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Systematic analysis of the role of RNA-binding proteins in the regulation of RNA stability.

Hasan A, Cotobal C, Duncan CD, Mata J - PLoS Genet. (2014)

Bottom Line: We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities.Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains.This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
mRNA half-lives are transcript-specific and vary over a range of more than 100-fold in eukaryotic cells. mRNA stabilities can be regulated by sequence-specific RNA-binding proteins (RBPs), which bind to regulatory sequence elements and modulate the interaction of the mRNA with the cellular RNA degradation machinery. However, it is unclear if this kind of regulation is sufficient to explain the large range of mRNA stabilities. To address this question, we examined the transcriptome of 74 Schizosaccharomyces pombe strains carrying deletions in non-essential genes encoding predicted RBPs (86% of all such genes). We identified 25 strains that displayed changes in the levels of between 4 and 104 mRNAs. The putative targets of these RBPs formed biologically coherent groups, defining regulons involved in cell separation, ribosome biogenesis, meiotic progression, stress responses and mitochondrial function. Moreover, mRNAs in these groups were enriched in specific sequence motifs in their coding sequences and untranslated regions, suggesting that they are coregulated at the posttranscriptional level. We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities. Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains. This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.

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Characterization of the RNA sets regulated by RBPs.(A) Inactivation of pab2 and red1 causes overexpression of a subset of ncRNAs. The y and x axes show expression levels in red1 mutants and wild type cells, respectively. Each dot corresponds to a different ncRNA. ncRNAs that are overexpressed in pab2Δ cells are displayed in red and all other ncRNAs in blue. (B) Similar sets of ncRNAs are affected in pab2 and red1 mutants. The Venn diagram shows the overlap among the ncRNAs overexpressed in red1Δ and pab2Δ cells. The number in brackets corresponds to the expected overlap if randomly-generated lists of the corresponding sizes were used. The p value of the observed overlap is shown below the Venn diagram. (C) Scw1 potential targets are enriched in cell cycle-regulated genes. Cell cycle expression profiles of periodically expressed Scw1-regulated genes (data from [76]). The timing of the cell cycle phases is shown: M (mitosis), G1, S phase (S), and G2. Cell division takes place during G1 and S phase. (D) Overlap between cell-cycle regulated genes and Scw1-regulated mRNAs. Labelling as in B. (E) Comparison of genes down-regulated in rpm1 mutants and genes encoding proteins localized to the mitochondrial membrane. Labelling as in B. (F) Overlap between genes overexpressed in SPAC17H9.04c mutants and genes encoding proteins involved in ribosome biogenesis. Labelling as in B.
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pgen-1004684-g002: Characterization of the RNA sets regulated by RBPs.(A) Inactivation of pab2 and red1 causes overexpression of a subset of ncRNAs. The y and x axes show expression levels in red1 mutants and wild type cells, respectively. Each dot corresponds to a different ncRNA. ncRNAs that are overexpressed in pab2Δ cells are displayed in red and all other ncRNAs in blue. (B) Similar sets of ncRNAs are affected in pab2 and red1 mutants. The Venn diagram shows the overlap among the ncRNAs overexpressed in red1Δ and pab2Δ cells. The number in brackets corresponds to the expected overlap if randomly-generated lists of the corresponding sizes were used. The p value of the observed overlap is shown below the Venn diagram. (C) Scw1 potential targets are enriched in cell cycle-regulated genes. Cell cycle expression profiles of periodically expressed Scw1-regulated genes (data from [76]). The timing of the cell cycle phases is shown: M (mitosis), G1, S phase (S), and G2. Cell division takes place during G1 and S phase. (D) Overlap between cell-cycle regulated genes and Scw1-regulated mRNAs. Labelling as in B. (E) Comparison of genes down-regulated in rpm1 mutants and genes encoding proteins localized to the mitochondrial membrane. Labelling as in B. (F) Overlap between genes overexpressed in SPAC17H9.04c mutants and genes encoding proteins involved in ribosome biogenesis. Labelling as in B.

Mentions: Pab2 is also involved in the processing of snoRNAs [43]. The presence of long ncRNA probes in our microarray platform allowed us to investigate if Pab2 and Red1 are involved in the regulation of other non-coding transcripts. Indeed, we found 18 ncRNAs overexpressed in pab2Δ, and 35 in red1Δ (Figure 2A and Table S5). As was the case with coding genes, almost all ncRNAs up-regulated in pab2Δ were also induced in red1Δ (Figure 2B and Table S5). Some of these ncRNAs were overexpressed more strongly than most coding targets, suggesting that they may be functionally important. These results demonstrate that our strategy can identify both known targets and additional functions of RBPs. While this manuscript was in preparation, a transcriptome analysis of red1Δ mutants using high throughput sequencing was published [44]. This study reported overexpression of large numbers of ncRNAs in red1Δ cells, which showed a highly significant overlap with our data (Figure S1D).


Systematic analysis of the role of RNA-binding proteins in the regulation of RNA stability.

