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Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Published on: October 28, 2014
Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and
M Muckenthaler1, N Gunkel, R Stripecke
1Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Abstract:
The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.
Insights
This study reveals how mRNA poly(A) tail length is linked to its translation state in human cells. Translational repression triggers poly(A) tail shortening, impacting gene expression.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cell Biology
Background:
- The poly(A) tail is crucial for mRNA translation initiation.
- Cellular mechanisms linking mRNA translation and poly(A) tail length remain incompletely understood.
Purpose of the Study:
- To identify and characterize a mechanism coupling mRNA poly(A) tail length with its translation state in mammalian somatic cells.
- To investigate the role of iron-responsive elements (IREs) and iron regulatory proteins (IRPs) in this regulatory pathway.
Main Methods:
- Investigated the regulation of human ferritin L-chain mRNA using iron-responsive elements (IREs) and iron regulatory proteins (IRPs).
- Utilized general translation inhibitors (puromycin, verrucarin, cycloheximide, anisomycin) to assess the impact on poly(A) tail length.
- Examined the relationship between ribosome association and poly(A) tail metabolism.
Main Results:
- Translational repression of ferritin L-chain mRNA by IRP binding to the IRE induces poly(A) tail shortening.
- IRP binding alone is insufficient; translational repression is required for poly(A) tail shortening.
- Ribosome association with mRNA is a critical determinant of poly(A) tail length, with dissociation leading to shortening.
Conclusions:
- Identified a novel mechanism of regulated polyadenylation as a consequence of translational control.
- Demonstrated differences in poly(A) tail metabolism between polysomal and messenger ribonucleoprotein (mRNP)-associated mRNAs.
- Proposed a role for this mechanism in maintaining translational repression.
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