Gene model for the ortholog of Sik3 in Drosophila mojavensis

TL;DR

This paper describes the gene model for the Sik3 ortholog in Drosophila mojavensis as part of a study on the evolution of the IIS pathway.

Contribution

Provides a new gene model for Sik3 in Drosophila mojavensis for IIS pathway evolution research.

Findings

Identified the Sik3 ortholog in Drosophila mojavensis genome assembly.

Used the gene model to study the evolution of the IIS pathway in Drosophila.

Contributed to a dataset for Course-based Undergraduate Research Experiences.

Abstract

Gene model for the ortholog of
Salt-inducible kinase 3
(
Sik3
) in the May 2011 (Agencourt dmoj_caf1/DmojCAF1) Genome Assembly (GenBank Accession: GCA_000005175.1 ) of
Drosophila mojavensis
. This ortholog 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
using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Figures

B](#f1) ). 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

Tables

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).

Full text

Description

**: **

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.