Description
|
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)
.
|
We propose a gene model for the
D. ananassae
ortholog of the
D. melanogaster
Insulin-like peptide 5 (
Ilp5
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein
XP_001956270.2
(Locus ID
LOC6502822
) in the May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly of
D. ananassae
(
GCA_000005115.1
- Drosophila 12 Genomes Consortium et al., 2007). This model is based on RNA-Seq data from
D. ananassae
(
SRP006203
,
PRJNA257286
,
SRP007906
,
PRJNA388952
)
and
Ilp5
in
D. melanogaster
using FlyBase release FB2023_03 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022; Jenkins
et al., 2022).
In animals, the pathway plays a central role in growth, development, reproduction, in addition to metabolism
(Semaniuk et al., 2021)
. In
Drosophila
, signaling starts when Insulin-like peptides (Ilp), of which there are eight, bind to their receptors. Ilp5 shows particular conservation from
Drosophila
to mammals, binding to insect insulin-binding proteins, and also shown to lower blood glucose levels when administered to rats
(Sajid et al., 2011)
. In
Drosophila
,
Ilp5
expression is highest in the insulin-producing cells in the brain, but also found expressed in the gut and ovaries of larvae, and neuroendocrine cells of both adults and larva
(Semaniuk et al., 2021)
. Flies lacking Ilp5 function show defects in fertility,
(Grönke et al. 2010; Strilbystka et al. 2020)
, behavior
(Semaniuk et al. 2018; Strilbystka et al. 2020)
, metabolism
(Semaniuk et al. 2018; Strilbystka et al. 2020)
, and lifespan
(Grönke et al. 2010)
.
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 Doleschall (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)
.
Synteny
Ilp5
occurs on
chromosome 3L in
D. melanogaster
and is nested by
CG43897
,
flanked upstream by
I-2
and
Cdk8
,
and flanked downstream by
CG32052
and
Ilp1
.
CG32052
nests
Ilp4
,
Ilp3
,
and
Ilp2
.
We determined that the putative ortholog of
Ilp5
is found on scaffold 13337 (
CH902618.1
) in
D. ananassae
(CAF1 assembly
GCA_000005115.1
) with
LOC6502822
(
XP_001956270.2
) (via
blastp
search with an e-value of 8e-09 and percent identity of 39.51%), where it is nested by
LOC6507311
(
XP_032310388.1
), which corresponds to
CG43897
in
D. melanogaster
with an e-value of 0.0 and percent identity of 68.77%, as determined by
blastp
. The
Ilp5
ortholog is flanked upstream by
LOC6507750
(
XP_001956268.3
) and
LOC6507312
(
XP_001956267.1
), which correspond to
I-2
and
iPLA2-VIA
in
D. melanogaster
with e-values of 1e-84 and 0.0 respectively and percent identities of 72.50% and 90.87% respectively, as determined by
blastp
. The
Ilp5
ortholog is flanked downstream by
LOC6507310
(
XP_014765147.1
) and
LOC6507308
(
XP_001956275.2
), which correspond to
CG32052
and
Ilp1
in
D. melanogaster
with e-values of 0.0 and 4e-24 respectively and percent identities of 86.67% and 52.46% respectively, as determined by
blastp
. The
CG32052
ortholog (
LOC6507310
,
XP_014765147.1
) nests
LOC6507751
(
XP_032309882.1
),
LOC6507752
(
XP_001956273.1
), and
LOC6507309
(
XP_001956274.1
), which correspond to
Ilp4
,
Ilp3
,
and
Ilp2
in
D. melanogaster
with e-values of 7e-28, 1e-21, and 2e-27 respectively and percent identities of 48.91%, 46.32%, and 46.79% respectively, as determined by
blastp
(
Figure 1A, A
ltschul et al., 1990).
We argue this is the correct ortholog assignment for
Ilp5
in
D. ananassae
because all but one gene in the local genomic neighborhood are syntenic and all of the
blastp
results used to determine orthology were of high-quality.
Protein Model
Ilp5
in
D. ananassae
has one protein coding isoform, Ilp5-PB (
Figure 1B
). Isoform Ilp5-PB contains two CDSs. This is the same relative to the ortholog in
D. melanogaster
, which also has one protein-coding isoform with two CDSs. The sequence of
Ilp5
in
D. ananassae
has 35.3% identity with the
Ilp5
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 accession BK064649. These data are also available in Extended Data files below, which are archived in CaltechData.
Special characteristics of the Protein Model
The dot plot for the alignment of the Ilp5-PB sequences in
D. melanogaster
and
D. ananassae
(Figure C) does not show any regions of sequence similarity. This is due to the default parameters of the dot plot requiring a word size of six, which is not present. However, the protein alignment (Figure D) shows that while there are no consecutive identities of six or higher, there are regions of Ilp5-PB that are conserved between
D. ananassae
and
D. melanogaster.
The lack of a full six-length word could be the result of mutations accumulating over time, in combination with the small size of this gene.
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. ananassae;
GCA_000005115.1
) 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 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 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.