Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion

Hudson, Andrew M.; Mannix, Katelynn M.; Gerdes, Julianne A.; Kottemann, Molly C.; Cooley, Lynn

doi:10.1101/315077

Cited by 3 publications

(7 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We identified HtsRC in two independent mass spectrometry approaches designed to identify ring canal-associated proteins (HUDSON et al 2019;MANNIX et al 2019). Figure 1E, black box), consistent with cleavage prior to V693 in Ovhts.…”

Section: Ovhts Polyprotein Is Cleaved Within the Htsrc Exon-encoded Pmentioning

confidence: 67%

“…This result is consistent with our prior conclusion that HtsRC is encoded entirely within this exon. Furthermore, transgenes encoding either the entire ovhts cDNA (HUDSON et al 2019) or just the HtsRC-encoding exon (data not shown) rescued htsRC-specific mutants, confirming that HtsRC is produced from this exon. The following experiments were conducted with ovhts-based transgenes.…”

Section: Ovhts Polyprotein Is Cleaved Within the Htsrc Exon-encoded Pmentioning

confidence: 75%

“…Work in the 1990s and early 2000s uncovered multiple kinases involved in ring canal expansion, suggesting that post-translational modification of ring canal components is required in this process (DODSON et al 1998;GUARNIERI et al 1998;JACKSON AND BERG 2002). More recent work has also revealed a role for membrane tension sensing by Filamin in normal ring canal expansion (HUELSMANN et al 2016) and we have previously shown proteasome dependent HtsRC turnover of HtsRC is important for clearance of the ring canal lumen (HUDSON et al 2019).…”

Section: Introductionmentioning

confidence: 81%

“…Mass spectrometry data from Hudson et al (2019) and Mannix et al (2019) were searched for semi-tryptic peptides at MS Bioworks using Mascot (Matrix Science); searches were conducted against the UniProt Drosophila melanogaster database with common laboratory contaminants added as well as custom protein sequences for HtsRC that included known SNPs. The results were parsed into Scaffold (Proteome Software) for further analysis.…”

Section: Analysis Of Ms/ms Data For Semi-tryptic Peptidesmentioning

confidence: 99%

“…In addition to ring canals, HtsRC is also found on cytoplasmic actin bundles in stage 10B egg chambers and on the oocyte cortex in later stages (HUELSMANN et al 2013;POKRYWKA et al 2014). The level of HtsRC at ring canals is under the control of a Cullin3 RING Ubiquitin ligase (CRL3) in which Kelch is the substrate adaptor that binds HtsRC, leading to poly-ubiquitylation of HtsRC and its destruction by the proteasome (HUDSON et al 2019). Mutations in the kelch gene lead to increased levels of ring canal HtsRC and F-actin, consistent with HtsRC being the target of CRL3 Kelch .…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

et al. 2020

Self Cite

View full text Add to dashboard Cite

Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC, a component specific to female germline ring canals, is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ectopic actin structures in somatic follicle cells. Finally, we present findings which indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility, a result which is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

show abstract

Section: Ovhts Polyprotein Is Cleaved Within the Htsrc Exon-encoded Pmentioning

confidence: 67%

Section: Ovhts Polyprotein Is Cleaved Within the Htsrc Exon-encoded Pmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 81%

Section: Analysis Of Ms/ms Data For Semi-tryptic Peptidesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

Tissue-specific dynamic codon redefinition in Drosophila

Hudson

Szabo

Loughran

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously we reported tissue-specific regulation of readthrough of the first kelch stop codon. Here, we characterize major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3′ of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3′ of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, we show that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.

show abstract

Tissue-specific dynamic codon redefinition in Drosophila

Hudson

Loughran

Szabo

et al. 2020

Preprint

View full text Add to dashboard Cite

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3' of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue-and cell-specific dynamic stop codon redefinition in Drosophila. Stop codon readthrough | recoding | Drosophila | Kelch | central nervous system (CNS) †Correspondence: j.atkins@ucc.ie or lynn.cooley@yale.edu Hudson et al. | bioRχiv | June 22, 2020 | 1-11

show abstract

Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion

Cited by 3 publications

References 49 publications

HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

Tissue-specific dynamic codon redefinition in Drosophila

Tissue-specific dynamic codon redefinition in Drosophila

Contact Info

Product

Resources

About