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)
.
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We propose a gene model for the
D. miranda
ortholog of the
D. melanogaster
Pten
gene. The genomic region of the ortholog corresponds to the uncharacterized protein
LOC108161604
(RefSeq accession
XP_033249503.1
) in the Apr. 2013 (UC Berkeley DroMir_2.2/DmirGB2) Genome Assembly of
D. miranda
(
GCA_000269505.2
). This model is based on RNA-Seq data from
D. miranda
(
SRP009365
)
and
Pten
in
D. melanogaster
using FlyBase release FB2023_02 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022).
The
Drosophila
Phosphatase and tensin homolog
(
Pten
also known as
dPTEN
; FBgn0026379), identified due to is conservation to the human tumor suppressor gene acts as a protein and lipid phosphatase in the insulin signaling pathway
(Goberdhan et al., 1999; Huang et al., 1999)
. Pten is known to affect cell number and size through the inhibition of the phosphoinositide 3-kinase (PI3K) and AKT kinase pathways
(Goberdhan et al., 1999; Gao et al., 2000)
. Pten is involved in stabilizing cell junctions
(Bardet et al., 2013)
and regulates the cytoskeleton, controlling the localization and organization of actin
(Goberdhan et al., 1999; von Stein et al., 2005)
.
D. miranda
(NCBI:txid 7229) is part of the
pseudoobscura
species subgroup within the
obscura
species group in the subgenus
Sophophora
of the genus
Drosophila
(Sturtevant 1942; Buzzati-Traverso and Scossiroli 1955)
. It was first described by Dobzhansky in 1935. Like other members of the species subgroup, it is endemic to the New World distributed through Canada, the USA, and Mexico, sympatric with its sibling species
D. pseudoobscura
(Markow and O'Grady 2006), living in temperate forest environments.
Synteny
The reference gene,
Pten
,
occurs on
chromosome chr2L in
D. melanogaster
and is flanked upstream by
Repressor splicing factor 1
(
Rsf1
)
and
REPTOR-binding partner
(
REPTOR-BP
)
and downstream by
CG5676
and
Ror
(
Ror
). The
tblastn
search of
D. melanogaster
Pten-PB against the
D. miranda
(
GCA_000269505.2
) Genome Assembly (database) placed the putative ortholog of
Pten
within scaffold CM001520 (CM001520.2) at locus
LOC108161604
(
XP_033249503.1
)— with an E-value of 1e-36 and a percent identity of 86.15%. Furthermore, the putative ortholog is flanked upstream by
LOC108162205
(
XP_017152305.1
) and
LOC108162202
(
XP_017152302.2
), which correspond to
eukaryotic translation elongation factor 1 delta
(
eEF1delta
) and
Ror
in
D. melanogaster
(E-value: 2e-103 and 0.0; identity: 65.25% and 84.53%, respectively, as determined by
blastp
;
Figure 1A, A
ltschul et al., 1990). The putative ortholog of
Pten
is flanked downstream by
LOC108161611
(
XP_017151399.1
) and
LOC108161608
(
XP_017151393.1
), which correspond to
defective proboscis extension response 21
(
dpr21
) and
FERM domain containing
(
Frmd5
) in
D. melanogaster
(E-value: 4e-156 and 0.0; identity: 76.51% and 75.64%, respectively, as determined by
blastp
). The putative ortholog assignment for
Pten
in
D. miranda
is supported by the following evidence: The
tblastn
results are of very good quality, and all isoforms and CDSs present in
D. melanogaster
appear to be present in
D. miranda
as well. While local synteny is not well-conserved in this neighborhood, this is likely still the correct ortholog to
Pten
in
D. miranda
.
Additionally, while the nearby
Ror
gene is not syntenic across the two neighborhoods, its proximity to
Pten
in both species is further evidence that this is the correct ortholog assignment for
Pten
in
D. miranda
.
Protein Model
Pten
in
D. miranda
has seven protein coding isoforms, Pten-PA, Pten-PB, Pten-PC, Pten-PD, Pten-PE, Pten-PG, Pten-PH (
Figure 1B
). Protein isoforms Pten-PA and Pten-PE are identical, and are encoded by ten CDSs in the genome and translated from mRNAs Pten-RA and Pten-RE (which differ in their UTRs). Protein isoforms Pten-PB, Pten-PD, Pten-PG, and Pten-PH are identical, and are encoded by ten CDSs in the genome and translated from mRNAs Pten-RB, Pten-RD, Pten-RG, and Pten-RH (which differ in their UTRs). Protein isoform Pten-PC is encoded by nine CDSs in the genome, and translated from mRNA Pten-RC. Relative to the ortholog in
D. melanogaster
, the CDS number is conserved, as
D. melanogaster
also has seven mRNA isoforms, which translate into three unique protein sequences, and two encoded by ten CDSs and one encoded by nine CDSs. The sequence of
Pten-PB
in
D. miranda
has 64.99% identity (E-value: 0.0) with the
protein-coding isoform
Pten-PB
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). Some regions of low sequence similarity exist particularly in CDSs five through ten, with CDS eight being substantially shorter in the target gene relative to the reference gene. Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession
BK064496
,
BK064497
,
BK064498
,
BK064499
,
BK064500
,
BK064501
,
BK064502
)
. These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
Special characteristics of the protein model
Lack of Synteny
: The genomic neighborhood of
Pten
in
D. melanogaster
is not syntenic to the neighborhood of the
Pten
ortholog in
D. miranda
.
However, due to the high-quality matches from the blast searches of the target gene itself, as well as the high conservation of all isoforms and CDSs, this is likely still the correct ortholog of
Pten
in
D. miranda
,
and the lack of synteny is likely due to expected divergence between the two species.
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
target gene for a given isoform and run it using
tblastn
against their target
Drosophila
species genome assembly [
D. miranda
(
GCA_000269505.2
)] 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. Genomic structure information (e.g., CDSs, CDS 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 each gene are generated by students under mentorship of their faculty course instructors. These models are then reconciled by a third independent researcher mentored by the project leaders to produce a final model like the one presented here. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.