Full-Length Transcript-Based Proteogenomics of Rice Improves Its Genome and Proteome Annotation

Area of Science:

• Plant Molecular Biology
• Genomics
• Transcriptomics

Background:

• Inaccurate genome annotation in rice (Oryza sativa) hinders molecular breeding and functional studies.
• Understanding the full scope of the rice transcriptome is crucial for advancing research.

Purpose of the Study:

• To utilize single-molecule long-read RNA sequencing (lrRNA_seq)-based proteogenomics to comprehensively analyze the rice transcriptome.
• To reveal the complexity of rice gene expression, including natural antisense transcripts (NATs), fusion transcripts, and posttranscriptional modifications.

Main Methods:

• Application of single-molecule long-read RNA sequencing (lrRNA_seq) for high-resolution transcriptome analysis.
• Proteogenomic analysis to identify novel peptides and proteoforms.
• Comparison with short-read RNA sequencing to evaluate transcript identification capabilities.

Main Results:

• Identification of approximately 60% of loci associated with natural antisense transcripts (NATs), suggesting significant roles in gene regulation.
• Discovery of numerous fusion and intergenic transcripts, alongside 906,456 transcript isoforms, with 72.9% of genes generating splicing variants.
• Characterization of 706,075 posttranscriptional events and identification of over 190,000 unique peptides, expanding the known rice proteome.

Conclusions:

• The rice genome organization, transcriptome diversity, and coding potential are significantly more complex than previously understood.
• lrRNA_seq provides superior capabilities for identifying long transcripts compared to short-read sequencing.
• These findings provide a more complete picture of the rice transcriptome and proteome, essential for future molecular breeding and functional genomics.