1. Regulation of Yeast NRD1 Expression by Premature Transcription Termination 
John T. Arigo, Kristina L. Carroll, Jessica M. Ames, Jeffry L. Corden 
Molecular Cell 
Volume 21, Issue 5, Pages (March 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 NRD1 Autoregulatory Elements Are Present in the Coding Region
(A) Northern blot of NRD1 mRNA using a T7 NRD1 coding region probe reveals increased expression of nrd1-102 mRNA at the nonpermissive temperature of 37°C (2 hr) (lanes 1–4). Replacement of the NRD1 promoter and 5′UTR with the ADH1 promoter and 5′UTR (lanes 5–8) shows that an autoregulatory element in the coding region functions at the permissive temperature. Positions of mRNAs are indicated by arrows. The ADH1:NRD1 mRNA is shorter than the NRD1 mRNA because the ADH1 5′ UTR is shorter than the NRD1 5′ UTR. SCR1 is a RNA Pol III product that serves as a loading control.
(B) Northern Blot of ADH1 mRNA in the NRD1 and nrd1-102 ts mutant. Strains were shifted to 37°C for 2 hr. SCR1 is a RNA Pol III product that serves as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Nrd1p and Nab3p Binding Motifs Located throughout the 5′ UTR and the Coding Region Are Responsible for the NRD1 Autoregulation
(A) Diagram of the NRD1 gene showing locations of either TCTT or GTA[AG] sequence elements that have been disrupted with silent mutations. A Northern blot of NRD1 mRNA containing GUA[AG] silent mutations is shown above the map, while a Northern blot of NRD1 mRNA containing UCUU silent mutations is shown below. Lines indicate single mutations, while multiple mutations are indicated by the numbers above or below the blot (referring to the lane numbers of the individual mutations in the multiple mutant). Quantitation of mRNA is under each NRD1 Northern blot. Values are normalized for SCR1 loading control, and values are relative to wt NRD1, which is set to 1.0. RNA from nrd1-102 and nab3-11 ts mutants was isolated from cells grown at the nonpermissive temperature for 2 hr. A T7 RNA probe to the 3′ end of the NRD1 gene, downstream of the silent mutations, was used in Northern blot.
(B) Western Blot showing Nrd1 protein levels from wt NRD1 and nrd1-218 mutant. Yeast extract (25 μg) was loaded in each lane. All strains are haploid.
(C) Graphical representation of filter binding data from purified Nrd1-Nab3 protein binding to in vitro-transcribed NRD1 (closed circles) and nrd1-218 (open circles) RNAs. Fraction of bound RNA is plotted against the concentration of protein (μM).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Chromatin Immunoprecipitation Reveals a Decrease in RNA Polymerase II Density at the 3′ End of the NRD1 Gene
(A) Diagram of NRD1 gene showing the positions of PCR primer pairs used in ChIP.
(B) Acrylamide gel analysis of ChIP PCR products on wt and nrd1-218 strains. PCR products from immunoprecipitated samples are shown to the left, while control PCR products from total chromatin samples are shown to the right. Asterisk indicates a no-ORF primer pair derived from chromosome 5.
(C) RNA Pol II occupancy values quantitated as described in Experimental Procedures. Values are averaged from four experiments. Error bars represent standard deviation.
(D) Northern blot of NRD1 or nrd1-218 mRNA in the NRD1 or nrd1-218 genomic integration strains used for ChIP experiments. SCR1 is a RNA Pol III product that serves as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 Transcription Run-On Analysis of NRD1 and nrd1-218
(A) Diagram of the NRD1 gene showing positions of the phagemid probes.
(B) Representative transcription run-on experiment. The ACT1 probe is derived from the 3′ end of the ACT1 gene. Negative control is empty pCR2.1 phagemid vector.
(C) Quantitative analysis of run-on experiments. Triplicate values were averaged, and ratios of 5′ phagemid probes to 3′ phagemid probes are shown for each NRD1 allele. 5′ and 3′ phagemid probes were normalized to the ACT1 probe. Values represent the amount of polymerase present in the 5′ region of the gene as compared to the 3′ region of the gene for each strain. Error bars represent standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Overexpressing NRD1 Reveals a 1 kb Short Transcript
(A) Northern Blot of NRD1 and nrd1-218 mRNA transcribed from a CEN/ARS plasmid (pRS415) or a 2 μm plasmid (pRS425). Blot was probed with a T7 probe including the 5′ UTR and coding region of the nrd1-218 gene. Probe template was derived from the nrd1-218 gene. SCR1 is a RNA Pol III product that serves as a loading control.
(B) Northern Blot of NRD1 mRNA transcribed from a CEN/ARS plasmid (pRS415) or a 2 μm plasmid (pRS425). A T7 RNA probe to the 3′ end of the NRD1 gene, downstream of the silent mutations, was used in Northern blot. SCR1 is a RNA Pol III product that serves as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6
Components of the Nuclear Exosome Are Involved in NRD1 Autoregulation
(A) Northern blot of NRD1 mRNA in NRD1, trf4Δ, rrp47/lrp1Δ, and nrd1-102 at 37°C (2 hr) and a mtr4 strain. Blot was probed with a T7 probe including the 5′ UTR and coding region of the NRD1 gene to show the 1 kb short NRD1 transcript. 12 hr indicates the time that the wt and mtr4 strains were exposed to doxycycline. NRD1 is being expressed from its genomic locus.
(B) Northern blot of NRD1 mRNA in trf4Δ, rrp47/lrp1Δ, and rrp6Δ, and an mtr4 strain. Blot was probed with a T7 probe specific to the 3′ end of the NRD1 gene. SCR1 is a RNA Pol III product that serves as a loading control. 12 hr indicates the time that the wt and mtr4 strains were exposed to doxycycline. NRD1 is being expressed from its genomic locus.
(C) ChIP of RNA pol II in TRF4 and trf4Δ strains. Diagram of the NRD1 gene showing the positions of PCR primer pairs used in ChIP. RNA Pol II occupancy values quantitated as described in Experimental Procedures. Values are averaged from three experiments. Error bars represent standard deviation.
(D) Transcription run-on analysis of TRF4 and trf4Δ strains. Diagram of the NRD1 gene showing positions of the RNA probes. Representative transcription run-on experiment is shown to the left. The ACT1 probe is derived from the 3′ end of the ACT1 gene. Negative control is a T7 GFP transcript. Quantitative analysis of run-on experiments is shown on the right. Triplicate values were averaged, and ratios of hybridization to the 5′ RNA probe and to the 3′ RNA probe are shown for the NRD1 gene in either TRF4 or trf4Δ strains. 5′ and 3′ RNA signals were normalized to the ACT1 probe. Error bars represent standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions