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Additional file 20: Fig. S11. Temperature dependence of transcript levels of candidate regulators of pst_E1n enhancer activity and dependence on Pvf/Pvr. (A-C) Transcript levels at the indicated temperatures in S2R+ cells as detected by 3′ RNA-Seq. Bar diagrams display log2 of the average of the read counts (after normalization) and s.d. (n = 3 replicate experiments). (A) Transcript levels of TFs with predicted binding sites within pst_E1n and detectable expression in S2R+ cells. (B) Transcript levels of JAK/STAT signal transduction proteins. Expression of upd1 is marginal at most in S2R+ cells and hence not included. (C) Transcript levels of the genes encoding the receptor tyrosine kinase Pvr and its known ligands Pvf2 and Pvf3. Expression of Pvf1 is marginal at most in S2R+ cells and hence not included. (D) Dependence of pst_E1n enhancer activity on Pvfs and Pvr. The indicated dsRNAs were used for depletion in SR9rg > pst_E1n (mRuby−, GFP+) cells, which were then shifted in aliquots to the indicated temperatures and analyzed eventually by flow cytometry. Bars display median GFP signal intensity. For depletion of Pvr and Pvfs, two dsRNA preparations (1 or 2) generated from distinct amplicons, were used in multiple experiments (indicated by a, b or c) in some cases.

Additional file 14: Fig. S7. Evaluation of the role of histone H2Av (His2Av) in transcriptional control of temperature-regulated genes in S2R+ cells. (A) To address the role of His2Av in temperature-dependent regulation of gene expression in S2R+ cells, we added His2Av dsRNA or lacZ dsRNA for control to culture aliquots followed by a shift to the indicated temperatures after four days. Twenty-four hours after the shift, we isolated total RNA for analysis with microarrays. Three replicate experiments were performed. (B) Analysis of transcript levels of His2Av and Act5C (for control) confirmed successful depletion of His2Av transcripts. Bar diagrams display transcript levels (average of three biological replicates and s.d.) at the analyzed temperatures. Average expression at the three temperatures observed after treatment with lacZ dsRNA was set to 100%. (C) Analysis of His2Av depletion by immunoblotting. S2R + _MtnAp-His2Av-mRFP cells (Lidsky et al. 2013) were treated with dsRNA derived from lacZ (for control) and two distinct His2Av amplicons (H2Av_1 and H2Av_2) for three and 5 days as indicated. Cell extracts were analyzed by immunoblotting with anti-mCherry (detecting His2Av-mRFP) and anti-PSTAIR (loading control). The combined treatment with both His2Av dsRNA preparations (H2Av_1 + 2) for 5 days was found to reduce His2Av-mRFP levels to 40% of controls according to quantification of signal intensities and normalization based on anti-PSTAIR signals. (D) Effects of His2Av depletion on the S2R+ cell transcriptome. A volcano plot is displayed with probes associated with signal intensities that were significantly (FDR

Additional file 4: Fig. S2. Increased use of distal polyadenylation sites at low temperature. (A-C) Microarray probes with pronounced CoolUp signals were observed to detect specifically the annotated transcripts with the longest 3′ untranslated region, as illustrated in case of the gene (A) RpLP1, (B) Kap-α3 and (C) ewg. The positions recognized by different probes are indicated (vertical bars of light grey shading) in a scheme with the transcribed region and the annotated transcripts. The bar diagrams below display signal intensities (mean of three biological replicates) observed with these probes after incubation of S2R+ cells at different temperatures (11, 14, 25 and 30 °C).

