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Differential polysomal localization of human insulin-like-growth-factor-2 mRNAs in cell lines and foetal liver
C H De Moor1, M Jansen, J S Sussenbach
1Department of Pediatrics, Utrecht University, The Netherlands.
Abstract:
Examination of the association of insulin-like-growth-factor-2 mRNAs with polyribosomes in five cell lines revealed that greater than 50% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin-like-growth-factor-2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20-30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin-like-growth-factor-2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin-like-growth-factor-2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin-like-growth-factor-2 mRNAs.
Insights
Insulin-like-growth-factor-2 (IGF-2) mRNA translation is regulated by its sequestration in untranslated fractions and the efficiency of translation initiation. Different IGF-2 mRNA leaders affect polysome association and translation efficiency in cell lines and fetal liver.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
Background:
- Insulin-like-growth-factor-2 (IGF-2) plays crucial roles in growth and development.
- The regulation of IGF-2 mRNA translation is not fully understood.
- Different IGF-2 mRNA variants exist, differing in their untranslated regions (UTRs).
Purpose of the Study:
- To investigate the association of different IGF-2 mRNA variants with polyribosomes.
- To understand how mRNA structure influences IGF-2 translation efficiency.
- To explore the role of mRNP sequestration in IGF-2 gene expression.
Main Methods:
- Analysis of IGF-2 mRNA association with polyribosomes in five cell lines and fetal liver.
- Sucrose gradient centrifugation to separate polysomal and untranslated fractions.
- Assessment of translation efficiency based on ribosome loading.
Main Results:
- Over 50% of total IGF-2 mRNA was found in untranslated fractions across cell lines.
- Least abundant IGF-2 mRNAs (leader 2 and 4) were primarily in polysomes, while the most abundant (leader 3) was mostly untranslated.
- Leader 4 mRNA showed higher ribosome association, suggesting greater translation efficiency.
- Cycloheximide treatment altered polysome distribution, indicating translational control.
Conclusions:
- IGF-2 translation is regulated by both mRNA sequestration in untranslated mRNPs and differential translation initiation efficiency.
- The specific leader sequence of IGF-2 mRNA significantly impacts its translational control.
- These regulatory mechanisms are conserved in both cell lines and fetal liver.