Hasan A, Cotobal C, Duncan CD, Mata J - PLoS Genet. (2014)

Characterization of the RNA sets regulated by RBPs.(A) Inactivation of pab2 and red1 causes overexpression of a subset of ncRNAs. The y and x axes show expression levels in red1 mutants and wild type cells, respectively. Each dot corresponds to a different ncRNA. ncRNAs that are overexpressed in pab2Δ cells are displayed in red and all other ncRNAs in blue. (B) Similar sets of ncRNAs are affected in pab2 and red1 mutants. The Venn diagram shows the overlap among the ncRNAs overexpressed in red1Δ and pab2Δ cells. The number in brackets corresponds to the expected overlap if randomly-generated lists of the corresponding sizes were used. The p value of the observed overlap is shown below the Venn diagram. (C) Scw1 potential targets are enriched in cell cycle-regulated genes. Cell cycle expression profiles of periodically expressed Scw1-regulated genes (data from [76]). The timing of the cell cycle phases is shown: M (mitosis), G1, S phase (S), and G2. Cell division takes place during G1 and S phase. (D) Overlap between cell-cycle regulated genes and Scw1-regulated mRNAs. Labelling as in B. (E) Comparison of genes down-regulated in rpm1 mutants and genes encoding proteins localized to the mitochondrial membrane. Labelling as in B. (F) Overlap between genes overexpressed in SPAC17H9.04c mutants and genes encoding proteins involved in ribosome biogenesis. Labelling as in B.
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pgen-1004684-g002: Characterization of the RNA sets regulated by RBPs.(A) Inactivation of pab2 and red1 causes overexpression of a subset of ncRNAs. The y and x axes show expression levels in red1 mutants and wild type cells, respectively. Each dot corresponds to a different ncRNA. ncRNAs that are overexpressed in pab2Δ cells are displayed in red and all other ncRNAs in blue. (B) Similar sets of ncRNAs are affected in pab2 and red1 mutants. The Venn diagram shows the overlap among the ncRNAs overexpressed in red1Δ and pab2Δ cells. The number in brackets corresponds to the expected overlap if randomly-generated lists of the corresponding sizes were used. The p value of the observed overlap is shown below the Venn diagram. (C) Scw1 potential targets are enriched in cell cycle-regulated genes. Cell cycle expression profiles of periodically expressed Scw1-regulated genes (data from [76]). The timing of the cell cycle phases is shown: M (mitosis), G1, S phase (S), and G2. Cell division takes place during G1 and S phase. (D) Overlap between cell-cycle regulated genes and Scw1-regulated mRNAs. Labelling as in B. (E) Comparison of genes down-regulated in rpm1 mutants and genes encoding proteins localized to the mitochondrial membrane. Labelling as in B. (F) Overlap between genes overexpressed in SPAC17H9.04c mutants and genes encoding proteins involved in ribosome biogenesis. Labelling as in B.
Mentions: Pab2 is also involved in the processing of snoRNAs [43]. The presence of long ncRNA probes in our microarray platform allowed us to investigate if Pab2 and Red1 are involved in the regulation of other non-coding transcripts. Indeed, we found 18 ncRNAs overexpressed in pab2Δ, and 35 in red1Δ (Figure 2A and Table S5). As was the case with coding genes, almost all ncRNAs up-regulated in pab2Δ were also induced in red1Δ (Figure 2B and Table S5). Some of these ncRNAs were overexpressed more strongly than most coding targets, suggesting that they may be functionally important. These results demonstrate that our strategy can identify both known targets and additional functions of RBPs. While this manuscript was in preparation, a transcriptome analysis of red1Δ mutants using high throughput sequencing was published [44]. This study reported overexpression of large numbers of ncRNAs in red1Δ cells, which showed a highly significant overlap with our data (Figure S1D).

Bottom Line: We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities.Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains.This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
mRNA half-lives are transcript-specific and vary over a range of more than 100-fold in eukaryotic cells. mRNA stabilities can be regulated by sequence-specific RNA-binding proteins (RBPs), which bind to regulatory sequence elements and modulate the interaction of the mRNA with the cellular RNA degradation machinery. However, it is unclear if this kind of regulation is sufficient to explain the large range of mRNA stabilities. To address this question, we examined the transcriptome of 74 Schizosaccharomyces pombe strains carrying deletions in non-essential genes encoding predicted RBPs (86% of all such genes). We identified 25 strains that displayed changes in the levels of between 4 and 104 mRNAs. The putative targets of these RBPs formed biologically coherent groups, defining regulons involved in cell separation, ribosome biogenesis, meiotic progression, stress responses and mitochondrial function. Moreover, mRNAs in these groups were enriched in specific sequence motifs in their coding sequences and untranslated regions, suggesting that they are coregulated at the posttranscriptional level. We performed genome-wide RNA stability measurements for several RBP mutants, and confirmed that the altered mRNA levels were caused by changes in their stabilities. Although RBPs regulate the decay rates of multiple regulons, only 16% of all S. pombe mRNAs were affected in any of the 74 deletion strains. This suggests that other players or mechanisms are required to generate the observed range of RNA half-lives of a eukaryotic transcriptome.

Show MeSH
Related in: MedlinePlus