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).
“
Roc1a
(
also known as
Rbx1
) is a member of the SCF E3 ubiquitin ligase complex and was originally identified through sequence similarity with vertebrate and yeast homologs and biochemical interaction studies (Bocca et al., 2001).
Roc1a
deletion mutants are lethal between the first and second larval instars and
Roc1a
mutant clones in imaginal discs have cell proliferation defects (Noureddine et al., 2002). In addition, Roc1a and other members of the SCF E3 ubiquitin ligase complex function in the pruning of larval neurons by targeting the insulin-responsive kinase Akt for ubiquitination and degradation, thus inhibiting insulin signaling (Wong et al., 2013).” (Lawson et al., 2025).
“
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).
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We propose a gene model for the
D. ananassae
ortholog of the
D. melanogaster
Regulator of cullins 1a
(
Roc1a
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein
LOC6504852
(RefSeq accession
XP_014759830.1
) in the May 2011 (Agencourt dana_caf1/DanaCAF1) Genome Assembly of
D. ananassae
(GenBank Accession:
GCA_000005115.1
; Drosophila 12 Genomes Consortium et al., 2007). This model is based on RNA-Seq data from
D. ananassae
(Gravely et al. 2011;
SRP006203
,
PRJNA257286
,
SRP007906
,
PRJNA388952
)
and
Roc1a
in
D. melanogaster
using FlyBase release FB2023_02 (
GCA_000001215.4
; Larkin et al.
2021; Gramates et al., 2022; Jenkins et al., 2022).
Synteny
The reference gene,
Roc1a
,
occurs on
chromosome X in
D. melanogaster
and is flanked upstream by
CG13367
and
suppressor of sable
(
Su(sable)
) and downstream by
Histone methyltransferase 4-20
(
Hmt4-20
)
and
SKP1-related A
(
SkpA
)
. The
tblastn
search of
D. melanogaster
Roc1a-PC (query) against the
D. ananassae
(GenBank Accession:
GCA_000005115.1
) Genome Assembly (database) placed the putative ortholog of
Roc1a
within scaffold_12929 (
CH902632.1
) at locus
LOC6504852
(
XP_014759830.1
)— with an E-value of 2e-63 and a percent identity of 68.29%. Furthermore, the putative ortholog is flanked upstream by
LOC6504851
(
XP_001966451.1
) and
LOC6504662
(
XP_001966450.2
), which correspond to
CG13367
and
CG5815
in
D. melanogaster
(E-value: 8e-176 and 0.0; identity: 68.19% and 67.55%, respectively, as determined by
blastp
;
Figure 1A,
Altschul et al., 1990). The putative ortholog of
Roc1a
is flanked downstream by
LOC6504661
(
XP_032309073.1
) and
LOC26514483
(
XP_014759887.1
), which correspond to
Hmt4-20
and
Ocrl
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 67.40% and 69.68%, respectively, as determined by
blastp
). The putative ortholog assignment for
Roc1a
in
D. ananassae
is supported by the partial conservation of the synteny of this genomic neighborhood, and all
BLAST
results indicate good-quality matches.
Protein Model
Roc1a
in
D. ananassae
has two unique protein-coding isoforms: Roc1a-PA (identical to Roc1a-PD) and Roc1a-PC (
Figure 1B
). mRNA isoforms
Roc1a-RA
and
Roc1a-RD
contain three protein-coding CDSs. mRNA isoform
Roc1a-RC
has two CDSs, with its first CDS (FlyBase ID: 1_2094_0) spanning the length of the first two CDSs of
Roc1a-RA
and
Roc1a-RD
(FlyBase IDs: 2_2094_0 and 3_2094_0) combined (all CDS IDs based on FlyBase release FB2023_02;
GCA_000001215.4
). Relative to the ortholog in
D. melanogaster
, the RNA CDS number and isoform structure is conserved
.
The sequence of
Roc1a-PC
in
D. ananassae
has 79.43% identity (E-value: 2e-76) with the
protein-coding isoform
Roc1a-PC
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). Coordinates of this curated gene model (Roc1a-PC, Roc1a-PA, Roc1a-PD) are stored by NCBI at GenBank/BankIt (accession
BK064627
,
BK064628
,
BK064629
, respectively). These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
Special characteristics of the protein model
Low sequence similarity in the latter portion of the first CDS of
Roc1a-RC
Boxes 1C-A and 1D-B highlight a region of decreased sequence similarity in the alignment of the first CDS of
Roc1a-RC
in
D. melanogaster
and
D. ananassae
. As shown in the dot plot and protein alignment (
Figure 1C-
A; 1D-B), many of the dissimilarities between the
Roc1a-RC
protein sequence in
D. melanogaster
and
D. ananassae
are found in the latter portion of the first CDS, which is unique to only isoform
Roc1a-RC
, as isoforms
Roc1a-RA
and
Roc1a-RD
have spliced out this region. A
blastp
search of of
Roc1a-PA
in
D. ananassae
has 96.30% identity (E-value: 5e-76) with the
protein-coding isoform
Roc1a-PA
in
D. melanogaster
, which is much higher relative to the 79.43% identity of the same search using Roc1a-PC. The significantly higher identity value for the other isoforms of
Roc1a
in
D. ananassae,
taken in the context of the protein alignment shown in figure 1D, provides further evidence that this is likely the correct ortholog, and that while the portion of the protein alignment unique to Roc1a-PC contains low sequence similarity, the remaining regions of
Roc1a
are very well-conserved.
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.