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. 2017 Nov 27;8:1787. doi: 10.1038/s41467-017-01786-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Complete cycle of in vitro ITS2 processing by the combined activities of the Las1 complex and nuclear exosome. a Analysis of the protein samples used in the complete in vitro ITS2-processing assay. Substrate 27SB pre-rRNA was introduced by addition of pre-60S particles affinity-purified via TAPF-Rsa4 from a LAS1-AID degron strain. The yeast exosome subunits and Mtr4 were expressed in E. coli and affinity-purified from bacterial cell lysates. Exo(10) comprises the exosome core plus Rrp44. The Las1 complex (Rat1, Grc3, Las1, Rai1, from top to bottom) was overexpressed in yeast and TAP-purified via the split-tag approach (Las1-TpA/Grc3-Flag), yielding a wt and a mutant Las1 complex with a defective Grc3 kinase. The indicated eluates were analysed by SDS-PAGE and Coomassie staining. b Primer extension for detection of 27SB pre-rRNA, 26S pre-rRNA, 25S’ pre-rRNA and 25S rRNA. c Northern blotting for detection of 7S pre-rRNA, 5.8S rRNA and 5S rRNA after the in vitro processing reaction. Processing of 27SB substrate pre-rRNA present in pre-60S particles (affinity-purified via TAPF-Rsa4 from LAS1-AID degron strain) using the recombinant exosome and its cofactor Mtr4, and affinity-purified Las1 complex, either wt or harbouring the Grc3 kinase mutant. The in vitro reactions contained the purified proteins and ATP as indicated. 7S pre-rRNA was detected with two different probes. 5S rRNA served as a loading control