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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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Gel filtration profile of affinity-purified pQE70-PMM1501 protein. Sizes of RNase E conformers were estimated using Bio-Rad’s gel filtration standard (black arrows) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Elution peaks of RNase E conformers are indicated by black arrows. Peaks at elution fraction 15 and 21 correspond to buffer components. The inset on the right shows a 10% SDS–PAGE gel that was loaded with elution fractions of the gel filtration.
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Figure 1: Gel filtration profile of affinity-purified pQE70-PMM1501 protein. Sizes of RNase E conformers were estimated using Bio-Rad’s gel filtration standard (black arrows) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Elution peaks of RNase E conformers are indicated by black arrows. Peaks at elution fraction 15 and 21 correspond to buffer components. The inset on the right shows a 10% SDS–PAGE gel that was loaded with elution fractions of the gel filtration.

Mentions: Expression of recombinant Prochlorococcus MED4 RNase E (PMM1501) protein required the adjustment of the codon usage of the first 63 nt and the last 42 nt of PMM1501 for sufficient expression in E. coli. C-terminally His-tagged pQE70-PMM1501 was overexpressed in E. coli strain Arctic Express (DE3) RIL and purified under native conditions by affinity column chromatography. Recombinant PMM1501 protein migrated according to its theoretical molecular weight of 70 kDa in SDS–polyacrylamide gels (Figure 1 and Supplementary Figure S1). Mass spectrometry analysis of the purified protein fraction did not reveal any contamination with E. coli-specific RNase E protein or other host ribonucleases (data not shown). Furthermore, western blots of the purified protein fraction were immunodecorated with antibodies targeting E. coli-specific RNase E, RNase R or PNPase. None of these antibodies detected measurable amounts of the respective target protein in the elution fraction used for cleavage assays (Supplementary Figure S1A). To test for the presence of other sources of E. coli-specific exonuclease activity in the purified protein fraction, we in vitro transcribed a poly-A RNA, a substrate for exonucleases in general (RNase R, PNPase, RNase II) but not for RNase E, and incubated it with RNase R or recombinant Prochlorococcus RNase E. Cleavage activity was only detected with RNase R but not with recombinant PMM1501 (Supplementary Figure S1B) verifying that the recombinant Prochlorococcus RNase E protein was not contaminated with nucleases from E. coli.Figure 1.


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)

Gel filtration profile of affinity-purified pQE70-PMM1501 protein. Sizes of RNase E conformers were estimated using Bio-Rad’s gel filtration standard (black arrows) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Elution peaks of RNase E conformers are indicated by black arrows. Peaks at elution fraction 15 and 21 correspond to buffer components. The inset on the right shows a 10% SDS–PAGE gel that was loaded with elution fractions of the gel filtration.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Gel filtration profile of affinity-purified pQE70-PMM1501 protein. Sizes of RNase E conformers were estimated using Bio-Rad’s gel filtration standard (black arrows) containing thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Elution peaks of RNase E conformers are indicated by black arrows. Peaks at elution fraction 15 and 21 correspond to buffer components. The inset on the right shows a 10% SDS–PAGE gel that was loaded with elution fractions of the gel filtration.
Mentions: Expression of recombinant Prochlorococcus MED4 RNase E (PMM1501) protein required the adjustment of the codon usage of the first 63 nt and the last 42 nt of PMM1501 for sufficient expression in E. coli. C-terminally His-tagged pQE70-PMM1501 was overexpressed in E. coli strain Arctic Express (DE3) RIL and purified under native conditions by affinity column chromatography. Recombinant PMM1501 protein migrated according to its theoretical molecular weight of 70 kDa in SDS–polyacrylamide gels (Figure 1 and Supplementary Figure S1). Mass spectrometry analysis of the purified protein fraction did not reveal any contamination with E. coli-specific RNase E protein or other host ribonucleases (data not shown). Furthermore, western blots of the purified protein fraction were immunodecorated with antibodies targeting E. coli-specific RNase E, RNase R or PNPase. None of these antibodies detected measurable amounts of the respective target protein in the elution fraction used for cleavage assays (Supplementary Figure S1A). To test for the presence of other sources of E. coli-specific exonuclease activity in the purified protein fraction, we in vitro transcribed a poly-A RNA, a substrate for exonucleases in general (RNase R, PNPase, RNase II) but not for RNase E, and incubated it with RNase R or recombinant Prochlorococcus RNase E. Cleavage activity was only detected with RNase R but not with recombinant PMM1501 (Supplementary Figure S1B) verifying that the recombinant Prochlorococcus RNase E protein was not contaminated with nucleases from E. coli.Figure 1.

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