microPublication Biology 2578-9430 Caltech Library 10.17912/micropub.biology.000677 new finding genotype data drosophila Drosophila mojavensis – chico Congleton Hannah 1 Kiser Cole A. 2 Colom Diaz Patricia A. 3 Schlichting Elizabeth 2 Walton Dorothy A. 1 Long Lindsey J. 1 Reed Laura K. 2 Martinez-Cruzado Juan Carlos 3 Rele Chinmay P. 2 § Oklahoma Christian University, Edmond, OK USA The University of Alabama, Tuscaloosa, AL USA University of Puerto Rico at Mayagüez, Mayaguez, PR Puerto Rico GEP Review Panel Correspondence to: Chinmay P. Rele ( cprele@ua.edu )

HC: Formal analysis, Validation, Writing - original draft, Writing - review & editing

CK: Formal analysis, Validation, Writing - original draft, Writing - review & editing

PC: Formal analysis, Writing - review & editing

ES: Formal analysis, Writing - review & editing

DW: Formal analysis, Writing - review & editing

LL: Supervision, Writing - review & editing

LR: Supervision, Writing - review & editing

JM: Supervision, Writing - review & editing

CR: Data curation, Formal analysis, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing - review & editing

 15 11 2022 2022 2022 10.17912/micropub.biology.000677 30 6 2021 1 1 1970 10 11 2022 Copyright: © 2022 by the authors 2022 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

(A) Synteny comparison of the genomic neighborhoods for chico in Drosophila melanogaster and D. mojavensis . Thin underlying arrows indicate the DNA strand within which the target gene– chico –is located on the negative strand in D. melanogaster (top) and D. mojavensis (bottom). The wide gene arrows pointing in the same direction as chico are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of chico are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. mojavensis indicate orthology to the corresponding gene in D. melanogaster , while black gene arrows indicate non-orthology. Gene symbols given in the D. mojavensis gene arrows indicate the orthologous gene in D. melanogaster , while the locus identifiers are specific to D. mojavensis . (B) Gene Model in GEP UCSC Track Data Hub (Raney et al ., 2014). The coding-regions of chico in D. mojavensis are displayed in the User Supplied Track (black); coding exons 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. mojavensis ), 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 and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. mojavensis ), and Splice Junctions Predicted by regtools using D. mojavensis RNA-Seq (Chen et al. , 2014; SRP006203). Splice junctions shown have a read-depth of 10-49 and 100-499 supporting reads in blue and pink. (C) Dot Plot of chico-PA in D. melanogaster ( x -axis) vs. the new PAA isoform in D. mojavensis ( y -axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. The red circle labeled 1, Regions which indicate a lack of similarity between the two sequences are highlighted in red (Region 1; Region 2). (D) Gene model in GEP UCSC displaying RNA-Seq and Splice Junctions data of exons six and seven of the ortholog in D. mojavensis . Boxes A and B indicate RNA-Seq drop-offs, boxes C and D indicate their corresponding splice acceptors and the region in between the boxes represent introns. The increase of RNA-Seq data in exons 6 and 7 and the existence of splice junction JUNC00059058 support that an additional isoform is present in D. mojavensis (chico-PAA).

Description

Introduction The insulin signaling pathway is a highly conserved pathway in animals and is central to nutrient uptake (Hietakangas and Cohen 2009; Grewal 2009). A key component of the pathway, encoding a substrate of the Insulin Receptor (InR) protein, chico binds to the InR and thus is also referred to as the Insulin Receptor Substrate (IRS). The IRS chico is essential to the control of cell and organ size (Goderdhan et al. 1999; Bohni et al. 1999), overall growth (Bakopoulos et al. 2020), including the sex-biased control of cell size and growth (Millington et al. 2021; Kim and O’Connor 2021), and is involved in lifespan (Clancy et al. 2001). We propose a gene model for the D. mojavensis ortholog of the D. melanogaster chico (chico) gene. The genomic region of the ortholog corresponds to the uncharacterized protein LOC6576893 (RefSeq accession XP_032585220.1) in the dmoj_caf1 Genome Assembly of D. mojavensis (GenBank Accession: GCA_000005175.1 - Chen et al. , 2014; SRP006203). This model is based on RNA-Seq data from D. mojavensis (Chen et al., 2014; SRP006203) and chico in D. melanogaster using FlyBase release FB2022_04 (GCA_000001215.4; Larkin et al. , 2021). D. mojavensis is part of the mulleri complex in the repleta species group within the subgenus Drosophila of the genus Drosophila (Wasserman 1992, Durando et al, 2000 ). It was first described by Patterson (Patterson and Crow, 1940). D. mojavensis specializes on rotting cactus as its host and is found in the Mojave and Sonoran Deserts of the southwestern United States and northwestern Mexico including the Baja Peninsula, as well as on the channel-islands off the coast of California (https://www.taxodros.uzh.ch). The Genomics Education Partnership maintains a mirror of the UCSC Genome Browser (Kent WJ et al. , 2002; Gonzalez et al. , 2021), which is available at https://gander.wustl.edu .

