Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation  Ruchi Jain, Nahid Iglesias, Danesh Moazed  Molecular Cell  Volume 63, Issue 2, Pages 191-205 (July 2016) DOI: 10.1016/j.molcel.2016.05.039 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 priRNA1 Loads onto the RITS Complex and Can Trigger Dcr1-Independent ura4+ Silencing (A) Left: structure of the S. pombe rDNA unit. Precursor rRNA is processed into mature 18S, 5.8S, and 28S rRNAs (gray boxes) through the removal of external and internal transcribed spacer regions (ETS and ITS, respectively; white boxes). priRNA1 sequence and location (magenta box) relative to the 3′ end of 28S rRNA are indicated. Relative abundance of priRNA1 (magenta) and other sequences (gray) reads in an Ago1-associated sRNA library (Figure S1D). (B and C) Native (B) and denaturing (C) northern blot analyses of priRNA1 associated with 3xFLAG-Ago1 (wild-type [WT] or D580A mutant proteins) or Tas3-TAP, respectively. Membranes were probed with an end-labeled DNA oligo specific for priRNA1. 21-nt-long double-stranded (ds) or single-stranded (ss) RNA oligos were run as size markers. IgG, immunoglobulin G; IP, immunoprecipitation; OE, overexpressed. (D) Schematic of ura4+::kanR and ura4+::priRNA1. Detailed description is given in main text. Cyan box, ura4+ gene; magenta box, six priRNA1 binding sites. The position of the insertions relative to the ura4+ stop codon is shown. Black bars, locations of ura4+ ChIP-qPCR and qRT-PCR primers. (E–G) Analysis of ura4+ silencing triggered by priRNA1 insertion in cells transformed with 3xFLAG-Ago1-overexpressing plasmid (Ago1OE +). (E) Silencing of ura4+ results in colony growth on plate containing 5-fluoroorotic acid (FOA). (F and G) ChIP-qPCR analyses of H3K9me2 levels at ura4+ (F) and centromeric dg (G). Means of three independent experiments with SD are shown as enrichment over clr4Δ. See also Figure S1 and Tables S1–S3. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Genetic Requirements for priRNA1-Dependent ura4+ Silencing (A–E) Analysis of cells transformed with 3xFLAG-Ago1-overexpressing plasmid (Ago1OE +). (A–C) Silencing of ura4+ results in colony growth on plate containing 5-fluoroorotic acid (FOA). (D and E) ChIP-qPCR analyses of H3K9me2 levels at ura4+ (D) and centromeric dg (E). Means of three independent experiments with SD are shown as enrichment over ura4+::kanR. WT, wild-type. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Ago1 Slicing Activity Is Not Required for priRNA1-Dependent ura4+ Silencing and H3K9me2 (A–D) Analysis of ura4+ silencing triggered by priRNA1 insertion in cells carrying an empty vector (−) or a plasmid overexpressing 3xFLAG-Ago1 (Ago1OE) wild-type (WT) (+), ago1-D580A mutant (D580A), or ago1-F276A/Y513A/K517A mutant (3A). (A) Silencing of ura4+ results in colony growth on a plate containing 5-fluoroorotic acid (FOA). (B and C) ChIP-qPCR analyses of H3K9me2 levels at ura4+ (B) and centromeric dg (C). Means of three independent experiments with SD are shown as enrichment over clr4Δ carrying Ago1OE +. (D) ChIP-seq analyses showing that priRNA1 (highlighted in magenta) induces H3K9me2 domains surrounding the site of insertion. See also Figure S2. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Ago1 Slicing Activity Is Not Required for Secondary siRNA Generation (A and B) Denaturing (A) and native (B) northern blot analysis of sRNAs associated with wild-type 3xFLAG-Ago1 (WT) (+) or D580A mutant (D580A) proteins from cells with the indicated genotypes. The membrane in (A) was sequentially probed with end-labeled DNA oligos specific for ura4+ siRNAs and priRNA1. snoRNA snoR69 serves as a loading control. Native northern blot analysis (B) separates double-stranded (ds) from single-stranded (ss) siRNAs. 