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[Preprint]. 2026 Jul 16:2026.07.10.737807. [Version 1] doi: 10.64898/2026.07.10.737807

Gene model for the ortholog of raptor in Drosophila eugracilis

Anne E Backlund 1, Jonas Nielsen 2, John Pulford 2, Janelle Suriaga 3, Jordan Pyle 3, Shadrian McDaniel 3, Jeffrey S Thompson 2, Chinmay P Rele 1, Jacqueline K Wittke-Thompson 3,Ω
PMCID: PMC13404653  PMID: 42523285

Abstract

Gene model for the ortholog of raptor in the D. eugracilis Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of Drosophila eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Metadata: Bioinformatics, Genomics, Drosophila, Genotype Data, New Finding

Introduction

This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP; thegep.org) for Course-based Undergraduate Research Experience (CURE). The following information in quotes may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster genes in the insulin signaling pathway.

“In this GEP CURE protocol students use web-based tools to manually annotate genes in non-model Drosophila species based on orthology to genes in the well-annotated model organism fruit fly Drosophila melanogaster. The GEP uses web-based tools to allow undergraduates to participate in course-based research by generating manual annotations of genes in non-model species (Rele et al., 2023). Computational-based gene predictions in any organism are often improved by careful manual annotation and curation, allowing for more accurate analyses of gene and genome evolution (Mudge and Harrow 2016; Tello-Ruiz et al., 2019). These models of orthologous genes across species, such as the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community.” (Myers et al., 2024).

“The particular gene ortholog described here was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila. The Insulin/insulin-like growth factor signaling pathway (IIS) is a highly conserved signaling pathway in animals and is central to mediating organismal responses to nutrients (Hietakangas and Cohen 2009; Grewal 2009).” (Myers et al., 2024).

“raptor positively regulates (Target of Rapamycin) TOR-mediated cell apoptosis and growth control by differentially regulating S6K-dependent signaling pathways, and is a crucial regulator of cell growth and metabolism in Drosophila (Lee and Chung 2007; Hatfield et al., 2015). raptor is orthologous to the human RPTOR gene (regulatory associated protein of raptor complex 1) and is a well conserved component of the TORC1 complex (Wang et al., 2012).” (Backlund et al., 2025).

“D. eugracilis is part of the melanogaster species group within the subgenus Sophophora of the genus Drosophila (Pélandakis et al., 1993). It was first described as Tanygastrella gracilis by Duda (1924) and revised to Drosophila eugracilis by Bock and Wheeler (1972). D. eugracilis is found in humid tropical and subtropical forests across southeast Asia (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).” (Morgan et al., 2022).

We propose a gene model for the D. eugracilis ortholog of the D. melanogaster raptor (raptor) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_017064130.1 (Locus ID LOC108103241) in the D. eugracilis Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly of D. eugracilis (GCA_000236325.2 – Chen et al., 2014). This model is based on RNA-Seq data from D. eugracilis (PRJNA63469) and raptor in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021).

Synteny

The reference gene, raptor, occurs on chromosome X in D. melanogaster and is nested by Multiple inositol polyphosphate phosphatase 2 (Mipp2), flanked upstream by Ca2+-channel protein α1 subunit T (Ca-α1T) and Neprilysin 1 (Nep1), and flanked downstream by CG4660 and CG4666. The tblastn search of D. melanogaster raptor-PB (query) against the D. eugracilis (GenBank Accession: GCA_000236325.2 Genome Assembly (database) placed the putative ortholog of raptor within scaffold AFPQ02004929 (AFPQ02004929.1) at locus LOC108103241 (XP_017064130.1)— with an E-value of 0.0 and a percent identity of 79.27%. Furthermore, the putative ortholog is nested by LOC108103242 (XP_017064132.1), which corresponds to Mipp2 in D. melanogaster (E-value: 0.0; identity: 86.34% as determined by blastp; Figure 1A, Altschul et al., 1990). The putative ortholog is flanked upstream by LOC108103196 (XP_041674378.1) and LOC108103207 (XP_017064086.1), which correspond to Ca-α1T and Nep1 in D. melanogaster (E-value: 0.0 and 0.0; identity: 89.10% and 93.95%, respectively, as determined by blastp). Additionally, LOC108103207 appears to be nesting LOC108103208 (XP_017064087.1), however tblastn did not map any D. melanogaster CDS to this locus, and upon a blastp search, no peptide in D. melanogaster was found to align with its protein sequence. Therefore we conclude that LOC108103208 is an ORF with misaligned RNA-Seq data and does not exist in D. eugracilis. The putative ortholog of raptor is flanked downstream by LOC108103198 (XP_017064075.1) and LOC108103202 (XP_017064080.1), which correspond to CG4660 and CG4666 in D. melanogaster (E-value: 7e-173 and 2e-144; identity: 90.42% and 99.48%, respectively, as determined by blastp). The putative ortholog assignment for raptor in D. eugracilis is supported by the following evidence: The genes surrounding the raptor ortholog are orthologous to the genes at the same locus in D. melanogaster and local synteny is completely conserved, supported by E-values and percent identities, so we conclude that LOC108103241 is the correct ortholog of raptor in D. eugracilis (Figure 1A).

