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Tissue-specific dynamic codon redefinition in Drosophila.

Andrew M Hudson1, Nicholas L Szabo1, Gary Loughran2

  • 1Department of Genetics, Yale School of Medicine, New Haven, CT 06520.

Proceedings of the National Academy of Sciences of the United States of America
|January 27, 2021
PubMed
Summary
This summary is machine-generated.

Translational stop codon readthrough in Drosophila produces C-terminally extended proteins. This readthrough is highly efficient in the central nervous system (CNS) neurons, but not in glia.

Keywords:
DrosophilaKelchcentral nervous system (CNS)recodingstop codon readthrough

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Area of Science:

  • Molecular Biology
  • Genetics
  • Neuroscience

Background:

  • Translational stop codon readthrough allows C-terminal protein extension from a single gene.
  • This recoding mechanism is observed across diverse organisms, including Drosophila and viruses.
  • The Kelch protein in Drosophila exists in both unextended (76 kDa) and extended (160 kDa) forms due to readthrough.

Purpose of the Study:

  • To investigate tissue-specific differences in translational stop codon readthrough efficiency in Drosophila.
  • To identify sequences that regulate readthrough efficiency.
  • To determine the prevalence of readthrough in neuronal proteins within the Drosophila central nervous system (CNS).

Main Methods:

  • Immunoblotting to detect protein levels in various Drosophila tissues.
  • Reporter assays to quantify readthrough efficiency.
  • Transgenic Drosophila and human cell culture to analyze regulatory sequences.
  • Confocal imaging to visualize readthrough in specific cell types (neurons vs. glia).

Main Results:

  • Readthrough was abundant in larval and adult CNS tissue, but low in malpighian tubules, ovary, and testis.
  • Adult brain readthrough efficiency exceeded 30%, occurring in neurons but not glia.
  • A 99-nucleotide mRNA sequence forming a stem-loop structure 3' of the stop codon enhanced readthrough efficiency.
  • Even minimal 3' sequences (6 nucleotides) maintained significant readthrough (6%) in adult neurons.

Conclusions:

  • Drosophila CNS exhibits high-efficiency translational stop codon readthrough, particularly in neurons.
  • Specific mRNA sequences, including potential stem-loops, significantly influence readthrough efficiency.
  • C-terminally extended forms of neuronal proteins are likely common in the Drosophila CNS.