Nebulous without <i>white</i>: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in <i>Drosophila nebulosa</i>

Sottolano, Christopher J; Revaitis, Nicole T.; Geneva, Anthony J.; Yakoby, Nir

doi:10.1093/g3journal/jkac231

Cited by 3 publications

(7 citation statements)

References 111 publications

(150 reference statements)

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“…Probe designs for nos were based on the coding sequence for all species, FBgn0002962 for D. mel (FlyBase), UniProt entry A0A6I8V5B3 for D. pse , and Q24710 for D. vir . For D. neb , the nos and pgc sequences were identified using the D. neb annotated long-read sequenced genome ( Sottolano, et al 2022 ). For D. mel , cycB and gcl probes targeting their coding sequence were generated based on FlyBase sequences FBgn0000405, labeled with CAL Fluor Red 590 dye, and FBgn0005695, labeled with CAL Fluor Red 610 Dye, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…For osk sequences, UniProt entry numbers B4LXK5 and A0A6I8URE4 were used for D. vir and D. pse. For D. neb , rpl7 and osk sequences were determined using the D. neb annotated long-read sequenced genome ( Sottolano, et al 2022 ). For all expression assays, the presented fold gene expression levels are displayed as values relative to D. mel and were calculated using the 2(-Delta Delta C(T)) method ( Livak and Schmittgen 2001 ) using rpl7 as an internal control ( Valentino, et al 2022 ).…”

Section: Methodsmentioning

confidence: 99%

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Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development

Doyle,

Burian,

Aharoni

et al. 2023

Molecular Biology and Evolution

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The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate germline mRNAs. In D. melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3′ UTR of germ granule mRNAs. Interestingly, the 3′ UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species’ homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3′ UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development

Doyle,

Burian,

Aharoni

et al. 2023

Molecular Biology and Evolution

Self Cite

View full text Add to dashboard Cite

show abstract

“…Probe designs for nos were based on the coding sequence for all species, FBgn0002962 for D. mel (FlyBase), UniProt entry A0A6I8V5B3 for D. pse , and Q24710 for D. vir . For D. neb , the nos and pgc sequences were identified using the D. neb annotated long-read sequenced genome [46]. For D. mel, cycB , and gcl probes targeting their coding sequence were generated based on FlyBase sequences FBgn0000405, labeled with CAL Fluor Red 590 dye, and sequence FBgn0005695, labeled with CAL Fluor Red 610 Dye, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…For osk sequences, UniProt entry numbers B4LXK5 and A0A6I8URE4 were used for D. vir and D. pse . For D. neb , rpl7 and osk sequences were determined using the D. neb annotated long-read sequenced genome [46]. For all expression assays, the presented fold gene expression levels are relative to D. mel and were calculated using the 2(-Delta Delta C(T)) method [49] using rpl7 as an internal control [20].…”

Section: Methodsmentioning

confidence: 99%

“…The D. mel neb nos 3′ UTR and D. mel pse nos 3′ UTR transgenes were created by first isolating species specific nos 3′ UTR from genomic DNA using PCR with primers with EcoRI and XhoI cut sites engineered at the ends of the forward and reverse primers and amplified with Phusion DNA Polymerase (NEB). Primers were designed based on the D. pse nos sequence used to designed the smFISH probe and the nos sequence for D. neb was identified from a long-read sequenced genome [46]. Next, we removed the nos 3′ UTR from a 4.3-kb D. mel genomic nos rescue fragment (a gift from the Gavis Lab [60]), using EcoRI and XhoI (NEB) and ligated species specific 3′ UTR with T4 DNA ligase (NEB).…”

Section: Fly Strains and Cloningmentioning

confidence: 99%

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Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms inDrosophila

Doyle

Burian

Aharoni

et al. 2023

Preprint

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The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate mRNAs that function in germline development and maintenance. In D. melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates that contain multiple transcripts from a specific gene. Nucleated by Oskar (Osk), homotypic clusters in D. melanogaster are generated through a stochastic seeding and self-recruitment process that requires the 3'UTR of germ granule mRNAs. Interestingly, the 3'UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species. Thus, we hypothesized that evolutionary changes in the 3'UTR influences germ granule development. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that homotypic clustering is a conserved developmental process used to enrich germ granule mRNAs. Additionally, we discovered that the number of transcripts found in nos and/or pgc clusters could vary significantly among species. By integrating biological data with computational modeling, we determined that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Finally, we found that the nos 3'UTR from different species can alter the efficacy of nos homotypic clustering, resulting in germ granules with reduced nos accumulation. Our findings highlight the impact that evolution has on the development of germ granules and may provide insight into processes that modify the content of other classes of biomolecular condensates.

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Decoupled evolution of theSex Peptidegene family andSex Peptide ReceptorinDrosophilidae

Hopkins,

Angus-Henry,

Kim

et al. 2023

Preprint

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Across internally fertilising species, males transfer proteins in their ejaculate that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 199, mostly drosophilid, species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses. We infer that SP has followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa radiation, SP exists largely as a single-copy gene that has been independently lost in several lineages. In contrast, within the Sophophora-Lordiphosa radiation SP has repeatedly and independently duplicated. Up to seven copies, collectively displaying extensive variation in sequence, are present in some species. We use cross-species RNA-seq data to provide evidence that this lineage-specific burst in evolutionary activity did not follow a significant shift in the sex or tissue specificity of SP's expression. We also document considerable interspecific variation in accessory gland microcarriers that appears to be independent of SP presence or sequence. We end by showing that the mode of SP's evolution is decoupled from that of its receptor, SPR, in which we detect no evidence of correlated diversifying selection in its coding sequence. Collectively, our work describes the divergent evolutionary trajectories that an apparently novel drosophilid gene has followed in different branches of the phylogeny and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor.

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Nebulous without white: annotated long-read genome assembly and CRISPR/Cas9 genome engineering in Drosophila nebulosa

Cited by 3 publications

References 111 publications

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development

Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms inDrosophila

Decoupled evolution of theSex Peptidegene family andSex Peptide ReceptorinDrosophilidae

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