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GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

Nature Microbiology volume 2, Article number: 16239 (2017) Cite this article

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Abstract

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

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Figure 1: T4 RNA ligase-catalysed in vivo linking of sRNA to mRNAs.
Figure 2: Factors influencing the ligation of PrrF1 sRNA to the sodB and PA4880 target mRNAs.
Figure 3: Overview of the GRIL-seq method.
Figure 4: Identification of targets of PrrF1 sRNA using GRIL-seq.
Figure 5: Comparison of the list of genes identified by GRIL-seq and RNA-seq following overexpression of PrrF1.

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  • 14 July 2017

    In the PDF version of this article previously published, the year of publication provided in the footer of each page and in the 'How to cite' section was erroneously given as 2017, it should have been 2016. This error has now been corrected. The HTML version of the article was not affected.

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Acknowledgements

We thank U. Das and S. Shuman for the gift of a recombinant plasmid with the T4 RNA ligase gene and W. Robins for help with Illumina sequencing. We thank T. Dougherty for critical reading of the manuscript. This work was supported by National Institutes of Health grant R37 AI021451 to S.L. and R15 GM102755 to B.T.

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Authors and Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Kook Han & Stephen Lory

  2. Computer Science Department, Wellesley College, Wellesley, 02481, Massachusetts, USA

    Brian Tjaden

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  1. Kook Han
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Contributions

S.L. and K.H. conceived the approach. K.H. carried out all of the experiments. S.L., K.H. and B.T. analysed the expression and GRIL-seq data. S.L., K.H. and B.T. wrote the paper.

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Correspondence to Stephen Lory.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Notes 1-5, Supplementary Methods, Supplementary Figures 1–15, Supplementary Tables 3–5, Supplementary References. (PDF 9943 kb)

Supplementary Tables 1 and 2

Supplementary Table 1: RNA-seq analysis, Supplementary Table 2: GRIL-seq analysis. (XLSX 800 kb)

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Han, K., Tjaden, B. & Lory, S. GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation. Nat Microbiol 2, 16239 (2017). https://doi.org/10.1038/nmicrobiol.2016.239

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