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Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins.

Scherrer T, Femmer C, Schiess R, Aebersold R, Gerber AP - Genome Biol. (2011)

Bottom Line: Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases.We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT

Background: Glucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression.

Results: Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.

Conclusions: This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models.

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Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA. RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA)5 was used as positive control (lane 5), and (GAUGCU)5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA)5 RNA (lane 6). A reaction without RNA is shown in lane 8.
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Figure 3: Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA. RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA)5 was used as positive control (lane 5), and (GAUGCU)5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA)5 RNA (lane 6). A reaction without RNA is shown in lane 8.

Mentions: Since yeast Gis2p and human ZNF9 are well conserved across the seven ZnFs, we wondered whether the respective RNA binding preferences are conserved as well. To examine this idea and to gain a more detailed insight into the RNA-binding specificities of the yeast and human protein, we tested a series of short 30-mer RNA oligonucleotides, comprising ten trinucleotide (triplet) repeats in RNA pull-down assays. We therefore incubated the RNAs with yeast extracts containing either tagged protein (eGis2-TAP, eZNF9-TAP) or with partially purified proteins expressed in E. coli (pGis2-His, pZNF9-His; see Materials and methods) to evaluate whether the observed interactions are direct. Biotinylated (GAUGAA)5 efficiently pulled-down Gis2p as well as ZNF9, which is in agreement with our postulated necessity of GAN repeats for protein interaction (Figure 3a). Thereby, guanosine at the first position of the triplet is essential for interaction as no binding was seen with short RNAs in which half of the Gs were changed to uridine (GAUUAA)5. We further analyzed binding selectivity for the second and third position of the triplet repeats: RNA oligos (GAUGUU)5 and (GUUGUU)5, in which adenosines at the second and/or third position were changed to uridine, still bound Gis2p and ZNF9. However, changing the adenosines at the second position to cytosine (GAUGCU)5 or to guanosine (GUGGUG)5 almost completely abrogated binding. Likewise, we observed substantially weaker binding to (GUGGUG)5 and (GACGAC)5, where the third position of the triplet was changed to guanosine and cytosine, respectively (Figure 3a, and data not shown). In conclusion, these experiments demonstrate that both the yeast and human protein bind specifically to GWW repeats (W = A/U). Noteworthy, this consensus is somewhat different to the GAN repeats enriched among the experimentally defined Gis2p targets. A search among all yeast ORFs for the presence of (GUN)3 trinucleotide repeat sequences - which includes the potential GUA and GUU binding sites for Gis2p - revealed that these sequences are simply not present among yeast ORFs. Thus, although Gis2p has broader specificity for GWW repeats, it can only associate with GA(A/U) repeats in yeast ORFs because of restrictions set by the genome.


Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins.

Scherrer T, Femmer C, Schiess R, Aebersold R, Gerber AP - Genome Biol. (2011)

Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA. RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA)5 was used as positive control (lane 5), and (GAUGCU)5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA)5 RNA (lane 6). A reaction without RNA is shown in lane 8.
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Figure 3: Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA. RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA)5 was used as positive control (lane 5), and (GAUGCU)5 as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA)5 RNA (lane 6). A reaction without RNA is shown in lane 8.
Mentions: Since yeast Gis2p and human ZNF9 are well conserved across the seven ZnFs, we wondered whether the respective RNA binding preferences are conserved as well. To examine this idea and to gain a more detailed insight into the RNA-binding specificities of the yeast and human protein, we tested a series of short 30-mer RNA oligonucleotides, comprising ten trinucleotide (triplet) repeats in RNA pull-down assays. We therefore incubated the RNAs with yeast extracts containing either tagged protein (eGis2-TAP, eZNF9-TAP) or with partially purified proteins expressed in E. coli (pGis2-His, pZNF9-His; see Materials and methods) to evaluate whether the observed interactions are direct. Biotinylated (GAUGAA)5 efficiently pulled-down Gis2p as well as ZNF9, which is in agreement with our postulated necessity of GAN repeats for protein interaction (Figure 3a). Thereby, guanosine at the first position of the triplet is essential for interaction as no binding was seen with short RNAs in which half of the Gs were changed to uridine (GAUUAA)5. We further analyzed binding selectivity for the second and third position of the triplet repeats: RNA oligos (GAUGUU)5 and (GUUGUU)5, in which adenosines at the second and/or third position were changed to uridine, still bound Gis2p and ZNF9. However, changing the adenosines at the second position to cytosine (GAUGCU)5 or to guanosine (GUGGUG)5 almost completely abrogated binding. Likewise, we observed substantially weaker binding to (GUGGUG)5 and (GACGAC)5, where the third position of the triplet was changed to guanosine and cytosine, respectively (Figure 3a, and data not shown). In conclusion, these experiments demonstrate that both the yeast and human protein bind specifically to GWW repeats (W = A/U). Noteworthy, this consensus is somewhat different to the GAN repeats enriched among the experimentally defined Gis2p targets. A search among all yeast ORFs for the presence of (GUN)3 trinucleotide repeat sequences - which includes the potential GUA and GUU binding sites for Gis2p - revealed that these sequences are simply not present among yeast ORFs. Thus, although Gis2p has broader specificity for GWW repeats, it can only associate with GA(A/U) repeats in yeast ORFs because of restrictions set by the genome.

Bottom Line: Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases.We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT

Background: Glucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression.

Results: Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.

Conclusions: This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models.

Show MeSH
Related in: MedlinePlus