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The rnb gene of Synechocystis PCC6803 encodes a RNA hydrolase displaying RNase II and not RNase R enzymatic properties.

Matos RG, Fialho AM, Giloh M, Schuster G, Arraiano CM - PLoS ONE (2012)

Bottom Line: PCC6803.The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates.When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cyanobacteria are photosynthetic prokaryotic organisms that share characteristics with bacteria and chloroplasts regarding mRNA degradation. Synechocystis sp. PCC6803 is a model organism for cyanobacteria, but not much is known about the mechanism of RNA degradation. Only one member of the RNase II-family is present in the genome of Synechocystis sp PCC6803. This protein was shown to be essential for its viability, which indicates that it may have a crucial role in the metabolism of Synechocystis RNA. The aim of this work was to characterize the activity of the RNase II/R homologue present in Synechocystis sp. PCC6803. The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates. When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics. This is the first reported case where when only one member of the RNase II/R family exists it displays RNase II and not RNase R characteristics.

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Modelling the RNase II protein from Synechocystis.(A) Representation of the predictive 3D model from Synechocystis RNase II (red) (B) and E. coli RNase II crystal structure (green) (PDB 2IX0 and 2IX1), with the RNA molecule inside (blue). (C) Superposition of E. coli RNase II structure and Synechocystis RNase II model. (D) In the catalytic cavity, the residues important for the activity of E. coli RNase II are shown in green, while the ones from Synechocystis protein are indicated in red.
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pone-0032690-g008: Modelling the RNase II protein from Synechocystis.(A) Representation of the predictive 3D model from Synechocystis RNase II (red) (B) and E. coli RNase II crystal structure (green) (PDB 2IX0 and 2IX1), with the RNA molecule inside (blue). (C) Superposition of E. coli RNase II structure and Synechocystis RNase II model. (D) In the catalytic cavity, the residues important for the activity of E. coli RNase II are shown in green, while the ones from Synechocystis protein are indicated in red.

Mentions: We also tested the activity of the protein against the synthetic double-stranded substrate, 16–30 ds. E. coli RNase II is sensitive to secondary structures, and is not able to degrade this RNA, stalling 7 nt before it reaches the double-stranded region, releasing a 23 nt fragment (Figure 5) [30], [31]. In contrast, E. coli RNase R is able to overcome the secondary structures, releasing the typical 2 nt fragments (Figure 5) [28]. When we tested the synRNB we could observe that it was not able to overcome secondary structures. It stalled 4 nt before it reached the double-stranded region, releasing a fragment of 20 nt, which is shorter when compared to the 23 nt fragment released by E. coli RNase II (Figure 5). The resolution of the crystal structure from E. coli RNase II showed us that the catalytic cavity of the protein is only accessible to single stranded RNA due to the steric hindrance at its entrance caused by the RNA-binding domains [11]. The synRNB showed to be able to move closer to the double-stranded junction when compared to the E. coli protein, since that the final product released is shorter (20 nt vs. 23 nt, respectively). This may indicate that the RNA-binding domains may have a different rearrangement in this protein. In order to address this question, we modelled synRNB and compared it with E. coli RNase II 3D structure (Figure 8). Both proteins seemed to have a similar overall structure arrangement, with the important residues for catalysis located in the same spatial position (Figure 8D). The active site of RNase II is composed by four highly conserved aspartates and an arginine, which are important for catalysis [13], [15], [17]. Tyrosine is a residue responsible for setting the final product in the RNases from this family [13], [15], [17] (Figure 8D). In synRNB, these residues can also be found and are located in an equivalent position (Figure 8D). If we look closer to the RNA binding domains, it is possible to see that the CSD1 of synRNB (Figure 8A) is quite different from the one from E. coli RNase II (Figure 8B). When we superposed both structures, that difference is more noticeable (Figure 8C). Moreover, the superposition of both structures also showed that the S1 domain from SynRNB (red) lacks at least two β-sheets when compared to the one from RNase II (green) (Figure 8C). Therefore, the CSD1 from Synechocystis protein is more distant from the S1 domain, which could result in a wider anchoring region which in turn might allow the substrate to move nearer to the catalytic cavity, explaining why this protein is able to get closer to the double-stranded junction (Figure 8C). The activity of this protein was also determined with the 16–30 ds at both temperatures, 30°C and 37°C. Similarly to what was observed to the single-stranded substrates, the activity of the protein for the 16–30 ds is the same as observed for the other substrates and does not change with temperature (Figure 6). At 30°C, the degradation pattern of the protein remained unaltered, and the protein was still not able to overcome secondary structures, releasing a final product of 20 nt similarly to what was observed at 37°C (Figures 5 and S1). It is known that, at lower temperatures, the RNA molecules form more stable secondary structures. In Synechocystis PCC6803, low temperatures highly induce the expression of an RNA helicase, CrhR [32], which may be involved in the degradation of the transcripts at these temperatures by helping to unwind the secondary structures. In a strain defective in this helicase, the PNPase levels are increased up to ∼2-fold. This would help to eliminate the transcripts with cold-induced excessive secondary structures [33]. No changes were observed for synRNB protein. Together with the results described here, these findings indicate that this protein may not be involved in the degradation of double-stranded substrates at environmental temperatures. Moreover, when we determined the dissociation constants for this substrate, the value is very similar to the ones obtained for the single-stranded substrates (Table 1). However, the protein associates and dissociates more rapidly to the double-stranded substrate when compared to the other two (Table 1, ka and kd values).


