Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression

Transcriptional readthrough at the <i>VHR2-THO1</i> locus.

<p><b>A:</b> Expression (II-III) and polymerase occupancy (I, IV) at the <i>VHR2-THO1</i> locus on chromosome V, in WT and <i>npl3</i>Δ mutant yeast. Expression was determined using strand-specific tiling arrays, and two biological replicates are shown for both yeast strains (tracks labeled 1 and 2). Expression from the Watson strand is shown above the genomic co-ordinate information, and expression from the Crick strand is shown below. Polymerase (Rpo21) occupancy on each strand is shown in blue (WT) or red (<i>npl3</i>Δ). <b>B:</b> Confirmation of transcriptional readthrough of <i>VHR2</i> (VHR2 RT) and down-regulation of <i>THO1</i> in <i>npl3</i>Δ using strand- specific reverse transcription followed by qPCR. Approximate locations of primers used for reverse-transcription are shown in 3(A) (green arrows). <i>VHR2</i> readthrough is measured by performing a reverse transcription reaction using an oligo that primes from ~500 nt downstream from the <i>VHR2</i> 3' end. qPCR primers are located towards the 3’ end of <i>THO1</i> and <i>VHR2</i>, respectively. The histogram shows changes in the <i>npl3Δ</i> mutant, compared to the WT levels (which were set to 1). <b>C:</b> Comparison of tiling array expression data and polymerase occupancy at regions across the <i>VHR2</i>-<i>THO1</i> locus, in WT and <i>npl3</i>Δ. Numbers represent change in the <i>npl3</i>Δ mutant relative to WT. <b>D</b>: Induction of <i>CYC1</i> in glycerol. Induction of <i>CYC1</i> expression in WT and <i>npl3</i>Δ yeast transferred from glucose to glycerol medium, determined by reverse-transcription followed by qPCR. <i>THO1</i> expression was measured in both conditions, as a negative control.</p

Distribution of Npl3 across RNAs is suggestive of a role in termination and surveillance.

<p><b>A:</b> Comparison of Npl3 binding across different RNA classes in two replicate CRAC experiments. Hits were assigned to RNA classes according to gene annotations from Ensembl (EF4.74) supplemented with additional features including ncRNAs, UTRs and antisense transcripts, as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005735#pgen.1005735.ref037" target="_blank">37</a>]. Antisense, intergenic and ncRNA RNA classes are defined as follows. Antisense: a hit will be assigned as 'antisense' if it maps to regions of published antisense transcription [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005735#pgen.1005735.ref044" target="_blank">44</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005735#pgen.1005735.ref045" target="_blank">45</a>] or to the opposite strand of a known feature. Intergenic: these are hits that map to any region of the genome where no other feature has been annotated. NcRNA: this category comprises 15 ncRNAs that don't fall into other categories, including scR1, SRG1 and TLC1. <b>B:</b> Distribution of Npl3 binding over mRNAs. Average distribution of Npl3 around the 5’ and 3’ ends of mRNAs. Transcripts are aligned at the transcription start sites (TSS) and polyA (pA) site. Grey dots depict precise number of hits at particular nucleotide positions and the dark green shows a line of best fit. Hits are normalized to a total of 1 across all mRNAs. <b>C:</b> Npl3 association with oligoadenylated mRNA fragments. As for panel A, but with data filtered for the presence of non-templated oligo(A) sequences (A2 or greater) recovered on target RNAs. <b>D</b>: Distribution of Npl3 binding over mRNAs. As for 1(B), but including reads containing oligoadenylated sequences only. <b>E</b>: Metagene analysis of Npl3 binding across CUTs. Distribution of Npl3 at all CUTs aligned by the TSS and 3' ends, with 100 nt flanks extending 100 nt into the 5' and 3' ends of transcripts. Only CUTs >150 nt in length were included in the analysis. <b>F</b>: Metagene analysis of Npl3 binding across snoRNAs. Distribution of Npl3 at all snoRNAs aligned by the TSS and 3' ends, with 100 nt flanks extending 50 nt into the 5' and 3' ends of transcripts.</p

Features of readthrough genes.

