Description
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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. simulans
(NCBI:txid7240) 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 Sturtevant (1919).
D. simulans
is a sibling species to
D. melanogaster
, thus extensively studied in the context of speciation genetics and evolutionary ecology (Powell 1990). Historically,
D. simulans
was a tropical species native to sub-Saharan Africa (Lemeunier et al., 1986) where figs served as a primary host (Lachaise and Tsacas 1983). However,
D. simulans's
range has expanded worldwide within the last century as a human commensal using a broad range of rotting fruits as breeding sites (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).” (Lawson et al., 2024).
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We propose a gene model for the
D. simulans
ortholog of the
D. melanogaster
lin-28
gene. The genomic region of the ortholog corresponds to the uncharacterized protein
LOC27209473
(RefSeq accession
XP_016030549.1
) in the May 2017 (Princeton ASM75419v2/DsimGB2) Genome Assembly of
D. simulans
(GenBank Accession:
GCA_000754195.3
). This model is based on RNA-Seq data from
D. simulans
(
SRP006203
; Graveley et al., 2011)
and
lin-28
in
D. melanogaster
using FlyBase release FB2023_02 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022; Jenkins et al., 2022).
lin-28
(
lin-28
) is a positive regulator of the insulin signaling (Zhu et al., 2011) and JAK-STAT (Sreejith et al., 2019) pathways.
lin-28
was discovered in
Caenorhabditis elegans
by mutations that produce heterochronic shifts in cell fate specification (Ambros and Horvitz 1984). Homologs were identified later in other animals, including
Drosophila
,
Xenopus
, mouse, and human (Moss and Tang 2003). lin-28 binds to many mRNA molecules to regulate their translation or stability (Balzer and Moss 2007; Cho et al., 2012). In
Drosophila
, lin-28 binds to the
insulin-like receptor (InR)
mRNA and stimulates the symmetric division of intestinal stem cells in response to nutrients (Chen et al., 2015; Luhur and Sokol 2015). In mammals, LIN28, in combination with OCT4, SOX2, and NANOG, can reprogram differentiated somatic cells to pluripotency (Yu et al., 2007).
Synteny
The referece gene,
lin-28
,
occurs on
chromosome 3L in
D. melanogaster
and is flanked upstream by
Blimp-1
and
Esa1-associated factor 6
(
Eaf6
)
and downstream by
Separase
(
Sse
) and
CG46320
.
The
tblastn
search of
D. melanogaster
lin-28-PA (query) against the
D. simulans
(GenBank Accession:
GCA_000754195.3
) Genome Assembly (database) placed the putative ortholog of
lin-28
within scaffold CM002912 (CM002912.1) at locus
LOC27209473
(
XP_016030549.1
)— with an E-value of 1e-42 and a percent identity of 97.67%. Furthermore, the putative ortholog is flanked upstream by
LOC27208506
(
XP_016030546.1
) and
LOC6736898
(
XP_002083751.1
), which correspond to
Blimp-1
and
Eaf6
in
D. melanogaster
(E-value: 0.0 and 2e-162; identity: 98.68% and 98.22%, respectively, as determined by
blastp
;
Figure 1A,
Altschul et al., 1990). The putative ortholog of
lin-28
is flanked downstream by
LOC6736901
(
XP_002083754.1
) and
LOC6736902
(
XP_016030550.1
), which correspond to
Sse
and
CG46320
in
D. melanogaster
(E-value: 0.0 and 2e-34; identity: 95.90% and 100.00%, respectively, as determined by
blastp
). The ortholog assignment for
lin-28
in
D. simulans
is supported by the following evidence: the synteny of the genomic neighborhood is completely conserved across both species, and all
BLAST
results used to determine orthology indicate very high-quality matches.
Protein Model
lin-28
in
D. simulans
has one protein-coding isoform, lin-28-PA (
Figure 1B
). mRNA isoform
lin-28-RA
contains five CDSs. Relative to the ortholog in
D. melanogaster
, the CDS number is conserved, as
lin-28
in
D. melanogaster
also has only one RNA isoform with five CDSs. The sequence of
lin-28-PA
in
D. simulans
has 95.90% identity (E-value: 1e-139) with the
protein-coding isoform
lin-28-PA
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession
BK064527
). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
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.