Additional file 9: Fig. S3. Temperature-regulated alternative polyadenylation. (A-D) 3′ RNA-Seq data obtained from S2R+ cells (A, C) and adult male flies (B, D) after 24 h of incubation at different temperatures (11, 14, 25 and 30 °C) was used for an analysis of temperature effects on the choice of polyadenylation sites (PASs). (A,B) Histograms illustrate the frequency of alternative polyadenylation (APA) in S2R+ cells (A) and adult male flies (B), as detected after pooling all data obtained at the different temperatures from the three biological replicates. In total, 14,669 PASs were detected in S2R+ cells, and 22,378 in adult male flies. These were assigned (see Materials and Methods) to a total of 7495 and 10,757 expressed genes in S2R+ cells and adult male flies, respectively. (C,D) Temperature effects on APA. For genes with multiple PASs, we compared the two PASs with highest presence (read counts) across all conditions and required the log2 fold change to be in opposite directions when comparing the read counts at the high (25 and 30 °C) with those at the low (11 and 14 °C) temperatures. The scatter plots display the fold changes at the proximal and distal of these two most strongly affected PASs in S2R+ cells (C) and adult flies (D). Blue dots represent genes (class I), where preference of the distal over the proximal PAS at low temperature is significant in comparison to the high temperature. Red dots represent genes (class II), where temperature change has a significant effect in opposite direction.

Additional file 3: Fig. S1. Concordance of transcript levels after quantification with different methods. (A-C) Temperature effects on transcript levels in S2R+ cells were analyzed using either qRT-PCR (A), microarrays (B) or 3′ RNA-Seq (C). The bar diagram displays relative expression levels at the different temperatures, as detected by the different methods. Expression at 25 °C was set to 1. For each analysis, culture aliquots were shifted for 24 h to the indicated temperatures (11, 25 and 30 °C) before RNA isolation. Representative CoolUp genes (CG13694, CG6321, CG32944, Orct2, Lip4) and CoolDown genes (Hsp23, Hsp22, DNApol-α50), as well as genes (Atg1, Pten), which were barely temperature regulated, were selected for validation by qRT-PCR. In case of qRT-PCR, mean and s.d. of three technical replicates are displayed. In case of the microarray data, mean and s.d. of three biological replicates and multiple probes, if present, are shown. In case of the 3′ RNA-Seq data, mean and s.d. of three biological replicates are presented.

Our group has been studying the hop-Verticillium pathosystem in order to decipher hop resistance towards Verticillium albo-atrum, the main causative agent of the devastating hop wilt spread through European hop gardens, and to search for virulence-associated factors that might explain the increased aggressiveness of the V.albo-atrum lethal pathotype. Various research approaches have been employed in these studies, from genetic mapping of resistance gene(s) and QTLs, gene expression studies of compatible and incompatible interactions (proteomics and transcriptomics approach) to whole genome sequencing of six different V.albo-atrum pathotypes. A short overview of our work will be given in this presentation, with an emphasis on the gene(s) conferring resistance to hop wilt and on studies of V.albo-atrum virulence factors.

RNA-binding proteins (RBPs) regulate splicing according to position-dependent principles, which can be exploited for analysis of regulatory motifs. Here we present RNAmotifs, a method that evaluates the sequence around differentially regulated alternative exons to identify clusters of short and degenerate sequences, referred to as multivalent RNA motifs. We show that diverse RBPs share basic positional principles, but differ in their propensity to enhance or repress exon inclusion. We assess exons differentially spliced between brain and heart, identifying known and new regulatory motifs, and predict the expression pattern of RBPs that bind these motifs. RNAmotifs is available at https://bitbucket.org/rogrro/rna_motifs.

Summary Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3′ mRNA sequencing. This reveals at nucleotide resolution the “RNA maps” describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles., Graphical Abstract, Highlights • TDP-43 regulates competing poly(A) sites in a highly position-dependent manner • expressRNA is a new platform for analysis of alternative polyadenylation and splicing • RNAmotifs2 is a cluster motif analysis platform integrated with expressRNA • Regulation of pre-mRNA processing might follow common position-dependent principles, Rot et al. investigate how TDP-43 regulates alternative polyadenylation in HEK293 cells. This defined position-dependent regulatory principles with high-resolution RNA maps. The authors provide an integrative computational platform for comprehensive analysis of alternative splicing and alternative polyadenylation (expressRNA), as well as software for positional analysis of clustered sequence motifs (RNAmotifs2).