<p><b>A:</b> Scatterplot comparing readthrough and expression, including only those genes that passed the filters (see bioinformatics methods in supplementary information for description of filters). Points shown in red (709/2234) represent those genes showing significant readthrough in both experiments (FDR = 0.05). <b>B:</b> Polymerase occupancy around the 3' ends of 200 genes with the highest readthrough rank, and 1200 genes with the lowest rank. <b>C:</b> Polymerase occupancy around the 3' ends of genes, as in 7(B), measured in the <i>npl3</i>Δ background. <b>D:</b> Enrichment scores of 6-mer motifs in the 3’ region of low readthrough genes, relative to high readthrough genes. The 3’ region was defined as -80 to -20 nt from the polyadenylation site [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005735#pgen.1005735.ref037" target="_blank">37</a>]. Low and high readthrough genes were defined as the bottom and top quartiles of mRNAs in the dataset. UAUAUA and UAAAUA are the most enriched 6-mer motif in low-readthrough genes. <b>E:</b> Localization of UAUAUA motifs around the polyadenylation sites of low readthrough genes (red) and high readthrough genes (black), aligned by the polyA site.</p

Altered RNA levels in strains lacking Npl3.

<p>Tiling microarray data were compared to assess relative RNA levels in the WT and <i>npl3Δ</i> strains for selected RNA classes; protein coding genes (A-B), CUT lncRNAs (C-D) and snoRNAs (E-F). The scatter plots (Fig 2A, 2C and 2E) display the total signal for each gene in the class for which the microarray signal was sufficiently high for quantification. Intensities are plotted on a log2 scale and the red line indicates equal intensity in the WT and <i>npl3</i>Δ mutant strains. The 3 most under-represented genes in the mutant strain–<i>THO1</i>, <i>PTC7</i> and <i>NPL3</i> –are highlighted in red. The volcano plots (Fig 2B, 2D and 2F) display differentially expressed RNAs in <i>npl3</i>Δ, with fold change plotted against p-value. The y-axis is the negative log10 p-value, adjusted to false discovery rate (FDR). The x-axis is log2 fold-change. The points in red are those that with significantly differential expression in the mutant (adj p-value <0.05). These include genes with both reduced expression (negative log2 fold change) and increased expression (positive log2 fold change).</p

Transcription termination defects at snoRNAs in <i>npl3</i>Δ.

<p><b>A</b>: Metagene analysis of Rpo21 binding across snoRNAs. Distribution of Rpo21 at all snoRNAs in WT (blue) and <i>npl3</i>Δ (red) yeast, aligned by the TSS and 3' ends, with 100 nt flanks extending 50 nt into the 5' and 3' ends of transcripts. <b>B:</b> Expression and polymerase occupancy at SNR60. Data displayed as in 4(A). <b>C:</b> Northern blot to detect extended SNR60 in <i>npl3</i>Δ (right panel). The left panel displays total RNA. Two biological replicates are shown for each strain, WT and <i>npl3</i>Δ. <b>D</b>: Polymerase occupancy within and flanking selected snoRNAs.</p

Transcription termination defects at CUTs in <i>npl3</i>Δ.

<p><b>A:</b> Expression and polymerase occupancy at the CUT680/URA2 locus, in WT and <i>npl3</i>Δ mutant yeast. Expression is determined using strand-specific tiling arrays, and two biological replicates are shown for each yeast strain (heat map tracks labeled 1 and 2). Polymerase occupancy was determined by CRAC, and is shown for WT (blue) and <i>npl3</i>Δ (red) yeast. Note there is an intron at the 5' end of <i>URA2</i>. <b>B:</b> Expression and polymerase occupancy at the <i>CUT324/CUT325/ADE12</i> locus. Data displayed as in panel A. <b>C:</b> Comparison of tiling array expression data and polymerase occupancy at regions across the <i>URA2</i> and <i>ADE12</i> loci, in WT and <i>npl3</i>Δ. Numbers represent change in the <i>npl3</i>Δ mutant relative to WT. <b>D</b>: Metagene analysis of Rpo21 binding across CUTs. Distribution of Rpo21 at all CUTs in WT (blue) and <i>npl3</i>Δ (red) yeast, aligned by the TSS and 3' ends, with 100 nt flanks extending 100 nt into the 5' and 3' ends of transcripts. Only CUTs >150 nt were included in the analysis.</p