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Published on: February 5, 2021
Transcriptome-wide analysis of pseudouridylation in Drosophila melanogaster
Wan Song1,2, Ram Podicheti3, Douglas B Rusch3
1Gill Center for Biomolecular Research, Indiana University, Bloomington, IN 47405, USA.
Abstract:
Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, and non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The 3 mRNAs most enriched for Psi encode highly expressed yolk proteins (Yp1, Yp2, and Yp3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery.
Insights
Researchers optimized RNA pseudouridine (Psi) detection in Drosophila melanogaster, finding thousands of Psi modifications across the transcriptome. This study highlights Psi
Area of Science:
- Molecular Biology
- Genomics
- RNA Biology
Background:
- Pseudouridine (Psi) is a prevalent RNA modification found across various RNA types, including mRNA, rRNA, and non-coding RNAs.
- Understanding Psi distribution and function is crucial for comprehending gene regulation and cellular processes.
- Previous transcriptome-wide Psi detection (Psi-seq) had not been established for the model organism Drosophila melanogaster.
Purpose of the Study:
- To optimize and implement Psi-seq for transcriptome-wide pseudouridine profiling in Drosophila melanogaster.
- To identify and characterize pseudouridine modification sites in the female fly head transcriptome.
- To investigate the role of pseudouridylation in ribosomal and mitochondrial function, and identify potential targets of the RluA-2 enzyme.
Main Methods:
- Optimization of the Psi-seq protocol for Drosophila melanogaster.
- High-throughput sequencing to map pseudouridine modifications across the transcriptome.
- Comparative analysis between RluA-2 mutant and control (w1118) genotypes.
- Differential gene expression analysis.
Main Results:
- Thousands of pseudouridine modification sites were identified throughout the female fly head transcriptome.
- Pseudouridine modifications are widespread in both cellular and mitochondrial rRNAs, and over a thousand sites were found on mRNAs.
- Genes encoding ribosomal proteins and mitochondrial RNAs are frequently pseudouridylated.
- The 7SLRNA and yolk protein mRNAs (Yp1, Yp2, Yp3) were identified as highly enriched for pseudouridine.
- Several pseudouridine sites were found to be missing in the RluA-2 mutant, suggesting they are RluA-2 targets.
- Loss of RluA-2 significantly impacts the ribosome and translational machinery.
Conclusions:
- The optimized Psi-seq method enables comprehensive pseudouridine profiling in Drosophila melanogaster.
- Pseudouridylation plays a significant role in ribosome biogenesis, mitochondrial function, and translational regulation in flies.
- RluA-2 is implicated as a key enzyme in pseudouridine modification with broad effects on the transcriptome and translational machinery.
