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APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression.

Huh SU, Paek KH - BMC Plant Biol. (2014)

Bottom Line: We found that APUM5 was associated with both biotic and abiotic stress responses.The APUM5-Pumilio homology domain (PHD) protein bound to the 3' untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3' UTR.These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3' UTRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life Sciences and Biotechnology, Korea University, 1, 5-ga, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea. khpaek95@korea.ac.kr.

ABSTRACT

Background: A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening.

Results: APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3' untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3' UTR.

Conclusions: These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3' UTRs.

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Related in: MedlinePlus

Gel mobility shift assay of the recombinant containing APUM5-PHD for 3′ UTR motifs of abiotic stress-responsive genes. (A-D). Indicated 32P-labeled RNA probes were incubated with recombinant GST-APUM5-PHD (1, 10, 50, and 100 nM) for 30 min in 20 μl RNA binding buffer at room temperature. RNA-protein complexes were separated on a 5% native gel and analyzed by autoradiography. GST protein (1000 nM) was used as a negative control. (E) 32P-labeled hbNRE2 RNA probes were incubated with recombinant GST-APUM5-PHD (1 and 10 nM) and used as a positive binding control.
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Figure 8: Gel mobility shift assay of the recombinant containing APUM5-PHD for 3′ UTR motifs of abiotic stress-responsive genes. (A-D). Indicated 32P-labeled RNA probes were incubated with recombinant GST-APUM5-PHD (1, 10, 50, and 100 nM) for 30 min in 20 μl RNA binding buffer at room temperature. RNA-protein complexes were separated on a 5% native gel and analyzed by autoradiography. GST protein (1000 nM) was used as a negative control. (E) 32P-labeled hbNRE2 RNA probes were incubated with recombinant GST-APUM5-PHD (1 and 10 nM) and used as a positive binding control.

Mentions: An electrophoretic mobility shift assay (EMSA) was performed to determine if the APUM5-PHD binds to the putative target RNAs. Genes that were highly down-regulated by APUM5 and had a ‘UGUA’ binding motif were selected. 32P-labeled synthetic 30 nucleotide RNAs along with ‘UGUA’ core sequence mutants were incubated with recombinant GST-APUM5-PHD protein. GST was used as a negative control. The EMSA results revealed that GST-APUM5-PHD bound effectively to DREB2A, RD22, COR15, and RAB18 but not to mutant RNAs, whereas the GST protein did not interact with these RNAs (Figure 8A–D). Furthermore, APUM5-PHD also showed strong binding affinity for hbNRE2 RNA (Figure 8E). APUM5 recognized the 8–10 nucleotide ‘UGUA’ core motifs. These results indicate that APUM5 binding affinity might be flexible for target binding motif recognition and this flexibility could contribute to multi-regulation of abiotic stress-responsive genes by destabilizing target mRNAs. This result confirmed that the Arabidopsis APUM5 protein has RNA binding activity and that the binding is important for regulating putative target 3′ UTRs.


APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression.

Huh SU, Paek KH - BMC Plant Biol. (2014)

Gel mobility shift assay of the recombinant containing APUM5-PHD for 3′ UTR motifs of abiotic stress-responsive genes. (A-D). Indicated 32P-labeled RNA probes were incubated with recombinant GST-APUM5-PHD (1, 10, 50, and 100 nM) for 30 min in 20 μl RNA binding buffer at room temperature. RNA-protein complexes were separated on a 5% native gel and analyzed by autoradiography. GST protein (1000 nM) was used as a negative control. (E) 32P-labeled hbNRE2 RNA probes were incubated with recombinant GST-APUM5-PHD (1 and 10 nM) and used as a positive binding control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3986970&req=5

Figure 8: Gel mobility shift assay of the recombinant containing APUM5-PHD for 3′ UTR motifs of abiotic stress-responsive genes. (A-D). Indicated 32P-labeled RNA probes were incubated with recombinant GST-APUM5-PHD (1, 10, 50, and 100 nM) for 30 min in 20 μl RNA binding buffer at room temperature. RNA-protein complexes were separated on a 5% native gel and analyzed by autoradiography. GST protein (1000 nM) was used as a negative control. (E) 32P-labeled hbNRE2 RNA probes were incubated with recombinant GST-APUM5-PHD (1 and 10 nM) and used as a positive binding control.
Mentions: An electrophoretic mobility shift assay (EMSA) was performed to determine if the APUM5-PHD binds to the putative target RNAs. Genes that were highly down-regulated by APUM5 and had a ‘UGUA’ binding motif were selected. 32P-labeled synthetic 30 nucleotide RNAs along with ‘UGUA’ core sequence mutants were incubated with recombinant GST-APUM5-PHD protein. GST was used as a negative control. The EMSA results revealed that GST-APUM5-PHD bound effectively to DREB2A, RD22, COR15, and RAB18 but not to mutant RNAs, whereas the GST protein did not interact with these RNAs (Figure 8A–D). Furthermore, APUM5-PHD also showed strong binding affinity for hbNRE2 RNA (Figure 8E). APUM5 recognized the 8–10 nucleotide ‘UGUA’ core motifs. These results indicate that APUM5 binding affinity might be flexible for target binding motif recognition and this flexibility could contribute to multi-regulation of abiotic stress-responsive genes by destabilizing target mRNAs. This result confirmed that the Arabidopsis APUM5 protein has RNA binding activity and that the binding is important for regulating putative target 3′ UTRs.

Bottom Line: We found that APUM5 was associated with both biotic and abiotic stress responses.The APUM5-Pumilio homology domain (PHD) protein bound to the 3' untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3' UTR.These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3' UTRs.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Life Sciences and Biotechnology, Korea University, 1, 5-ga, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea. khpaek95@korea.ac.kr.

ABSTRACT

Background: A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening.

Results: APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3' untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3' UTR.

Conclusions: These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3' UTRs.

Show MeSH
Related in: MedlinePlus