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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Published on: September 15, 2021

5'-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the

Jing Chen1, Julianne T Yang, Dana L Doctor

  • 1Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, OH 45056, USA.

Gene
|July 30, 2013
PubMed

Insights

The RNP-4F spliceosome factor

Area of Science:

  • Molecular Biology
  • Genetics
  • Developmental Biology

Background:

  • RNP-4F is a conserved spliceosome assembly factor essential for pre-mRNA splicing.
  • Two developmentally regulated isoforms of rnp-4f mRNA exist in Drosophila, differing in a 177-nucleotide 5 -untranslated region (UTR).
  • This 5 -UTR region potentially forms a stable stem-loop structure, raising questions about its functional significance.

Purpose of the Study:

  • To investigate the functional role of the evolutionarily conserved 5 -UTR stem-loop in modulating rnp-4f gene expression.
  • To determine if the 5 -UTR feature affects gene expression at the transcriptional or post-transcriptional level.
  • To assess the impact of RNP-4F translational regulation in the developing Drosophila central nervous system.

Main Methods:

  • Construction of a new GFP reporter vector, pUAS-Neostinger.
  • Utilized the UAS-GAL4 system with tissue-specific drivers for controlled GFP expression in Drosophila embryos.
  • Quantified GFP reporter mRNA and protein levels using fluorescence microscopy, Western blotting, and real-time qRT-PCR.

Main Results:

  • The wild-type 177-nt stem-loop sequence significantly increased GFP reporter protein levels without altering mRNA levels, indicating post-transcriptional enhancement.
  • Mutational analysis of the stem-loop identified potential cis-acting elements involved in this regulation.
  • RNP-4F translational regulation was found to be highly sensitive in the developing fly central nervous system.

Conclusions:

  • The 5 -UTR stem-loop of rnp-4f plays a crucial role in post-transcriptional gene regulation, specifically enhancing translation.
  • This regulatory mechanism is particularly important in the developing Drosophila central nervous system.
  • The findings provide insights into the molecular mechanisms governing RNP-4F translational control during development.
Keywords:
3′-UTR3′-untranslated mRNA region5′-UTR5′-untranslated mRNA regionAlternative splicingCNSDNA complementary to RNADrosophila adenosine deaminase acting on RNAGFPGFP reporterIRESPCRPosttranscriptional regulationRNP-4FRT-PCRU-series small nuclear ribonucleoproteinU-snRNPbase pairbpcDNAcentral nervous systemdADARgreen fluorescent proteininternal ribosomal entry sitemRNAmessenger RNAntnucleotidepolymerase chain reactionreverse transcriptase-polymerase chain reactionribonucleoprotein encoded by rnp-4f gene in polytene band 4Frnp-4f gene

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