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Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
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5'-UTR and ORF elements, as well as the 3'-UTR regulate the translation of Cyclin

Boram Kim1, Hye Min Kim2, Min Kook Kang3

  • 1Department of Biological Science, Inje University, 197 Inje-ro, Gimhae, Gyeongnam, 50834, Republic of Korea.

Biochemical and Biophysical Research Communications
|May 23, 2020
PubMed

Insights

Monitoring mouse oocyte cell cycle progression requires measuring gene translation. Researchers found that both the 5' untranslated region (UTR) and open reading frame (ORF), not just the 3' UTR, influence Ccnb1 and Ccnb2 gene expression.

Area of Science:

  • * Molecular and Cellular Biology
  • * Developmental Biology
  • * Gene Expression Regulation

Background:

  • * Mammalian oocyte maturation relies on maternal transcript translation.
  • * Cyclin B1 (Ccnb1) and Cyclin B2 (Ccnb2) are crucial for oocyte cell cycle progression in mice.
  • * Previous studies primarily used 3'-untranslated region (UTR) reporter constructs to assess gene translation, assuming it's the main regulatory element.

Purpose of the Study:

  • * To investigate the impact of 5' UTR and open reading frame (ORF) on Ccnb1 and Ccnb2 gene expression in mouse oocytes and somatic cells.
  • * To compare the translational efficiency of different reporter constructs, including those with 5' UTR, ORF, and 3' UTR combinations.
  • * To determine if the 3' UTR alone is sufficient for accurately monitoring gene expression during oocyte maturation.

Main Methods:

  • * Construction and expression analysis of Renilla luciferase (RL) reporter genes incorporating various combinations of Ccnb1 and Ccnb2 5' UTR, ORF, and 3' UTR in HEK293T cells and mouse oocytes.
  • * Measurement of RL expression levels to quantify translational activity.
  • * Comparison of reporter expression patterns during oocyte maturation.

Main Results:

  • * The 5' UTR and/or ORF of Ccnb significantly altered RL reporter expression in both cell types.
  • * The ORF generally suppressed RL expression, while the 5' UTR enhanced it in most cases.
  • * Reporter constructs solely containing the 3' UTR showed the highest expression increase for Ccnb1, but other reporters, including those with ORF, demonstrated significant modulation during oocyte maturation, with varying effects for Ccnb1 and Ccnb2.

Conclusions:

  • * Gene expression, particularly for Ccnb1 and Ccnb2 during mouse oocyte maturation, is regulated by elements beyond the 3' UTR, including the 5' UTR and ORF.
  • * Reliance solely on 3' UTR reporter constructs may lead to inaccurate assessments of gene expression and translation.
  • * Caution is advised when using 3' UTR-only reporters for monitoring gene expression, necessitating consideration of other regulatory regions.
Keywords:
Cyclin BMouse oocyteOpen reading frameTranslational regulationUntranslated region

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