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. 2021 Mar 25;12:1859. doi: 10.1038/s41467-021-22077-4

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© The Author(s) 2021

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. 3 — a RNA immunoprecipitation (RIP) assays were performed on extracts prepared from RTT106-TAP; GAL1:3HA-BCD1 cells disrupted (rsa1Δ) or not of the RSA1 ORF. The cells were cultivated in the presence of galactose (YPG) or shifted to a glucose-containing medium (YPD) for 6 h before preparation of the extract. IP was performed using IgG-sepharose beads. RNAs retained on beads were quantified by RT-qPCR as described in the Methods section. The snoRNAs and pre-snoRNAs analyzed are indicated. b Chromatin immunoprecipitation (ChIP) assays were performed on yeast GAL1:3HA-BCD1 cells transformed with recombinant plasmid FLAG-RTT106; with (rsa1Δ) or without the disruption of the RSA1 ORF. Cells were cultivated in YPG or shifted to YPD medium before ChIP assays. Cells expressing nontagged Rtt106p (GAL1:3HA-BCD1 and GAL1:3HA-BCD1; rsa1Δ) were used as controls and IP was performed using anti-FLAG antibody. The loci analyzed by qPCR analysis are indicated. c Chip assays performed as in panel b on GAL1:3HA-BCD1 cells transformed with recombinant plasmid FLAG-RTT106. Primers were used for qPCR analysis on both side and across the U3 encoding gene (SNR17A). Data reported in the panels a and c are mean values plus standard error of the mean of three biological replicates (n = 3). Data reported in panel b are mean values plus standard error of the mean of three (for GAL1::3HA-BCD1, rsa1Δ + p413 (−) and GAL1::3HA-BCD1, rsa1Δ + p413::FLAG-RTT106; n = 3) or four (for GAL1::3HA-BCD1 + p413 (−) and GAL1::3HA-BCD1 + p413::FLAG-RTT106; n = 4) biological replicates. Two-tailed t-tests: *P < 0.05, **P < 0.01, ***P < 0.001. In panel b, for FLAG-RTT106 YPG versus YPD: * = 0.012 for HTA/B1, *** = 0.0006 for SNR128 (U14), * = 0.045 for SNR52, ** = 0.002 for SNR32, * = 0.032 for HMR a1. Note that SNR17A (U3) is close to significance: P = 0,056. For FLAG-RTT106 YPG versus rsa1Δ; FLAG-RTT106 YPG: * = 0.02 for SNR128 (U14) and * = 0.017 for SNR32. In panel c, for FLAG-RTT106 YPG versus YPD: ** = 0.006, * = 0.031, and ** = 0.001 from the left to the right. Source data for all these panels are provided as Source Data files.