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Mammalian microRNAs predominantly act to decrease target mRNA levels.

Guo H, Ingolia NT, Weissman JS, Bartel DP - Nature (2010)

Bottom Line: Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels.Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels.These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

ABSTRACT
MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (>/=84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

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Ribosome profiling in human cells captured features of translationa, Schematic diagram of ribosome profiling. Sequencing reproducibility and evidence for mapping to the correct mRNA isoforms are illustrated (Supplementary Fig. 1a, b). b, RPF density near the ends of ORFs, combining data from all quantified genes. Plotted are RPF 5′ termini, as reads per million reads mapping to genes (rpM). Illustrated below the graph are the inferred ribosome positions corresponding to peak RPF densities, at which the start codon was in the P site (left) and the stop codon was in the A site (right). The offset between the 5′ terminus of an RPF and the first nucleotide in the human ribosome A site was typically 15 nucleotides (nt). c, Density of RPFs and mRNA-Seq tags near the ends of ORFs in HeLa cells. RPF density is plotted as in panel b, except positions are shifted +15 nucleotides to reflect the position of the first nucleotide in the ribosome A site. Composite data are shown for ≥600-nucleotide ORFs that passed our threshold for quantification (≥100 RPFs and ≥100 mRNA-Seq tags). d, Fraction of RPFs and mRNA-Seq tags mapping to each of the three codon nucleotides in panel c.
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Figure 1: Ribosome profiling in human cells captured features of translationa, Schematic diagram of ribosome profiling. Sequencing reproducibility and evidence for mapping to the correct mRNA isoforms are illustrated (Supplementary Fig. 1a, b). b, RPF density near the ends of ORFs, combining data from all quantified genes. Plotted are RPF 5′ termini, as reads per million reads mapping to genes (rpM). Illustrated below the graph are the inferred ribosome positions corresponding to peak RPF densities, at which the start codon was in the P site (left) and the stop codon was in the A site (right). The offset between the 5′ terminus of an RPF and the first nucleotide in the human ribosome A site was typically 15 nucleotides (nt). c, Density of RPFs and mRNA-Seq tags near the ends of ORFs in HeLa cells. RPF density is plotted as in panel b, except positions are shifted +15 nucleotides to reflect the position of the first nucleotide in the ribosome A site. Composite data are shown for ≥600-nucleotide ORFs that passed our threshold for quantification (≥100 RPFs and ≥100 mRNA-Seq tags). d, Fraction of RPFs and mRNA-Seq tags mapping to each of the three codon nucleotides in panel c.

Mentions: Ribosome profiling generates short sequence tags that each mark the mRNA coordinates of one bound ribosome19. The outline of our protocol for mammalian cells paralleled that used for yeast (Fig. 1a). Cells were treated with cycloheximide to arrest translating ribosomes. Extracts from these cells were then treated with RNase I to degrade regions of mRNAs not protected by ribosomes. The resulting 80S monosomes, many of which contained a ∼30-nucleotide RPF, were purified on sucrose gradients and then treated to release the RPFs, which were processed for Illumina high-throughput sequencing.


Mammalian microRNAs predominantly act to decrease target mRNA levels.

Guo H, Ingolia NT, Weissman JS, Bartel DP - Nature (2010)

Ribosome profiling in human cells captured features of translationa, Schematic diagram of ribosome profiling. Sequencing reproducibility and evidence for mapping to the correct mRNA isoforms are illustrated (Supplementary Fig. 1a, b). b, RPF density near the ends of ORFs, combining data from all quantified genes. Plotted are RPF 5′ termini, as reads per million reads mapping to genes (rpM). Illustrated below the graph are the inferred ribosome positions corresponding to peak RPF densities, at which the start codon was in the P site (left) and the stop codon was in the A site (right). The offset between the 5′ terminus of an RPF and the first nucleotide in the human ribosome A site was typically 15 nucleotides (nt). c, Density of RPFs and mRNA-Seq tags near the ends of ORFs in HeLa cells. RPF density is plotted as in panel b, except positions are shifted +15 nucleotides to reflect the position of the first nucleotide in the ribosome A site. Composite data are shown for ≥600-nucleotide ORFs that passed our threshold for quantification (≥100 RPFs and ≥100 mRNA-Seq tags). d, Fraction of RPFs and mRNA-Seq tags mapping to each of the three codon nucleotides in panel c.
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Figure 1: Ribosome profiling in human cells captured features of translationa, Schematic diagram of ribosome profiling. Sequencing reproducibility and evidence for mapping to the correct mRNA isoforms are illustrated (Supplementary Fig. 1a, b). b, RPF density near the ends of ORFs, combining data from all quantified genes. Plotted are RPF 5′ termini, as reads per million reads mapping to genes (rpM). Illustrated below the graph are the inferred ribosome positions corresponding to peak RPF densities, at which the start codon was in the P site (left) and the stop codon was in the A site (right). The offset between the 5′ terminus of an RPF and the first nucleotide in the human ribosome A site was typically 15 nucleotides (nt). c, Density of RPFs and mRNA-Seq tags near the ends of ORFs in HeLa cells. RPF density is plotted as in panel b, except positions are shifted +15 nucleotides to reflect the position of the first nucleotide in the ribosome A site. Composite data are shown for ≥600-nucleotide ORFs that passed our threshold for quantification (≥100 RPFs and ≥100 mRNA-Seq tags). d, Fraction of RPFs and mRNA-Seq tags mapping to each of the three codon nucleotides in panel c.
Mentions: Ribosome profiling generates short sequence tags that each mark the mRNA coordinates of one bound ribosome19. The outline of our protocol for mammalian cells paralleled that used for yeast (Fig. 1a). Cells were treated with cycloheximide to arrest translating ribosomes. Extracts from these cells were then treated with RNase I to degrade regions of mRNAs not protected by ribosomes. The resulting 80S monosomes, many of which contained a ∼30-nucleotide RPF, were purified on sucrose gradients and then treated to release the RPFs, which were processed for Illumina high-throughput sequencing.

Bottom Line: Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels.Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels.These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

ABSTRACT
MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (>/=84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

Show MeSH
Related in: MedlinePlus