The rnb gene of Synechocystis PCC6803 encodes a RNA hydrolase displaying RNase II and not RNase R enzymatic properties.

Matos RG, Fialho AM, Giloh M, Schuster G, Arraiano CM - PLoS ONE (2012)

Modelling the RNase II protein from Synechocystis.(A) Representation of the predictive 3D model from Synechocystis RNase II (red) (B) and E. coli RNase II crystal structure (green) (PDB 2IX0 and 2IX1), with the RNA molecule inside (blue). (C) Superposition of E. coli RNase II structure and Synechocystis RNase II model. (D) In the catalytic cavity, the residues important for the activity of E. coli RNase II are shown in green, while the ones from Synechocystis protein are indicated in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293843&req=5

pone-0032690-g008: Modelling the RNase II protein from Synechocystis.(A) Representation of the predictive 3D model from Synechocystis RNase II (red) (B) and E. coli RNase II crystal structure (green) (PDB 2IX0 and 2IX1), with the RNA molecule inside (blue). (C) Superposition of E. coli RNase II structure and Synechocystis RNase II model. (D) In the catalytic cavity, the residues important for the activity of E. coli RNase II are shown in green, while the ones from Synechocystis protein are indicated in red.
Mentions: We also tested the activity of the protein against the synthetic double-stranded substrate, 16–30 ds. E. coli RNase II is sensitive to secondary structures, and is not able to degrade this RNA, stalling 7 nt before it reaches the double-stranded region, releasing a 23 nt fragment (Figure 5) [30], [31]. In contrast, E. coli RNase R is able to overcome the secondary structures, releasing the typical 2 nt fragments (Figure 5) [28]. When we tested the synRNB we could observe that it was not able to overcome secondary structures. It stalled 4 nt before it reached the double-stranded region, releasing a fragment of 20 nt, which is shorter when compared to the 23 nt fragment released by E. coli RNase II (Figure 5). The resolution of the crystal structure from E. coli RNase II showed us that the catalytic cavity of the protein is only accessible to single stranded RNA due to the steric hindrance at its entrance caused by the RNA-binding domains [11]. The synRNB showed to be able to move closer to the double-stranded junction when compared to the E. coli protein, since that the final product released is shorter (20 nt vs. 23 nt, respectively). This may indicate that the RNA-binding domains may have a different rearrangement in this protein. In order to address this question, we modelled synRNB and compared it with E. coli RNase II 3D structure (Figure 8). Both proteins seemed to have a similar overall structure arrangement, with the important residues for catalysis located in the same spatial position (Figure 8D). The active site of RNase II is composed by four highly conserved aspartates and an arginine, which are important for catalysis [13], [15], [17]. Tyrosine is a residue responsible for setting the final product in the RNases from this family [13], [15], [17] (Figure 8D). In synRNB, these residues can also be found and are located in an equivalent position (Figure 8D). If we look closer to the RNA binding domains, it is possible to see that the CSD1 of synRNB (Figure 8A) is quite different from the one from E. coli RNase II (Figure 8B). When we superposed both structures, that difference is more noticeable (Figure 8C). Moreover, the superposition of both structures also showed that the S1 domain from SynRNB (red) lacks at least two β-sheets when compared to the one from RNase II (green) (Figure 8C). Therefore, the CSD1 from Synechocystis protein is more distant from the S1 domain, which could result in a wider anchoring region which in turn might allow the substrate to move nearer to the catalytic cavity, explaining why this protein is able to get closer to the double-stranded junction (Figure 8C). The activity of this protein was also determined with the 16–30 ds at both temperatures, 30°C and 37°C. Similarly to what was observed to the single-stranded substrates, the activity of the protein for the 16–30 ds is the same as observed for the other substrates and does not change with temperature (Figure 6). At 30°C, the degradation pattern of the protein remained unaltered, and the protein was still not able to overcome secondary structures, releasing a final product of 20 nt similarly to what was observed at 37°C (Figures 5 and S1). It is known that, at lower temperatures, the RNA molecules form more stable secondary structures. In Synechocystis PCC6803, low temperatures highly induce the expression of an RNA helicase, CrhR [32], which may be involved in the degradation of the transcripts at these temperatures by helping to unwind the secondary structures. In a strain defective in this helicase, the PNPase levels are increased up to ∼2-fold. This would help to eliminate the transcripts with cold-induced excessive secondary structures [33]. No changes were observed for synRNB protein. Together with the results described here, these findings indicate that this protein may not be involved in the degradation of double-stranded substrates at environmental temperatures. Moreover, when we determined the dissociation constants for this substrate, the value is very similar to the ones obtained for the single-stranded substrates (Table 1). However, the protein associates and dissociates more rapidly to the double-stranded substrate when compared to the other two (Table 1, ka and kd values).

Bottom Line: PCC6803.The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates.When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, Oeiras, Portugal.

ABSTRACT
Cyanobacteria are photosynthetic prokaryotic organisms that share characteristics with bacteria and chloroplasts regarding mRNA degradation. Synechocystis sp. PCC6803 is a model organism for cyanobacteria, but not much is known about the mechanism of RNA degradation. Only one member of the RNase II-family is present in the genome of Synechocystis sp PCC6803. This protein was shown to be essential for its viability, which indicates that it may have a crucial role in the metabolism of Synechocystis RNA. The aim of this work was to characterize the activity of the RNase II/R homologue present in Synechocystis sp. PCC6803. The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates. When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics. This is the first reported case where when only one member of the RNase II/R family exists it displays RNase II and not RNase R characteristics.

Show MeSH