A hybrid transcriptome assembly reveals coding and non-coding landscapes in Drosophila hemocytes

Area of Science:

• * Molecular Biology
• * Immunology
• * Genomics

Background:

• * Drosophila larval hemocyte-mediated innate immunity is well-studied, but transcriptional and post-transcriptional regulation remains unclear.
• * Understanding these regulatory mechanisms is key to deciphering immune responses.

Purpose of the Study:

• * To comprehensively annotate the Drosophila larval transcriptome using a hybrid sequencing approach.
• * To identify novel long non-coding RNAs (lncRNAs) involved in the innate immune response.
• * To investigate the functional roles of identified lncRNAs in hemocyte differentiation and immune defense.

Main Methods:

• * Hybrid sequencing strategy combining Oxford Nanopore long-read and Illumina short-read sequencing.
• * Bioinformatic analysis to identify full-length transcripts and novel lncRNAs, including artifact removal.
• * Single-cell RNA sequencing to identify cell type-specific lncRNA markers.
• * RNA interference (RNAi) to assess the functional impact of lncRNAs.

Main Results:

• * A comprehensive transcriptome annotation revealing novel transcript boundaries.
• * Discovery of 349 unannotated long non-coding RNAs (lncRNAs) significantly induced during wasp infestation.
• * Identification of cell type-specific lncRNA markers in lamellocytes and crystal cells.
• * Functional validation showing that depletion of two lncRNAs impairs lamellocyte formation during infestation.

Conclusions:

• * The study provides high-confidence full-length transcript models of the Drosophila larval immune transcriptome.
• * Identified lncRNAs play critical regulatory roles in Drosophila innate immunity, particularly in response to parasitic wasp infestation.
• * This work highlights the importance of non-coding RNAs in immune defense mechanisms.