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Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

ABSTRACT: MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.

SUBMITTER: Bazzini AA

PROVIDER: S-EPMC3547538 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

Bazzini Ariel A AA Lee Miler T MT Giraldez Antonio J AJ

Science (New York, N.Y.) 20120315 6078

MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deade ...[more]

PMID: 22422859

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Project description:Decoding of aberrant mRNAs leads to unproductive ribosome stalling and sequestration of components of the translation machinery. Bacteria have evolved three seemingly independent pathways to resolve stalled translation complexes. The trans-translation process, orchestrated by the hybrid transfer-messenger RNA (tmRNA) and its essential protein co-factor, small protein B (SmpB), is the principal translation quality control system for rescuing unproductively stalled ribosomes. Two specialized alternative rescue pathways, coordinated by ArfA and ArfB, have been recently discovered. The SmpB-tmRNA mediated trans-translation pathway, in addition to re-mobilizing stalled translation complexes, co-translationally appends a degradation tag to the associated nascent polypeptides, marking them for proteolysis by various cellular proteases. Another unique feature of trans-translation, not shared by the alternative rescue pathways, is the facility to recruit ribonuclease R (RNase R) for targeted degradation of non-stop mRNAs, thus preventing further futile cycles of translation. The distinct C-terminal lysine-rich (K-rich) domain of RNase R is essential for its recruitment to stalled ribosomes. To gain new insights into the structure and function of RNase R, we investigated its global architecture, the spatial arrangement of its distinct domains, and the identities of key functional residues in its unique K-rich domain. Small-angle X-ray scattering models of RNase R reveal a tri-lobed structure with flexible N- and C-terminal domains, and suggest intimate contacts between the K-rich domain and the catalytic core of the enzyme. Alanine-scanning mutagenesis of the K-rich domain, in the region spanning residues 735 and 750, has uncovered the precise amino acid determinants required for the productive engagement of RNase R on tmRNA-rescued ribosomes. Theses analyses demonstrate that alanine substitution of conserved residues E740 and K741result in profound defects, not only in the recruitment of RNase R to rescued ribosomes but also in the targeted decay of non-stop mRNAs. Additionally, an RNase R variant with alanine substitution at residues K749 and K750 exhibits extensive defects in ribosome enrichment and non-stop mRNA decay. In contrast, alanine substitution of additional conserved residues in this region has no effect on the known functions of RNase R. In vitro RNA degradation assays demonstrate that the consequential substitutions (RNase R(E740A/K741A) and RNase R(K749A/K750A)) do not affect the ability of the enzyme to degrade structured RNAs, indicating that the observed defect is specific to the trans-translation related activities of RNase R. Taken together, these findings shed new light on the global architecture of RNase R and provide new details of how this versatile RNase effectuates non-stop mRNA decay on tmRNA-rescued ribosomes.

Dataset Information

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

Publications

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish.

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