Gene model for the ortholog of wrd in Drosophila ananassae

TL;DR

This paper presents a gene model for the wrd ortholog in Drosophila ananassae to study the evolution of a signaling pathway.

Contribution

The paper provides a new gene model for studying the evolution of the IIS pathway in Drosophila.

Findings

A gene model for the wrd ortholog was characterized in Drosophila ananassae.

The model is part of a dataset for studying the evolution of the IIS pathway.

It follows a gene annotation protocol for undergraduate research experiences.

Abstract

Gene model for the ortholog of
well-rounded
(
wrd
) in the May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly (GenBank Accession:
GCA_000005115.1
) of
Drosophila ananassae
. 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.

Figures

B](#f1) ). The protein isoform encoded by mRNAs *wrd-RD, wrd-RE, wrd-RM, wrd-RL* , and *wrd-RN* (which differ in their UTRs) contains seven CDSs. The protein isoform encoded by mRNAs *wrd-RK* and *wrd-RO* contain eight protein-coding CDSs. Isoform wrd-PJ, is encoded by *wrd-RJ * which contains eight CDSs. Protein isoform wrd-PI is encoded by five CDSs. Isoform wrd-PQ is encoded by nine CDSs. Isoform wrd-PP is encoded by eight CDSs. Isoform wrd-PC is encoded by six CDSs. Isoform wrd-PB is encoded by eight CDSs. Isoform wrd-PA is encoded by seven CDSs. This isoform strucutre is the same relative

Tables

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 this box 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 fruitfly 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). “ D . ananassae (NCBI:txid7217) is part of the melanogaster species group within the subgenus Sophophora of the genus Drosophila (Sturtevant 1939; Bock and Wheeler 1972). It was first described by Doeschall (1858). D. ananassae is circumtropical (Markow and O'Grady 2005; https://www.taxodros.uzh.ch , accessed 1 Feb 2023), and often associated with human settlement (Singh 2010). It has been extensively studied as a model for its cytogenetic and genetic characteristics, and in experimental evolution (Kikkawa 1938; Singh and Yadav 2015).” (Lawson et al., 2024).

Full text

Description

**: **

The model presented here is the ortholog of wrd in the May 2011 (Agencourt dana_caf1/DanaCAF1) assembly of D. ananassae ( GCA_000005115.1 – Drosophila 12 Genomes Consortium et al., 2007) and corresponds to the Gnomon Peptide ID ( XP_032305638.1 ) predicted model in

• D. ananassae * ( LOC6499555 ) . This gene model is based on RNA-Seq data from D. ananassae (Gravely et al . , 2011; SRP006203 , PRJNA257286 , SRP007906 , PRJNA388952 ) and the
• wrd * ( GCA_000001215.4 ) in *D. melanogaster * from FB2022_03 (Larkin et al *., * 2021; Gramates et al., 2022; Jenkins et al., 2022).

The well-rounded ( wrd ) gene was first identified in a gain-of-function screen for molecules that regulate synaptic development in the neuromuscular junction (NMJ) in Drosophila melanogaster (Viquez et al., 2006). Its gene product is one of the two B' regulatory subunits of protein phosphatase 2A (PP2A), which interacts with liprin-α to stabilize specification of the active zone, a site in the axonal plasma membrane that regulates endo- and exocytosis of synaptic vesicles (Li et al., 2014). Another interacting partner of wrd is S6K, an important kinase in the insulin/TOR signaling pathway that is targeted for dephosphorylation by the PP2A holoenzyme through its molecular interaction with wrd (Hahn et al., 2010). Loss-of-function wrd mutants are viable and have fewer synaptic boutons that are larger and have a smooth round contour, a phenotype which gave the gene its name (Viquez et al., 2006). Knockout flies for the *wrd * B' subunit of PP2A are lean, have a reduced lifespan, and elevated insulin signaling (Hahn et al., 2010), with tissue specificity fine-tuned by cyclin G (Fischer et al., 2016).

** Synteny **

*wrd * occurs on chromosome 3L in *D. melanogaster * and is flanked upstream by * CG7215 * and Peroxiredoxin 5 ( * Prx5 ) * , and downstream by cadmus ( * cdm ) * and * CG7208 . * We determined that the putative ortholog of wrd is found on scaffold 13340 ( CH902617.1 ) in D. ananassae with LOC6499555 ( XP_032305638.1 ) (via tblastn search with an e-value of 0.0 and percent identity of 88.81%). The *wrd * ortholog is flanked upstream by LOC6501022 ( XP_001954393.1 ) and LOC6501023 ( XP_001954394.2 ) which correspond to * CG7215 * and * Prx5 * in *D. melanogaster * with e-values of 7e-78 and 3e-127, respectively, and percent identities 82.31% and 93.68%, respectively, as determined by blastp . The *wrd * ortholog is flanked downstream by LOC6501021 ( XP_001954390.2 ) and LOC6499556 ( XP_001954389.1 ) which correspond to

• cdm * and

CG6142 * in *D. melanogaster * with e-values of 0.0 and 0.0, respectively, and percent identities of 87.44% and 88.64%, respectively, as determined by blastp ( Figure 1A, Altschul et al . , 1990). We believe this is the correct ortholog assignment for wrd in D. ananassae because synteny is well-conserved, with only one gene in the genomic neighborhood being non-orthologous, and because all of the BLAST searches used to determine orthology were very high quality.

** Protein Model **

*wrd * in

• D. ananassae * has nine unique protein-coding isoforms ( Figure 1B ). The protein isoform encoded by mRNAs wrd-RD, wrd-RE, wrd-RM, wrd-RL , and wrd-RN (which differ in their UTRs) contains seven CDSs. The protein isoform encoded by mRNAs wrd-RK and wrd-RO contain eight protein-coding CDSs. Isoform wrd-PJ, is encoded by *wrd-RJ * which contains eight CDSs. Protein isoform wrd-PI is encoded by five CDSs. Isoform wrd-PQ is encoded by nine CDSs. Isoform wrd-PP is encoded by eight CDSs. Isoform wrd-PC is encoded by six CDSs. Isoform wrd-PB is encoded by eight CDSs. Isoform wrd-PA is encoded by seven CDSs. This isoform strucutre is the same relative to the ortholog in D. melanogaster , which also has 9 unique protein-coding isoforms. All of the *D. melanogaster * isoforms have the same number of CDSs as their corresponding ortholog in *D. ananassae. * The amino acid sequence of
• wrd * in
• D. ananassae* has 83.0% identity with the wrd in *D. melanogaster * as determined by
• blastp* ( Figure 1C ). The coordinates of the curated gene models can be found in NCBI at GenBank/BankIt using the accessions BK064428 , BK064429 , BK064430 , BK064431 , BK064432 , BK064433 , BK064434 , BK064435 , BK064436 , BK064437 , BK064438 , BK064439 , BK064440 , and BK064441 . These data are also available in Extended Data files below, which are archived in CaltechData.

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 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.