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

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.