Figure 1. raptor gene model comparison between Drosophila eugracilis and Drosophila melanogaster orthologs.

Figure 1.

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(A) Synteny comparison of the genomic neighborhoods for raptor in Drosophila melanogaster and D. eugracilis. Thin underlying arrows indicate the DNA strand within which the target gene–raptor–is located in D. melanogaster (top) and D. eugracilis (bottom). The thin arrow pointing to the left indicate that raptor is on the negative (−) strand in D. melanogaster and D. eugracilis. The wide gene arrows pointing in the same direction as raptor are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of raptor are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. eugracilis indicate orthology to the corresponding gene in D. melanogaster. Gene symbols given in the D. eugracilis gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. eugracilis. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al., 2014). The coding-regions of raptor in D. eugracilis are displayed in the User Supplied Track (black); coding CDSs are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. eugracilis), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females, Adult Males, and Mixed Embreyos (red, light blue, and dark purple, respectively; alignment of Illumina RNA-Seq reads from D. eugracilis), and Splice Junctions Predicted by regtools using D. eugracilis RNA-Seq (PRJNA63469). The splice junctions pertaining to the raptor ortholog (JUNC00003875 and JUNC00003873) shown in pink have read-depths of 170 and 161, respectively. (C) Dot Plot of raptor-PB in D. melanogaster (x-axis) vs. the orthologous peptide in D. eugracilis (y-axis). Amino acid number is indicated along the left and bottom; CDS number is indicated along the top and right, and CDSs are also highlighted with alternating colors. Line breaks in the dot plot indicate mismatching amino acids at the specified location between species. Two regions lack sequence similarity and are highlighted by the purple box labeled I and the orange box labeled II.

Protein Model

raptor in D. eugracilis has three CDSs within the genome sequence. The protein sequence (raptor-PB and raptor-PC) is translated from two mRNA isoforms that differ in their UTRs (raptor-RB and raptor-RC; Figure 1B). Relative to the ortholog in D. melanogaster, the CDS number and protein isoform count are conserved. The sequence of raptor-PB in D. eugracilis has 87.29% identity (E-value: 0.0) with the protein-coding isoform raptor-PB in D. melanogaster, as determined by blastp (Figure 1C). Unusual characteristics of this model include two regions which lack sequence similarity between D. melanogaster and D. eugracilis (purple box I and orange box II; Figure 1C), and low RNA-Seq data throughout CDSs of the raptor ortholog. Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK064555). This gene model can also be seen within the target genome at this TrackHub.

Special characteristics of the protein model

Low RNA-Seq Data.

There is relatively low RNA-Seq data for the raptor ortholog in D. eugracilis, with peaks occurring downstream of the 3’ end of the gene (Figure 1B). These peaks can be attributed to the untranslated region of the Mipp2 ortholog, the nesting gene of raptor. In the future, long-read RNA-Seq data can be used to further investigate raptor in D. eugracilis.

Methods

“Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2023). Briefly, students use the GEP instance of the UCSC Genome Browser v.435 (https://gander.wustl.edu; Kent et al., 2002; Navarro Gonzalez et al., 2021) to examine the genomic neighborhood of their reference IIS gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students then retrieve the protein sequence for the D. melanogaster reference gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly on the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1990) to identify potential orthologs. To validate the potential ortholog, students compare the local genomic neighborhood of their potential ortholog with the genomic neighborhood of their reference gene in D. melanogaster. This local synteny analysis includes at minimum the two upstream and downstream genes relative to their putative ortholog. They also explore other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Detailed explanation of how these lines of genomic evidenced are leveraged by students in gene model development are described in Rele et al. (2023). Genomic structure information (e.g., CDSs, intron-exon number and boundaries, number of isoforms) for the D. melanogaster reference gene is retrieved through the Gene Record Finder (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023). Approximate splice sites within the target gene are determined using tblastn using the CDSs from the D. melanogaster reference gene. Coordinates of CDSs are then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirm the biological validity of their target gene model using the Gene Model Checker (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023), which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. At least two independent models for a gene are generated by students under mentorship of their faculty course instructors. Those models are then reconciled by a third independent researcher mentored by the project leaders to produce the final model. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.” (Gruys et al., 2025)

Supplementary Material

Supplemental Files

1. Zip file containing a FASTA, PEP, GFF files for the gene model

2. Figure 1 in high resolution

Acknowledgements

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models. Further, we would like to thank the editors and developers at the journal microPublication: Biology for assistance in developing the template for these single gene ortholog publications.

