We propose a gene model for the D. ananassae ortholog of the D. melanogaster Insulin-like peptide 3 ( Ilp3 ) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_001956273.1 (Locus ID LOC6507752 ) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1 Genome Assembly ( GCA_000005115.1 ; Drosophila 12 Genomes Consortium et al., 2007)). This model is based on RNA-Seq data from D. ananassae ( SRP006203 , SRP007906 ; PRJNA257286 , PRJNA388952 ; Graveley et al., 2011) and Ilp3 in D. melanogaster using FlyBase release FB2022_04 ( GCA_000001215.4 ; Larkin et al., 2021; Gramates et al., 2022; Jenkins et al., 2022).

Invertebrate insulins function similarly to metazoan insulin-like growth factors and play a role in cell and organ growth (Jin Chan and Steiner 2000). In Drosophila, seven insulin-like peptides (Ilp1-Ilp7) have a two-chain structure similar to vertebrate insulin and interact with the sole insulin-like receptor, InR, to initiate the insulin signaling cascade (Brogiolo et al., 2001; Nassel and Broeck 2016). Like the Ilp2 and Ilp5 genes, the Ilp3 gene is expressed in median neurosecretory cells (MNCs) in the brain (Ikeya et al., 2002). While the seven Ilps act redundantly with respect to promoting growth, they also have unique expression patterns and functions (Ikeya et al., 2002; Grönke et al., 2010). Ilp3 may act with the transcription factor dFOXO in a positive feedback loop to regulate Ilp2 and Ilp5 secretion from MNCs (Grönke et al., 2010). In female Drosophila , ablation of MNCs or knockout of Ilp3 have been shown to reduce fecundity and remating rates (Grönke et al., 2010; Wigby et al., 2011). Knockout of Ilp3 also results in sleep defects (Yamaguchi et al., 2022).

Synteny

The reference gene, Ilp3 , occurs on chromosome 3L in D. melanogaster and is nested within CG32052 , alongside Insulin-like peptide 4 ( Ilp4 ) (upstream) and Insulin-like peptide 2 ( Ilp2 ) (downstream). Ilp3 is flanked further upstream by L-2-hydroxyglutarate dehydrogenase ( L2HGDH ) and CG43897 ( CG43897 ) and further downstream by Insulin-like peptide 1 ( Ilp1 ) and Z band alternatively spliced PDZ-motif protein 67 ( Zasp67 ). The tblastn search of D. melanogaster Ilp3-PA (query) against the D. ananassae (GenBank Accession: GCA_000005115.1 ) Genome Assembly (database) placed the putative ortholog of Ilp3 within scaffold scaffold_13337 ( CH902618.1 ) at locus LOC6507752 ( XP_001956273.1 )— with an E-value of 5e-08 and a percent identity of 31.13%. Furthermore, the putative ortholog is nested within LOC6507310 ( XP_014765147.1 ) alongside LOC6507751 ( XP_032309882.1 ; upstream) and LOC6507309 ( XP_001956274.1 ; downstream), which correspond to CG32052 , Ilp4 and Ilp2 in D. melanogaster (E-value: 0.0, 3e-31 and 2e-27; identity: 86.67%, 51.52% and 46.79%, respectively, as determined by blastp ; Figure 1A, Altschul et al., 1990). The putative ortholog is flanked further upstream by LOC6507750 ( XP_001956268.3 ) and LOC6507311 ( XP_032310388.1 ), that nests LOC6502822 ( XP_001956270.2 ); which correspond to L2HGDH , CG43897 , and Ilp5 in D. melanogaster (E-value: 2e-111, 0.0 and 9e-09; identity: 68.06, 69.28% and 39.51%, respectively, as determined by blastp ). The putative ortholog of Ilp3 is flanked downstream by LOC6507308 ( XP_001956275.2 ) and LOC6507753 ( XP_014765448.1 ), which correspond to Ilp1 and Zasp67 in D. melanogaster (E-value: 6e-35 and 0.0; identity: 52.46% and 71.34%, respectively, as determined by blastp ). The putative ortholog assignment for Ilp3 in D. ananassae is supported by the following evidence: The genes surrounding the Ilp3 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 LOC6507752 contains the correct ortholog of Ilp3 in D. ananassae ( Figure 1A ).

Protein Model

Consistent with the blastp search result which shows 51.52% identity between D. melanogaster Ilp3-PA and the D. ananassae gene model, the dot plot features a few minor gaps along the diagonal, indicating significant conservation between the two protein sequences. Ilp3 in D. ananassae has one protein-coding isoforms (Ilp3-PA; Figure 1B ). Isoform (Ilp3-PA) contains two CDSs. Relative to the ortholog in D. melanogaster , the CDS number and isoform count are conserved. The sequence of Ilp3-PA in D. ananassae has 54.74% identity (E-value: 8e-31) with the protein-coding isoform Ilp3-PA in D. melanogaster , as determined by blastp ( Figure 1C ). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK064566 ). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).

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 WJ 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. melanogaste r 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.