Functional annotation of a divergent genome using sequence and structure-based similarity

Area of Science:

• Genomics
• Structural Biology
• Bioinformatics

Background:

• Microsporidia possess highly streamlined genomes, posing challenges for traditional gene annotation due to high sequence divergence.
• Many microsporidian genes remain functionally uncharacterized, hindering biological understanding.
• Advances in protein structure prediction and databases offer new avenues for functional gene annotation.

Purpose of the Study:

• To develop and validate a novel workflow for functional gene annotation in microsporidian genomes.
• To improve the accuracy and comprehensiveness of genome annotation for divergent organisms.
• To characterize the genome of the microsporidian Vairimorpha necatrix.

Main Methods:

• A workflow combining sequence and structure-based annotation using the ANNOTEX (Annotation Extension for ChimeraX) plugin was established.
• Protein structure predictions were generated using ColabFold and searched against the PDB and AlphaFold databases with Foldseek.
• Manual curation integrated sequence and structure-based evidence for enhanced annotation quality.

Main Results:

• The workflow was applied to the Vairimorpha necatrix genome, annotating 3080 protein-coding sequences with high RNA sequencing confirmation.
• Functional annotation accuracy and quality were significantly improved compared to traditional methods.
• The study provided a comprehensive genome description for V. necatrix and identified functions for previously uncharacterized genes in Encephalitozoon cuniculi.

Conclusions:

• A new functional annotation tool, ANNOTEX, was developed for divergent organisms like microsporidia.
• The structure-based annotation approach provides a valuable template for studying other divergent species.
• This work enhances the understanding of V. necatrix, an uncharacterized intracellular pathogen.