Termination of Cryptic Unstable Transcripts Is Directed by Yeast RNA-Binding Proteins Nrd1 and Nab3  John T. Arigo, Daniel E. Eyler, Kristina L. Carroll, Jeffry L. Corden  Molecular Cell  Volume 23, Issue 6, Pages 841-851 (September 2006) DOI: 10.1016/j.molcel.2006.07.024 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 3′-Extended Readthrough Products Accumulate in nrd1 and nab3 Mutants Analysis of genes upregulated in the nab3-11 microarray. Genomic maps are above the blots with RNAs indicated by long arrows. Enlarged open arrows represent a CUT promoter. Closed arrows represent the promoter of adjacent mRNAs. Temperature-sensitive mutants were shifted to 37°C for 2 hr. (A) Northern blot using a T7 probe specific for GRE1. (B) Northern blot showing transcripts from the 3′ end of FMP40 cloned in the antisense orientation into a GFP readthrough transcription reporter vector (Carroll et al., 2004). (C) Quantification of transcription run-on analysis of the antisense FMP40 region. Hybridization signals were averaged from three independent experiments. Signals from RNA probes A, B, and C were background corrected and normalized to signals from an RNA probe specific for ACT1. Error bars indicate standard deviation. (D) Northern blot using a T7 probe specific for GAT4. (E) Northern blot using a T7 probe specific for NEL025C. SCR1 is used as a loading control. Molecular Cell 2006 23, 841-851DOI: (10.1016/j.molcel.2006.07.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Involvement of the Nuclear Exosome in Stability of Cryptic Unstable Transcripts (A) Map showing the position of antisense RPR2 transcripts and the T7 probe used in the blots shown below. (B) Northern blot analysis of RNAs produced in various exosome mutant backgrounds. The ACT1 probe is used as a loading control. Temperature-sensitive strains were shifted to 37°C for 2 hr. Arrowheads indicate the position of the readthrough transcript described in Figure 1. (C) Ten-fold serial dilutions of yeast cultures identified at the left were grown on YPD media at the indicated temperatures. (D) Image Quant scans of lanes 12 and 13 from (B). The dashed line represents RNA from the rrp47Δ strain while the solid line represents the rrp47Δ, nrd1-102 strain. Both strains were grown at 25°C. The arrow below indicates the direction of electrophoretic mobility. Molecular Cell 2006 23, 841-851DOI: (10.1016/j.molcel.2006.07.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Chromatin Immunoprecipitation of TAP-Tagged Nrd1 and Nab3 (A) Nrd1 and Nab3 occupancy in Nrd1 and Nab3 TAP-tagged strains with PCR primers amplifying the NEL025C region. (B) Nrd1 and Nab3 occupancy in Nrd1 and Nab3 TAP-tagged strains with PCR primers amplifying the GRE1 region. Pol II was immunoprecipitated from a wild-type strain with an antibody to the Rpb3 subunit. (C) Nrd1 and Nab3 occupancy in Nrd1 and Nab3 TAP-tagged strains with PCR primers amplifying the GAT4 region. Quantification of occupancy value is explained in Experimental Procedures. Values for TAP-tagged strains were averaged from quadruplicate experiments. Pol II experiments were averaged from duplicate experiments. Error bars represent standard deviation with the exception of the Pol II data, which are the average of two experiments. Molecular Cell 2006 23, 841-851DOI: (10.1016/j.molcel.2006.07.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Dissecting a CUT Termination Element (A) Map of the FLC1-FMP40 intergenic region harboring a CUT. Potential Nab3 binding sites are indicated by asterisks. The position of the CUT transcript is indicated above the line, and regions (3×, 1×, and 0×) that represent potential termination elements analyzed in (C) are indicated below the line. Potential Nab3 binding site sequences (TCTT) are indicated by bold letters. (B) Northern blot with total RNA probed with a T7 probe specific for the intergenic region shown in (A). Temperature-sensitive strains were shifted to 37°C for 2 hr. An ACT1 probe is used as a loading control, while the TRS31 probe is used to verify that Nrd1 and Nab3 function have been compromised at the nonpermissive temperature. (C) Northern blot with various potential termination regions cloned into a GFP readthrough transcription vector. The T3 probe is specific for GFP. SCR1 is used as a loading control. (D) Northern blot as in (C) but with either the wild-type 1× or a mutated 1× in which the Nab3 binding site TCTT has been changed to TATT. Molecular Cell 2006 23, 841-851DOI: (10.1016/j.molcel.2006.07.024) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Nrd1 CTD Interaction Is Required for Termination of a CUT (A) Northern blot of total RNA hybridized with a T3 probe specific for GFP. The entire intergenic region was cloned upstream of a GFP readthrough reporter. (B) Northern blot with total RNA probed with a T3 probe specific for GFP. 3× TCTT element from Figure 4 is used under control of ADH1 promoter. Quantification of GFP corrected for SCR1 loading control is shown under SCR1 blot. (C) Northern blot with total RNA probed with a T7 probe specific for the TRS31 gene, which is downstream of snR13. SCR1 is used as a loading control. Temperature-sensitive strains were shifted to 37°C for 2 hr. Molecular Cell 2006 23, 841-851DOI: (10.1016/j.molcel.2006.07.024) Copyright © 2006 Elsevier Inc. Terms and Conditions