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Antisense RNA protects mRNA from RNase E degradation by RNA-RNA duplex formation during phage infection.

Stazic D, Lindell D, Steglich C - Nucleic Acids Res. (2011)

Bottom Line: The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs.These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs.Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany.

ABSTRACT
The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

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RNase E cleavage assay of four in vitro transcripts of genomic island II with (+) and without (−) recombinant Prochlorococcus RNase E. Cleavage fragments were separated on a 7 M urea–6% PAA gel and stained with ethidium bromide. Fragment sizes were estimated from an NEB ssRNA marker.
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Figure 3: RNase E cleavage assay of four in vitro transcripts of genomic island II with (+) and without (−) recombinant Prochlorococcus RNase E. Cleavage fragments were separated on a 7 M urea–6% PAA gel and stained with ethidium bromide. Fragment sizes were estimated from an NEB ssRNA marker.

Mentions: We established an in vitro RNase E cleavage assay to follow up our initial questions about how the activation of genes in genomic island II is regulated during phage infection and how these RNAs are protected from degradation in light of the enhanced transcription of RNase E. The mRNAs of four small ORFs of genomic island II (PMM0684, PMM0685, PMED4_07411, PMED4_7431), which were induced during phage infection (Figure 2B and Supplementary Figure S3), were synthesized in vitro and incubated with purified recombinant MED4 RNase E. All four transcripts were recognized by RNase E and were susceptible to specific cleavage (Figure 3). RNase E and RNase III cleavage sites of PMM0685 were investigated in more detail by northern hybridization (Supplementary Figure S4) using specific oligonucleotide probes targeting the 5′- and 3′-ends, respectively, of the ribonuclease-treated transcripts. The RNase E cleavage sites identified are listed in Table 1. All four cleavage sites were located in loop regions, indicating that the secondary structure is more relevant than sequence specificity for RNase E recognition (Figure 4B). RNase III cleavage sites were located in double-stranded RNA regions (Figure 4B) and are specified in Table 2. Figure 4A shows the results of RNase E, RNase III and RNase T1 digestions for single-stranded and duplex RNAs of PMM0685 in vitro-synthesized RNA. Both sense and antisense RNAs of duplex RNAs had identical sizes and an exact overlap. Incubation of single-stranded PMM0685 RNA with RNase E or RNase III yielded specific cleavage products, whereas RNase T1, which cleaves single-stranded RNA after guanine residues, led to complete digestion of PMM0685 in vitro-synthesized RNA. In contrast, the a priori duplex formation of sense and antisense RNA before RNase E treatment prevented RNase E digestion. RNase III treatment of duplex RNA resulted in complete digestion of the duplex, whereas treatment with RNase T1 did not show any cleavage products. These findings suggest that the secondary and tertiary structures of both RNA molecules were altered and hybridized to each other over their full-length. Clearly, the resultant structural changes hinder substrate accessibility of ribonucleases that recognize single-stranded regions (RNase E and RNase T1) and promote enzyme activity of RNA double strand-dependent enzymes like RNase III.Figure 3.


Antisense RNA protects mRNA from RNase E degradation by RNA-RNA duplex formation during phage infection.

Stazic D, Lindell D, Steglich C - Nucleic Acids Res. (2011)

RNase E cleavage assay of four in vitro transcripts of genomic island II with (+) and without (−) recombinant Prochlorococcus RNase E. Cleavage fragments were separated on a 7 M urea–6% PAA gel and stained with ethidium bromide. Fragment sizes were estimated from an NEB ssRNA marker.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113571&req=5

Figure 3: RNase E cleavage assay of four in vitro transcripts of genomic island II with (+) and without (−) recombinant Prochlorococcus RNase E. Cleavage fragments were separated on a 7 M urea–6% PAA gel and stained with ethidium bromide. Fragment sizes were estimated from an NEB ssRNA marker.
Mentions: We established an in vitro RNase E cleavage assay to follow up our initial questions about how the activation of genes in genomic island II is regulated during phage infection and how these RNAs are protected from degradation in light of the enhanced transcription of RNase E. The mRNAs of four small ORFs of genomic island II (PMM0684, PMM0685, PMED4_07411, PMED4_7431), which were induced during phage infection (Figure 2B and Supplementary Figure S3), were synthesized in vitro and incubated with purified recombinant MED4 RNase E. All four transcripts were recognized by RNase E and were susceptible to specific cleavage (Figure 3). RNase E and RNase III cleavage sites of PMM0685 were investigated in more detail by northern hybridization (Supplementary Figure S4) using specific oligonucleotide probes targeting the 5′- and 3′-ends, respectively, of the ribonuclease-treated transcripts. The RNase E cleavage sites identified are listed in Table 1. All four cleavage sites were located in loop regions, indicating that the secondary structure is more relevant than sequence specificity for RNase E recognition (Figure 4B). RNase III cleavage sites were located in double-stranded RNA regions (Figure 4B) and are specified in Table 2. Figure 4A shows the results of RNase E, RNase III and RNase T1 digestions for single-stranded and duplex RNAs of PMM0685 in vitro-synthesized RNA. Both sense and antisense RNAs of duplex RNAs had identical sizes and an exact overlap. Incubation of single-stranded PMM0685 RNA with RNase E or RNase III yielded specific cleavage products, whereas RNase T1, which cleaves single-stranded RNA after guanine residues, led to complete digestion of PMM0685 in vitro-synthesized RNA. In contrast, the a priori duplex formation of sense and antisense RNA before RNase E treatment prevented RNase E digestion. RNase III treatment of duplex RNA resulted in complete digestion of the duplex, whereas treatment with RNase T1 did not show any cleavage products. These findings suggest that the secondary and tertiary structures of both RNA molecules were altered and hybridized to each other over their full-length. Clearly, the resultant structural changes hinder substrate accessibility of ribonucleases that recognize single-stranded regions (RNase E and RNase T1) and promote enzyme activity of RNA double strand-dependent enzymes like RNase III.Figure 3.

Bottom Line: The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs.These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs.Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany.

ABSTRACT
The ecologically important cyanobacterium Prochlorococcus possesses the smallest genome among oxyphototrophs, with a reduced suite of protein regulators and a disproportionately high number of regulatory RNAs. Many of these are asRNAs, raising the question whether they modulate gene expression through the protection of mRNA from RNase E degradation. To address this question, we produced recombinant RNase E from Prochlorococcus sp. MED4, which functions optimally at 12 mM Mg(2+), pH 9 and 35°C. RNase E cleavage assays were performed with this recombinant protein to assess enzyme activity in the presence of single- or double-stranded RNA substrates. We found that extraordinarily long asRNAs of 3.5 and 7 kb protect a set of mRNAs from RNase E degradation that accumulate during phage infection. These asRNA-mRNA duplex formations mask single-stranded recognition sites of RNase E, leading to increased stability of the mRNAs. Such interactions directly modulate RNA stability and provide an explanation for enhanced transcript abundance of certain mRNAs during phage infection. Protection from RNase E-triggered RNA decay may constitute a hitherto unknown regulatory function of bacterial cis-asRNAs, impacting gene expression.

Show MeSH
Related in: MedlinePlus