Funding

This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215(3): 403–410. [DOI] [PubMed] [Google Scholar]
  2. Backlund AE, Laskowski LF, Trosdal E, Wittke-Thompson J, Yowler B, Rele CP. 2025. Gene model for the ortholog of raptor in Drosophila busckii. Manuscript submitted. [Google Scholar]
  3. Bock IR. and Wheeler MR. (1972). The Drosophila melanogaster species group. Univ Texas Publs Stud Genet 7(7213): 1–102. [Google Scholar]
  4. Chen ZX, Sturgill D, Qu J, Jiang H, Park S, Boley N, et al. , Richards S. 2014. Comparative validation of the D. melanogaster modENCODE transcriptome annotation. Genome Res 24: 1209–1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duda O. 1924. Revision der europäischen u. grönländischen sowie einiger sudostasiat. Arten der Gattung Piophila Fallén (Dipteren) [part]. Konowia 3: 97–113 [1924.07.10] [Google Scholar]
  6. Gramates LS, Agapite J, Attrill H, Calvi BR, Crosby M, dos Santos G Goodman JL, Goutte-Gattat D, Jenkins V, Kaufman T, Larkin A, Matthews B, Millburn G, Strelets VB, and the FlyBase Consortium. 2022. FlyBase: a guided tour of highlighted features. Genetics 220(4): iyac035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grewal SS. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. 2009. Int J Biochem Cell Biol 41(5):1006–1010. [DOI] [PubMed] [Google Scholar]
  8. Gruys ML, Sharp MA, Lill Z, Xiong C, Hark AT, Youngblom JJ, Rele CP, Reed LK. 2025. Gene model for the ortholog of Glys in Drosophila simulans. microPubl Biol: 10.17912/micropub.biology.001168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hatfield I, Harvey I, Yates ER, Redd JR, Reiter LT, Bridges D. 2015. The role of TORC1 in muscle development in Drosophila. Sci Rep 5: 9676. . [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hietakangas V, Cohen SM. Regulation of tissue growth through nutrient sensing. 2009. Annu Rev Genet 43:389–410. [DOI] [PubMed] [Google Scholar]
  11. Jenkins VK, Larkin A, Thurmond J, FlyBase Consortium. 2022. Using FlyBase: A Database of Drosophila Genes and Genetics. Methods Mol Biol 2540: 1–34. [DOI] [PubMed] [Google Scholar]
  12. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. 2002. The Human Genome Browser at UCSC. Genome Res 12: 996–1006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Larkin A, Marygold SJ, Antonazzo G, Attrill H, dos Santos G, Garapati PV, Goodman JL, Gramates LS, Millburn G, Strelets VB, Tabone CJ, and Thurmond J and the FlyBase Consortium. 2021. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res 49(D1): D899–D907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee G, Chung J. 2007. Discrete functions of rictor and raptor in cell growth regulation in Drosophila. Biochem Biophys Res Commun 357(4): 1154–1159. [DOI] [PubMed] [Google Scholar]
  15. Morgan A, Kiser CA, Bronson I, Lin H, Guillette N, McMahon R, Kennell JA, Long LJ, Reed LK, Rele CP. 2022. Drosophila eugracilis – Akt. microPubl Biol: 10.17912/micropub.biology.000544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mudge JM, Harrow J. 2016. The state of play in higher eukaryote gene annotation. Nat Rev Genet 17: 758–772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Myers A, Hoffman A, Natysin M, Arsham AM, Stamm J, Thompson JS, Rele CP, Reed LK. 2024. Gene model for the ortholog Myc in Drosophila ananassae. microPubl Biol: 10.17912/micropub.biology.000856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Navarro Gonzalez J, Zweig AS, Speir ML, Schmelter D, Rosenbloom KR, Raney BJ, Powell CC, Nassar LR, Maulding ND, Lee CM et al. 2021. The UCSC Genome Browser database: 2021 update. Nucleic Acids Res 49(1): 1046–1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pélandakis M, Solignac M. 1993. Molecular phylogeny of Drosophila based on ribosomal RNA sequences. J Mol Evol 37: 525–543. [DOI] [PubMed] [Google Scholar]
  20. Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik D, Kent WJ. 2014. Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7):1003–1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rele CP, Sandlin KM, Leung W, Reed LK. 2023. Manual annotation of Drosophila genes: a Genomics Education Partnership protocol. F1000Research 11: 1579. 10.12688/f1000research.126839.2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tello-Ruiz MK, Marco CF, Hsu FM, Khangura RS, Qiao P, Sapkota S, Stitzer MC, Wasikowski R, Wu H, Zhan J et al. 2019. Double triage to identify poorly annotated genes in maize: The missing link in community curation. PLoS One 14: e0224086–e0224013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang T, Blumhagen R, Lao U, Kuo Y, Edgar BA. 2012. LST8 Regulates Cell Growth via Target-of-Rapamycin Complex 2 (TORC2). Mol Cell Biol 32(12): 2203–2213. [DOI] [PMC free article] [PubMed] [Google Scholar]

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