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Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA

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  • 1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

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Summary

We developed Nanopore 3' end-capture sequencing (Nano3P-seq) to analyze RNA polyadenylation. This method quantifies RNA abundance, tail length, and composition, revealing dynamic regulation during development.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • RNA polyadenylation is crucial for RNA processing, influencing mRNA fate and translation efficiency.
  • PolyA tail length variations are observed in response to developmental signals, impacting gene expression.
  • Existing methods for analyzing polyadenylation have limitations in resolution and scope.

Purpose of the Study:

  • To introduce Nanopore 3' end-capture sequencing (Nano3P-seq), a novel method for comprehensive RNA tail analysis.
  • To demonstrate Nano3P-seq's capability in quantifying RNA abundance, tail length, and tail composition at single-read resolution.
  • To investigate the dynamics of polyadenylation during vertebrate embryogenesis and identify non-canonical tail features.

Main Methods:

  • Development of Nano3P-seq utilizing nanopore cDNA sequencing and a template-switching strategy.
  • Sequencing RNA molecules from the 3' end without PCR amplification or adapter ligation.
  • Application of Nano3P-seq to mouse and zebrafish models for transcriptomic analysis.

Main Results:

  • Nano3P-seq accurately quantifies RNA abundance and polyA tail lengths across diverse RNA biotypes.
  • PolyA tails were identified not only in mRNA and lncRNA but also in 16S mitochondrial ribosomal RNA.
  • Dynamic, isoform-specific regulation of mRNA tail lengths was observed during vertebrate embryogenesis, correlating with mRNA decay.
  • Non-A bases within polyA tails were detected and their distribution during embryogenesis characterized.

Conclusions:

  • Nano3P-seq is a robust and unbiased method for high-resolution analysis of RNA polyadenylation.
  • The method provides insights into the dynamic regulation of RNA tails and their role in gene expression.
  • Nano3P-seq expands the understanding of polyadenylation beyond canonical mRNAs to include other RNA species and non-canonical tail structures.