21-nt-long ds or ss RNA oligos complementary to the end-labeled DNA oligos were run as size markers. (C–E) High-throughput sequencing analyses of total sRNAs from ura4-priRNA1 ago1Δ cells carrying a plasmid overexpressing the wild-type 3xFLAG-Ago1 (WT) or the Slicer-incompetent ago1 mutant (D580A) proteins showing that priRNA1 (highlighted in magenta) induces secondary siRNAs generation surrounding the site of insertion. (C) Tracks show the normalized numbers of reads mapping to the ura4-priRNA1 locus in each library in a strand-specific way (blue, + strand; red, − strand). The two lower tracks represent the same data plotted on different scales. The location of the ura4-priRNA1 locus is indicated below the siRNA peaks, and the chromosome coordinates are indicated above the peaks. (D) Table summarizing primary or secondary sRNA read densities mapping to the indicated regions in Ago1 WT or D580A RNA-seq libraries, in reads per million. (E) Abundance of secondary siRNA reads (blue, mapping to ura4+ and kanR loci) relative to the number of priRNA1 reads (magenta, primary siRNAs) in each sequencing libraries. See also Figure S3 and Table S3. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Slicing Promotes Release of the RITS Complex from Chromatin (A) Schematic of RITS residence time experiment. Detailed description is given in the main text. WT, wild-type. (B and C) ChIP-qPCR analyses of Tas3-TAP levels at ura4+ (B) and centromeric dg locus (C) in the indicated cells carrying either a plasmid overexpressing the wild-type 3xFlag-ago1 allele (+), the Slicer-incompetent ago1-D580A mutant (D580A), or an empty 3xFLAG vector (−). The relative fold enrichment of Tas3-TAP was normalized to the untagged strain. The p value is based on a one-tailed, two-sample unequal variance Student’s t test. Error bars indicate SD; n = 3 biological replicates. See also Figure S4. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Maintenance of the Modified Minichromosome L5/priRNA1 in the Slicer-Incompetent Ago1 (A–C) Minichromosome stability analyses in wild-type (WT) cells containing an ade6-704 allele and carrying an empty plasmid or a plasmid overexpressing the 3xFLAG-Ago1 WT or D580A mutant allele were transformed with either MC-L5 or MC-L5/priRNA1. (A) Schematic diagrams of the minichromosome plasmids (MC) bearing the L5 fragment (left: MC-L5; Baum et al., 1994; Buscaino et al., 2013) or the modified L5/priRNA1 fragment (right: MC-L5/priRNA1). L5, 1.6 kb of the centromeric dg element (gray box); sup3-5-tRNA, suppressor of ade6-704. MC-L5/priRNA1 contains three insertions of six priRNA1 repeats (magenta boxes) in the L5 sequence. Detailed description is given in Experimental Procedures (Figure S5E). (B) Colony color assay on medium with limiting adenine to examine minichromosome stability. White-sectoring colonies indicate stable minichromosomes that are retained during mitosis (marked with white arrowheads in WT + Ago1OE D580A + MC-L5/priRNA1 cells); red colonies are indicative of minichromosome loss. (C) Percentage of MC-positive white-sectoring colonies in the indicated strains. Quantification of the colony color assay results shown in (B). Completely white colonies might contain integrated minichromosomes and were not included in the quantification. See also Figure S5 and Table S2. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Model for RNAi-Mediated Heterochromatin Assembly Is S. pombe Highlighting the Slicer-Dependent and -Independent Steps The slicing activity of Ago1 is critical for release of the siRNA passenger strand and generation of active RITS (Slicer I) and for efficient release and recycling of RITS coupled to nascent transcript degradation (Slicer II). The recruitment of CLRC, which leads to H3K9 methylation, and the recruitment of RDRC, which leads to dsRNA synthesis and secondary siRNA generation, are both independent of Ago1 Slicer activity (blue shade). RITS and the Ago1 siRNA-loading complex ARC form a minimal sRNA-directed pathway that can recruit downstream activities to promote heterochromatin assembly. Molecular Cell 2016 63, 191-205DOI: (10.1016/j.molcel.2016.05.039) Copyright © 2016 Elsevier Inc. Terms and Conditions