Figure 7

Nab3 is required to suppress cryptic transcriptional activities. (A) UCSC genome browser images of the region showing HHT1 and IPP1. ‘Selected intervals’ indicate genomic regions with a read coverage FDR =0.01 used for pyMotif analyses. See the legend to Figure 5 for additional details. Chromosomal positions of RT-PCR products and northern blot probes are also indicated. (B) Western blot displaying levels of 3HA-tagged Nrd1 and Nab3 proteins before and after the shift to glucose. Experimental details are provided in the Materials and methods. Proteins were detected using horse radish-conjugated anti-HA antibodies (Santa Cruz). (C) Schematic representation of transcripts generated in the SNR13-TRS31 region of yeast chromosome IV (adapted from [13]). About 1 to 4% of the SNR13 transcripts were read-through transcripts in Nab3 and Nrd1 depleted cells, respectively. (D) Northern blot analysis of IPP1, HHT1, snR13 and U2 snRNA and 3' extended species. Shown are phosphoimager scans of a blot probed with various oligonucleotides (indicated on the left of each panel). U2 snRNA levels were used as a loading control. (E) Depletion of Nrd1 and/or Nab3 results in a reduction of HHT1 and IPP1 mRNA levels. The mRNA levels were quantified using the AIDA software package and normalized to both the levels in the parental strain and the U2 snRNA. (F, G). Quantitative RT-PCR analysis of HHT1 and IPP1 transcription in coding sequences (exon) and downstream regions. Fold change in transcription downstream of these genes was calculated by normalizing the data of the downstream regions to the signals obtained for the exon region. Error bars indicate standard deviations (H) Detection of IPP1 read-through transcripts by end-point RT-PCR. The diagram indicates the regions amplified. The position of 3' extended products and exon fragments in the gel are indicated on the right of the gel image.