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A method for in vivo identification of bacterial small RNA-binding proteins.

Osborne J, Djapgne L, Tran BQ, Goo YA, Oglesby-Sherrouse AG - Microbiologyopen (2014)

Bottom Line: Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins.Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo.As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.

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Development of PrrF and PrrH probes for sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS). (A) Sequence of the prrF locus, with the PrrF1, PrrF2 and PrrH probe locations underlined. Asterisks indicate the transcriptional start site of PrrF1 and PrrH (*) and PrrF2 (**). The PrrF1 and PrrF2 transcribed sequences are in bold, and the PrrF1 and PrrF2 Rho-independent terminators are italicized. (B) Northern blots of PAO1 and the indicated prrF mutants grown in M9 minimal media for 18 h, with or without supplementation of 100 μmol/L FeCl3 as indicated, using the PrrF1, PrrF2, and PrrH probes shown in (A). (C) Expression of the PrrF and PrrH sRNAs was determined by qPCR after 4, 8, and 18 h of growth in M9 minimal media with no iron supplementation. Relative expression of each RNA was determined using a standard curve, and values for each time point were normalized to the 4 h time point. Error bars indicate the standard deviation of three independent experiments.
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fig01: Development of PrrF and PrrH probes for sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS). (A) Sequence of the prrF locus, with the PrrF1, PrrF2 and PrrH probe locations underlined. Asterisks indicate the transcriptional start site of PrrF1 and PrrH (*) and PrrF2 (**). The PrrF1 and PrrF2 transcribed sequences are in bold, and the PrrF1 and PrrF2 Rho-independent terminators are italicized. (B) Northern blots of PAO1 and the indicated prrF mutants grown in M9 minimal media for 18 h, with or without supplementation of 100 μmol/L FeCl3 as indicated, using the PrrF1, PrrF2, and PrrH probes shown in (A). (C) Expression of the PrrF and PrrH sRNAs was determined by qPCR after 4, 8, and 18 h of growth in M9 minimal media with no iron supplementation. Relative expression of each RNA was determined using a standard curve, and values for each time point were normalized to the 4 h time point. Error bars indicate the standard deviation of three independent experiments.

Mentions: Northern analysis of the PrrF and PrrH sRNAs was performed as previously described with some modifications (Oglesby-Sherrouse and Vasil 2010). Briefly, 10–20 μg of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7 mol/L urea) gel then transferred to a BrightStar membrane (Life Technologies, Grand Island, NY, USA) using a semi-dry transfer apparatus. Biotinylated oligonucleotides that were complementary to the regions of PrrF1, PrrF2, or PrrH as shown in Figure 1A were purchased from Integrated DNA Technologies (IDT) and hybridized to blots overnight at 42°C. The membrane was washed using the Northern Max Low Stringency and High Stringency wash solutions according to the manufacturer's instructions. Detection of the biotinylated probes was carried out using the BrightStar BioDetect nonisotopic detection kit (Life Technologies).


A method for in vivo identification of bacterial small RNA-binding proteins.

Osborne J, Djapgne L, Tran BQ, Goo YA, Oglesby-Sherrouse AG - Microbiologyopen (2014)

Development of PrrF and PrrH probes for sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS). (A) Sequence of the prrF locus, with the PrrF1, PrrF2 and PrrH probe locations underlined. Asterisks indicate the transcriptional start site of PrrF1 and PrrH (*) and PrrF2 (**). The PrrF1 and PrrF2 transcribed sequences are in bold, and the PrrF1 and PrrF2 Rho-independent terminators are italicized. (B) Northern blots of PAO1 and the indicated prrF mutants grown in M9 minimal media for 18 h, with or without supplementation of 100 μmol/L FeCl3 as indicated, using the PrrF1, PrrF2, and PrrH probes shown in (A). (C) Expression of the PrrF and PrrH sRNAs was determined by qPCR after 4, 8, and 18 h of growth in M9 minimal media with no iron supplementation. Relative expression of each RNA was determined using a standard curve, and values for each time point were normalized to the 4 h time point. Error bars indicate the standard deviation of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263517&req=5

fig01: Development of PrrF and PrrH probes for sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS). (A) Sequence of the prrF locus, with the PrrF1, PrrF2 and PrrH probe locations underlined. Asterisks indicate the transcriptional start site of PrrF1 and PrrH (*) and PrrF2 (**). The PrrF1 and PrrF2 transcribed sequences are in bold, and the PrrF1 and PrrF2 Rho-independent terminators are italicized. (B) Northern blots of PAO1 and the indicated prrF mutants grown in M9 minimal media for 18 h, with or without supplementation of 100 μmol/L FeCl3 as indicated, using the PrrF1, PrrF2, and PrrH probes shown in (A). (C) Expression of the PrrF and PrrH sRNAs was determined by qPCR after 4, 8, and 18 h of growth in M9 minimal media with no iron supplementation. Relative expression of each RNA was determined using a standard curve, and values for each time point were normalized to the 4 h time point. Error bars indicate the standard deviation of three independent experiments.
Mentions: Northern analysis of the PrrF and PrrH sRNAs was performed as previously described with some modifications (Oglesby-Sherrouse and Vasil 2010). Briefly, 10–20 μg of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7 mol/L urea) gel then transferred to a BrightStar membrane (Life Technologies, Grand Island, NY, USA) using a semi-dry transfer apparatus. Biotinylated oligonucleotides that were complementary to the regions of PrrF1, PrrF2, or PrrH as shown in Figure 1A were purchased from Integrated DNA Technologies (IDT) and hybridized to blots overnight at 42°C. The membrane was washed using the Northern Max Low Stringency and High Stringency wash solutions according to the manufacturer's instructions. Detection of the biotinylated probes was carried out using the BrightStar BioDetect nonisotopic detection kit (Life Technologies).

Bottom Line: Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins.Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo.As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.

Show MeSH