.
Main Methods:
- Long-read sequencing of the native transcriptome from a *smg1* knockout mutant of *Physcomitrella patens* (line 2) using Oxford Nanopore Technology (ONT).
- RNA isolation using Trizol from 5-day-old protonemata, followed by sequencing with kit SQK-RNA002 on a MinION device.
- Data processing included basecalling with Guppy v.4.0.15 for three biological replicates.
Main Results:
- Generation of high-quality native transcriptomes for the *smg1*Δ mutant of *Physcomitrella patens*.
- Provides a dataset advantageous for identifying and functionally characterizing direct NMD targets.
- Establishes a foundation for studying NMD pathway regulation in plants.
Conclusions:
- Long-read sequencing is effective for characterizing transcriptomes and identifying NMD targets.
- The generated data facilitates the study of mRNA quality control mechanisms in plants.
- This research contributes to understanding how NMD influences plant development and stress responses.
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