Synteny

The target gene, chico, occurs on chromosome 2L in D. melanogaster and is flanked upstream by CG31717 and basket (bsk) and downstream by maternal expression at 31B (me31B) and methuselah-like 15 (mthl15) . The tblastn search of D. melanogaster chico-PA (query) against the D. mojavensis (GenBank Accession: GCA_000005175.1) Genome Assembly (database) placed the putative ortholog of chico within scaffold scaffold_6500 (CH933807.1) at locus LOC6576893 (XP_032585220.1)— with an E-value of 0.0 and a percent identity of 44.97%. Furthermore, the putative ortholog is flanked upstream by LOC6576896 (XP_015020894.1) LOC6576895 (XP_002002875.4) and LOC6576894 (XP_015020893.1), which correspond to CG31717, Multi-substrate lipid kinase (Mulk) and trunk (trk) in D. melanogaster (E-value: 2e-77, 3e-155 and 3e-100; identity: 65.64%, 54.44% and 65.58%, respectively, as determined by blastp ; Figure 1A, Altschul et al. , 1990). The putative ortholog of chico is flanked downstream by LOC6576891 (XP_002002871.1) and LOC6576890 (XP_002002870.1), which correspond to CG33303 and CG5731 in D. melanogaster (E-value: 0.0 and 0.0; identity: 65.78% and 84.56%, respectively, as determined by blastp ). The putative ortholog assignment for chico in D. mojavensis is supported by the following evidence: Although the genes surrounding the chico ortholog are not orthologous to the genes at the same locus in D. melanogaster , we conclude that LOC6576893 is the correct ortholog of chico in D. mojavensis (Figure 1A), supported by strong e-values and percent identities and the lack of other matches while performing the blastp search.

Protein Model

Consistent with the blastp search result which shows 44.97% identity between D. melanogaster chico-PA and the D. mojavensis gene model as well as the low sensitivity parameters used to generate the dot plot (i.e., word size = 3; neighborhood threshold = 11), the dot plot of the two protein sequences contain multiple large gaps along the diagonal. chico in D. mojavensis has 3 identical protein-coding isoforms (chico-PA, chico-PB and chico-PC; Figure 1B). Isoform (chico-PA, chico-PB, chico-PC) contains 8 protein-coding exons. Relative to the ortholog in D. melanogaster , the coding-exon number is conserved. The sequence of chico-PA in D. mojavensis has 44.97% identity (E-value: 0.0) with the protein-coding isoform chico-PA in D. melanogaster , as determined by blastp (Figure 1C). While conducting our research, the presence of a new isoform of chico in D. mojavensis was discovered. The isoform identified as PA D. mojavensis is orthologous to the PA isoform in D. melanogaster and the PAA isoform is the novel isoform. Lower score Splice Junctions data due to lower levels of RNA-Seq data indicate the chico-PAA isoform is expressed at lower levels, or in fewer cells, than the chico-PA isoform (Figure 1D). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK059533). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).

Special characteristics of the protein model

Novel isoform: A new isoform of chico (chico-PAA) has been discovered based on the available RNA-Seq data and Splice Junctions data (Figure 1D). RNA-Seq data drops off in two regions, labeled A and B, and RNA-Seq data increase are displayed in two regions, labeled C and D. Blue and pink data tracks beneath the RNA-Seq data represent Splice Junctions, with pink splice junctions having a higher score than blue splice junctions (minimum read-depth scores found in above image caption of Figure 1A). The existence of splice junction JUNC00059058 as well as the decrease and sharp increase in RNA-Seq data in exons 6 and 7 which overlap the exons of isoform PA indicates an additional isoform (Figure 1D).

Extended Data

Description: FASTA. Resource Type: Model. DOI: 10.22002/n7ehe-xej65

Description: GFF. Resource Type: Model. DOI: 10.22002/26d2v-g8r63

Description: Peptide Sequence. Resource Type: Model. DOI: 10.22002/vbfq6-4z544

Acknowledgments

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model, and Madeline Gruys for retrofitting this model.

 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.

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