Limits...
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.

Show MeSH

Related in: MedlinePlus

Gis2p preferentially binds to coding sequences that bear GAN repeats. (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53. Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3091301&req=5

Figure 2: Gis2p preferentially binds to coding sequences that bear GAN repeats. (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53. Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).

Mentions: We next wondered whether there are common structural features within mRNA targets that could specify Gis2p interaction. We therefore retrieved the coding sequences (CDSs) of ORFs from the Saccharomyces Genome Database (SGD) [42], as well as 3'- and 5'-UTR sequences [43] for the 50 highest scored Gis2p mRNA targets, and we searched for common motifs using Multiple Expectation Maximization for Motif Elicitation (MEME) as an unbiased motif discovery tool [44] (see Materials and methods). MEME identified a consensus sequence composed of 14 GAN trinucleotide repeats (N refers to any of the four nucleotides) within ORFs (median approximately 7 GAN repeats) (Figure 2a). No motif was found among the 3'- and 5'-UTR sequences [43] or when searching 500 bp downstream or upstream of these ORFs covering UTRs (data not shown). Furthermore, GAN repeats are overrepresented in the ORFs of our experimentally defined Gis2p targets with FDR <5% (for example, 98 Gis2p targets among all genomically encoded 232 ORFs that bear at least one (GAN)7 sequence element; P < 10-22), and ORFs with greater numbers of GAN repeats tend to be more highly enriched in Gis2 affinity isolations (the distribution of (GAN)7-containing ORFs in Gis2-TAP affinity isolations is shown in Additional file 5). These results let us propose that Gis2p may bind to stretches of GAN repeats, which are preferentially located in ORFs/CDSs of mRNA targets.


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 preferentially binds to coding sequences that bear GAN repeats. (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53. Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091301&req=5

Figure 2: Gis2p preferentially binds to coding sequences that bear GAN repeats. (a) Conserved sequence element in the ORF of Gis2p targets identified with MEME. The E-value reflects the probability to detect the motif by chance. (b) RNA-protein complexes formed between biotinylated RNA fragments and Gis2-TAP were purified on streptavidin beads and monitored by immunoblot analysis. Representative experiments from at least three biological replicates are shown. Biotin labeled fragments comprising the 5'-UTRs (lanes 2 and 5), ORFs (lanes 3 and 6), and 3'-UTRs (lanes 4 and 7) of Erv25 and Fcy1 were incubated with extracts of Gis2-TAP expressing cells (lane 1). Eno2-5'UTR (lane 8) is a negative control RNA derived from the 'non-target' ENO2 (lane 8) and a sample without RNA (lane 9) was used to control for RNA-independent binding to the beads. (c) RNA pull-downs with RNA fragments derived from NOP53. Nop53-GAN (lanes 3 and 7 to 9) contains a GAN-rich sequence element whereas the similarly sized fragment Nop53-ctrl does not (lanes 4 and 10). Erv25-ORF (lane 2) and Eno2-ORF (lane 5) are positive and negative control RNAs, respectively. Binding of Gis2-TAP to Nop53-GAN was competed with a ten-fold excess of non-biotinylated Nop53-GAN (lane 8) but not with excess of Nop53-ctrl (lane 9). (d) RNA pull-downs with two fragments derived from the RAS2 ORF. Biotinylated RNAs were incubated with extracts from yeast cells expressing Gis2-TAP (eGis2-TAP, lanes 1 to 3) or with Gis2-His expressed and purified from Escherichia coli (pGis2-His, lanes 4 and 5). A fragment derived from the ORF of SNF5 was used as a negative control RNA (lane 3).
Mentions: We next wondered whether there are common structural features within mRNA targets that could specify Gis2p interaction. We therefore retrieved the coding sequences (CDSs) of ORFs from the Saccharomyces Genome Database (SGD) [42], as well as 3'- and 5'-UTR sequences [43] for the 50 highest scored Gis2p mRNA targets, and we searched for common motifs using Multiple Expectation Maximization for Motif Elicitation (MEME) as an unbiased motif discovery tool [44] (see Materials and methods). MEME identified a consensus sequence composed of 14 GAN trinucleotide repeats (N refers to any of the four nucleotides) within ORFs (median approximately 7 GAN repeats) (Figure 2a). No motif was found among the 3'- and 5'-UTR sequences [43] or when searching 500 bp downstream or upstream of these ORFs covering UTRs (data not shown). Furthermore, GAN repeats are overrepresented in the ORFs of our experimentally defined Gis2p targets with FDR <5% (for example, 98 Gis2p targets among all genomically encoded 232 ORFs that bear at least one (GAN)7 sequence element; P < 10-22), and ORFs with greater numbers of GAN repeats tend to be more highly enriched in Gis2 affinity isolations (the distribution of (GAN)7-containing ORFs in Gis2-TAP affinity isolations is shown in Additional file 5). These results let us propose that Gis2p may bind to stretches of GAN repeats, which are preferentially located in ORFs/CDSs of mRNA targets.

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