Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism
Figure 2
Trf4p promotes polyadenylation-independent degradation of introns.
(A) Microarray data for 18 introns that changed more than 2-fold (FDRs<5%) in trf4Δ or trf5Δ mutants. Overexpression of Trf4p-DADA in trf4Δ mutant cells reduced steady-state levels of most of these introns to WT levels. Microarrays are the same as shown in Figure 1. Introns containing snoRNAs (snR) are indicated. (B) Relative changes of the expression of RPL7B (left panel) and RPS9A (right panel) introns, pre-mRNAs, and mature mRNAs as revealed by exon (ex), intron (int), and intron-exon junction probes (pre) present on the microarrays. Int-1 and int-2 refer to the first and the second intron of the RPL7B gene; pre-1 and pre-2 refer to the respective intron-exon junction probes of the RPL7B pre-mRNA. The height of the bar represents average log2 ratios from triplicate microarray data (Dataset S1); error bars show the standard deviation. (C) Relative changes of expression of RPL16A (left panel) and RPL40A (right panel) introns (int), pre-mRNAs (pre) and mature mRNAs (ex). Data was extracted from triplicate microarray data as described above. (D) Depletion of Trf4p or Trf5p promotes intron stability in vivo. Total RNA was purified from trf4Δ trf5Δ mutant strains either expressing TRF4 or TRF5, which were transcribed under the pGAL1 promoter. Cells were initially grown in a galactose containing medium (TRF4 Gal; TRF5 Gal) and then shifted to a glucose containing medium (YPD) at 30°C (TRF4 Glc; TRF5 Glc) for 1 h. Accumulation of introns of RPS9A and RPL40A and of the first intron of RPL7B was determined by qRT-PCR with intron-specific primers. The steady-state levels of introns in each sample was calculated as log2 of normalized ratios relative to the t0 time point, which corresponds to the cultures immediately before the galactose to glucose shift (for details see Materials and Methods). The values represent averages from two independent qRT–PCR analyses.