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.
mojavensis
(NCBI:txid7230)
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, accessed 1 Feb 2023).” (Congleton et al., 2023).
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We propose a gene model for the
D. mojavensis
ortholog of the
D. melanogaster
Salt-inducible kinase 3
(
Sik3
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein
XP_015019060.1
(Locus ID
LOC6579294
) in the May 2011 (Agencourt dmoj_caf1/DmojCAF1) Genome Assembly of
D. mojavensis
(
GCA_000005175.1
). This model is based on RNA-Seq data from
D. mojavensis
(
SRP006203
- Chen et al., 2014)
and
Sik3
in
D. melanogaster
using FlyBase release FB2023_03 (
GCA_000001215.4
; Larkin et al.,
2021; Gramates et al., 2022; Jenkins et al., 2022).
The gene
Sik3
(
Salt-inducible kinase 3
) is related to the AMPK Ser/Thr class of kinases and was identified using sequence homology upon comparison of the human and mouse
SIK
genes with the
Drosophila melanogaster
genome (Okamoto et al., 2004).
Sik3
null mutants are not viable, so studies in
D. melanogaster
used hypomorphic alleles to characterize the role of Sik3 in the Insulin/TOR pathway (Wang et al., 2011). Under fed conditions, Sik3 is phosphorylated and activated downstream of Akt and liver kinase B1 (LKB1) where Sik3 promotes sequestration of Histone Deacetylase 4 (HDAC4) in the cytoplasm via phosphorylation (Wang et al., 2011; Choi et al., 2015). Under fasting conditions, Protein Kinase A (PKA) phosphorylates and inactivates Sik3, allowing HDAC4 to translocate to the nucleus and activate the transcription factor Forkhead Box, subgroup O (dFOXO) (Walkinshaw et al., 2013; Wang et al., 2011). Sik3 has also been shown to negatively regulate the Hippo signaling pathway in
Drosophila
and to be involved in circadian rhythm regulation in a range of species from flies to mice (Wehr et al., 2013; Funato et al., 2016; Liu et al., 2022).
Synteny
Sik3
occurs on
chromosome 2R in
D. melanogaster
and is flanked by upstream genes
CG44433
,
CG42855
,
and downstream genes
CG15073
, Vacuolar protein sorting 51
(
Vps51
)
.
Sik3
holds a nested gene of
CG15071
.
It has been determined that the putative ortholog of
Sik3
is found on scaffold
CH933808.1
(scaffold_6496) in
D. mojavensis
with
LOC6579294
(
XP_015019060.1
, via
tblastn
search with an e-value of 0.0 and percent identity of 67.19%), where it is surrounded by upstream genes
LOC116804149
(
XP_032586360.1
) and
LOC26528447
(
XP_015019062.1
), which correspond to
CG44433
and
CG42855
in
D. melanogaster
with e-values 0.48, 7e-11 and and percent identities of 65.00%, 52.63%, respectively, as determined by
blastp
(
Figure 1A,
Altschul et al., 1990).
The nested gene within the putative ortholog has a LOCID of
LOC26528208
(
XP_015019061.1
) and it corresponds to
CG15071
in
D. melanogaster
with an e-value of 4e-56 and a percent identity of 52.15%. The putative ortholog is flanked downstream by
LOC6579292
(
XP_002005188.1
) and
LOC6579291
(
XP_032586692.1
), which correspond to
CG15073
and
Vps51
in
D. melanogaster
with e-values of 0.0, and percent identities 60.22%, 91.87%, respectively, as determined by
blastp
. This is likely the correct ortholog assignment for
Sik3
in
D. mojavensis
for two reasons: 1) the best alignment indicated with a
blastp
search resulting in
Sik3
with an e-value of 0.0 and a percent identity of 78.35%; and 2) the local synteny is highly conserved, consisting of the upstream and downstream genes being orthologous to
D. melanogaster
(
Figure 1A
)
.
Protein Model
Sik3
in
D. mojavensis
has six protein coding isoforms (Sik3-PA, Sik3-PB, Sik3-PC, Sik3, PD, Sik3-PE, Sik3-PF) (
Figure 1B
). mRNA isoform
Sik3-RA
contains seven CDSs. mRNA isoforms
Sik3-RC
,
Sik3-RD
,
Sik3-RB
all contain eleven CDSs, and mRNA isoforms
Sik3-RF
and
Sik3-RE
contain ten CDSs. These isoforms are the same relative to the ortholog in
D. melanogaster
which contains six protein coding isoforms (Sik3-PA, Sik3-PB, Sik3-PC, Sik3, PD, Sik3-PE, Sik3-PF) with the same CDS structure. The dot plot that compares the protein alignment between
D. mojavensis
and
D. melanogaster
shows an indel within the 10th CDS, and the sequence alignment has a percent identity of 78.35% as determined by
blastp
(
Figure 1C
).
The coordinates of the curated gene models (Sik3-PC, Sik3-PB, Sik3-PD, Sik3-PE, Sik3-PF and Sik3-PA) can be found in NCBI at GenBank using the accessions
BK064485
,
BK064486
,
BK064487
,
BK064488
,
BK064489
and
BK064490
. 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.