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)
.
“
D. eugracilis
is part of the
melanogaste
r species group within the subgenus
Sophophora
of the genus
Drosophila
(Pélandakis et al., 1993)
. It was first described as
Tanygastrella gracilis
by Duda (1924) and revised to
Drosophila eugracilis
by Bock and Wheeler (1972).
D. eugracilis
is found in humid tropical and subtropical forests across southeast Asia (https://www.taxodros.uzh.ch, accessed 1 Feb 2023).”
(Morgan et al., 2022)
.
|
We propose a gene model for the
D. eugracilis
ortholog of the
D. melanogaster
Regulator of cullins 1a
(
Roc1a
) gene. The genomic region of the ortholog corresponds to the uncharacterized protein
LOC108105484
(RefSeq accession
XP_017067590.1
) in the Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly of
D. eugracilis
(GenBank Accession:
GCA_000236325.2
). This model is based on RNA-Seq data from
D. eugracilis
(
PRJNA63469
- Chen et al., 2014)
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).
Roc1a
(also known as
Rbx1, Ring-Box 1, dRbx1, EG:115C2.11 , ROC1
) 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)
.
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-PA (query) against the
D. eugracilis
(GenBank Accession:
GCA_000236325.2
) Genome Assembly (database) placed the putative ortholog of
Roc1a
within scaffold KB465011 (KB465011.1) at locus
LOC108105484
(
XP_017067590.1
)— with an E-value of 5e-76 and a percent identity of 96.30%. Furthermore, the putative ortholog is flanked upstream by
LOC108105536
(
XP_017067657.1
) and
LOC108105511
(
XP_017067625.1
), which correspond to
CG13367
and
CG5815
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 75.82% and 81.68%, respectively, as determined by
blastp
;
Figure 1A, A
ltschul et al. 1990). The putative ortholog of
Roc1a
is flanked downstream by
LOC108105483
(
XP_017067589.1
) and
LOC108105410
(
XP_017067446.1
), which correspond to
Hmt4-20
and
SkpA
in
D. melanogaster
(E-value: 0.0 and 4e-117; identity: 84.47% and 98.77%, respectively, as determined by
blastp
;
Figure 1A
). The putative ortholog assignment for
Roc1a
in
D. eugracilis
is supported by the following evidence: the genetic neighborhoods of
Roc1a
in
D. melanogaster
and
D. eugracilis
are completely syntenic with the exception of the second farthest upstream gene, and all
BLAST
search results indicate very high-quality matches.
Protein Model
Roc1a
in
D. eugracilis
has one unique protein-coding isoforms (Roc1a-PA and Roc1a-PD;
Figure 1B
) encoded by mRNA isoforms
Roc1a-RA
and
Roc1a-RD
containing three identical protein-coding CDSs. Relative to the ortholog in
D. melanogaster
, the RNA CDS number is conserved for these isoforms, as
Roc1a-RA
and
Roc1a-RD
in
D. melanogaster
also contain three CDSs. However,
D. melanogaster
also has a third isoform with two CDSs,
Roc1a-RC
, that is not present in
D. eugracilis
(see: “special characteristics”). The sequence of
Roc1a-PA
in
D. eugracilis
has 96.30% identity (E-value: 5e-76) with the
protein-coding isoform
Roc1a-PA
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
)
.
Coordinates of this curated gene model (Roc1a-PA, Roc1a-PD) are stored by NCBI at GenBank/BankIt (accession
BK064556
,
BK064557
, respectively
)
. These data are also archived in the CaltechDATA repository (see “Extended Data” section below).
Special characteristics of the protein model
mRNA isoform
Roc1a-RC
in
D. melanogaster
is very similar to the other two isoforms,
Roc1a-RA
and
Roc1a-RD
. The difference between these isoforms is that
Roc1a-RA
and
Roc1a-RD
have three CDSs whereas
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
; Larkin et al.,
2021). In
D. melanogaster
, the reading frames for the first and second CDSs of
Roc1a-RA
and
Roc1A-RD
are the same, so for isoform
Roc1a-RC
in which the two CDSs are combined, translation is not disrupted. However, in
D. eugracilis,
the reading frames for the first and second CDSs of
Roc1a-RA
and
Roc1A-RD
are different, so the continuous reading frame for the combined CDS in
Roc1a-RC
results in the incorrect reading frame later in translation, and thus the presence of in-frame stop codons in the final CDS of this isoform. This has been highlighted in
Figure 1D
through the differing CDS colors corresponding to their respective frames, and the white portion of the final CDS in
Roc1a-RC
indicating the presence of in-frame stop codons. This, in combination with the lack of RNA-Seq data and TransDecoder Transcript predictions supporting the existence of
Roc1a-RC
, suggests that
Roc1a-RC
is likely absent from
D. eugracilis
(
Figure 1D
)
.
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.