Npl3 Stimulates Elongation leads to more efficient termination [11]. In contrast, the npl3- S411A mutant had the opposite effect, showing increased hybridization at the 39 end, indicating the presence of readthrough transcription beyond the polyA site. A list of genes with a similar transcription readthrough defect for the npl3-S411A strain and additional sample plots are included in Table S2 and Figures S2, S3 and S4. For reference, the plot corresponding to the ratio of the rna15-2 vs. wild-type is included. The rna15-2 strain has a well-characterized termination defect that results in readthrough transcription [44–46]. At the FBA1 gene, changes were observed in the npl3-S411A and rna15-2 mutants at both ends of the gene denoting a terminating effect in the flanking MPE1 gene (Figure 5A). For two of the examples shown (FBA1 and MPE1), the ratio of rna15-2 strain was similar to that of the npl3- S411A (Figure 5A). The PDX1 gene lying upstream of TDH3, had a clear termination defect in the rna15-2 strain, but was unaffected by the S411A or npl3-120 mutations (Figure 5B). This suggests that the competition between Npl3 and Rna15 might be dependent on cis-elements; i.e., strong polyA signals may override the competition [12]. The npl3-S411A mutation resulted clearly in readthrough transcription of .800 genes (Table S2). A scatter- plot is provided for these genes using the mean value for the log2 ratio of npl3-S411A vs. wild-type for a 300 bp region within the ORF and at the 39UTR of each gene (Figure 5C). An increase in mean value of the probes was observed at the 39UTR. A scatter- plot in Figure 5D was also generated for the total number of genes in the Crick strand. The majority of these genes showed an increase mean value at the 39UTR relative to the region within the ORF for the npl3-S411A mutant (Figure 5D). Our results are consistent with the involvement of Npl3 in a widely distributed mechanism of transcription termination. Discussion Termination of transcription is a critical mechanistic step in regulating gene expression, particularly for genes with multiple polyadenylation sites. Here we report that the SR/hnRNP protein Npl3 directly enhances the elongation rate of RNAP II. Although Figure 5. Opposing effects in termination demonstrated using RNA-binding or phosphorylation defective npl3 alleles. RNA was extracted from NPL3, npl3-120 and npl3-S411A and whole transcript sense strand cDNA was synthesized and used for hybridization to tiled arrays. The hybridization signals for (A) FBA1, MPE1 and (B) TDH3 for two independent experiments were used to calculate the ratio of mutant vs. wild-type. The top panel shows the ratio of rna15-2/RNA15; bottom panel shows the ratio of each npl3 mutant/NPL3; a solid line for npl3-120/NPL3, or dashed line for npl3-S411A/NPL3. The corresponding position and orientation (Watson (W+), dashed bars, or Crick (C-) strand, black solid bars) of the genes is also shown. (C–D) A 300 bp region at the beginning of each ORF (+50 to +350 relative to the start codon), was selected for comparison to the same size region at the 39UTR (2100 to +200 relative to the stop codon) of the genes that showed increase readthrough for the npl3-S411A strain. The mean value of the log2 ratio of npl3-S411A/NPL3 was calculated and graphed in a scatter plot. (D) The total number of genes corresponding to the Crick strand were used to calculate the mean value of the same region described for (C). doi:10.1371/journal.pone.0003273.g005 PLoS ONE | www.plosone.org 7 September 2008 | Volume 3 | Issue 9 | e3273