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The RNA helicase Mtr4p is a duplex-sensing translocase

Nature Chemical Biology volume 13, pages 99–104 (2017)Cite this article

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Abstract

The conserved Saccharomyces cerevisiae Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3′-to-5′ translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p molecule performs consecutive unwinding steps. We further show that RNA unwinding by Mtr4p requires interaction with upstream RNA duplex. Inclusion of Mtr4p within the TRAMP complex increases the rate constant for unwinding initiation but does not change the characteristics of Mtr4p's helicase mechanism. Our data indicate that Mtr4p utilizes a previously unknown unwinding mode that combines aspects of canonical translocating helicases and non-canonical duplex-sensing helicases, thereby restricting directional translocation to duplex regions.

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Figure 1: Observation of Mtr4p unwinding RNA duplex via high-resolution optical trapping.
Figure 2: Mtr4p unwinding rates versus tether tension and nucleotide composition for unwinding the 16-bp RNA hairpin.
Figure 3: Mtr4p unwinding 32-bp-long RNA hairpins.
Figure 4: Observation of TRAMP unwinding RNA.
Figure 5: Mtr4p RNA unwinding model.

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Acknowledgements

We thank members of the Comstock and Jankowsky laboratories for scientific discussion. Funding was provided by US National Science Foundation grant MCB-1514706 (to M.J.C.) and US National Institutes of Health grants R01 GM118088 and GM099720 (to E.J.).

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Authors and Affiliations

  1. Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA

    Eric M Patrick & Matthew J Comstock

  2. Center for RNA Molecular Biology and Department of Biochemistry, Case Western University, Cleveland, Ohio, USA

    Sukanya Srinivasan & Eckhard Jankowsky

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  1. Eric M Patrick
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  4. Matthew J Comstock
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Contributions

M.J.C., E.J. and E.M.P. conceived and designed the high-resolution single-molecule trapping experiments. E.M.P. produced tether constructs and performed all single-molecule experiments. E.M.P. and M.J.C. performed single-molecule data analysis. S.S. produced Mtr4p and TRAMP samples and performed all ensemble experiments and data analysis. E.M.P., S.S., E.J. and M.J.C. wrote the paper.

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Correspondence to Matthew J Comstock.

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Patrick, E., Srinivasan, S., Jankowsky, E. et al. The RNA helicase Mtr4p is a duplex-sensing translocase. Nat Chem Biol 13, 99–104 (2017). https://doi.org/10.1038/nchembio.2234

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