Figure 1.
TAF6 is essential for human cell viability.
(A) PCR analysis of TAF6 mRNA levels 72 hours post-transfection with siRNAs targeting all TAF6 mRNAs. (B) Total cell extracts from HeLa cells transfected with the indicated siRNAs for 72 hours were separated by SDS-PAGE subject to Western blot analysis with monoclonal antibodies specific for TAF6α and tubulin as a loading control. (C) The viability of HeLa cells transfected with siRNA directed against TAF6 was measured 4 days post-transfection by methylene blue staining (see materials and methods). Viability (y-axis) is expressed relative to that of cells transfected with control siRNA. (D) TAF6 depletion results in loss of HeLa cell viability. HeLa cells were treated for 4 days with control siRNA (panel 1), or siRNA that target the TAF6 mRNA (panels 2 & 3). Cells were photographed with a phase-contrast microscope.
Figure 2.
Distinct impact of TAF6δ induction versus total TAF6 mRNA depletion on the transcriptome of HeLa cells.
(A) Heat map showing the impact of statistically significantly (p<0.05) changes in gene expression during TAF6 mRNA depletion by siRNA at 48 and 72 hours post transfection. Red indicates induction and blue repression. Genes were ordered according to fold change at 72 hours post transfection. (B) Distribution of expression profiles amongst the six possible outcomes. Genes upregulated or downregulated at the both time points are schematized with lines. (C) The previously defined TAF6δ transcriptome signature compared to the transcriptome resulting from depletion of total TAF6 mRNA. The heat map shows the gene expression during siRNA-mediated total TAF6 mRNA depletion for the 961 TAF6δ signature genes. (D) Venn diagrams depicting genes subsets statistically significantly regulated by total TAF6 mRNA depletion versus by TAF6δ induction. Upper diagram contains all regulated genes, middle diagram includes induces genes (upward arrow) and the lower Venn diagram includes repressed genes (downward arrow). (E) Gene ontology analysis of statistically significantly regulated genes during total TAF6 mRNA depletion. Enriched pathways are shown with their associated p-values.
Figure 3.
Development and validation of a minigene system to study the alternative splicing of TAF6.
(A) A schematic diagram showing the workflow used to study cis-acting RNA sequences in TAF6 alternative splicing using a TAF6 minigene plasmid. The plasmid containing an uninterrupted genomic sequence from the taf6 gene that includes portions of exons 2 and 3, as well as the natural intron 2 is depicted alone with the positions of primers used to detect exogenously expressed RNA species by RT-PCR. Minigenes were transfected into HeLa and 42 hours later total RNA was isolated for use in RT-PCR with primers from flanking plasmid sequences. PCR products were quantified by analysis using an Agilent Bioanalyzer. The percentage TAF6δ is expressed as a ratio of total spliced TAF6 mRNAs (δ + α). (B) Validation of the minigene system via mutagenesis. The proximal (P) 5′ splice site (SS) and distal (D) 5′ SS are illustrated. Mutations that knock-out (ko) SS or strengthen by creating consensus (cons) SS and their impact on the percentage of TAF6δ produced (x-axis), are indicated. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).
Figure 4.
Scanning mutagenesis of constitutive exon 2 and exon 3.
(A) Scanning mutations (black rectangles) in the TAF6 minigene were generated by PCR before transfection into HeLa. RNA was isolated and splice products were analysed by RT-PCR as in Figure 3. The percentage of exogenous TAF6δ mRNA produced by a given mutated construct are graphically shown (x-axis). (B) As in panel A, except that mutations were in exon 3. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).
Figure 5.
Targeted mutagenesis of intron 2 of the taf6 gene.
(A) Mutations of intron motifs are indicated with arrows in the TAF6 minigene constructs. HeLa cell transfection and splice product analysis was carried out as in Figure 3. The percentage of exogenous TAF6δ mRNA is graphically shown (x-axis). (B) As in panel A except mutations were focused on poly G motifs found within intron 2. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).
Figure 6.
RNA secondary structure at the proximal 5′ splice site can force the selection of TAF6δ.
(A) The sequence and names of mutations within alternative exon 2 (2a) of the TAF6 minigene constructs are indicated at the left. A potential SF2/ASF binding site is indicated by a blue box. HeLa cell transfection and splice product analysis was carried out as in Figure 3. The percentage of exogenous TAF6δ mRNA is graphically shown (x-axis). (B) As in panel A except that mutations (red nucleotides) are shown to the right in hypothetical RNA secondary structures generated using the M-Fold algorithm. The proximal 5′ splice site (SS) is indicated as green boxes. (C) As in B with further mutations. (D) As in B with further mutations. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).
Figure 7.
Evidence for an exonic splicing silencer in alternative exon 2.
(A) A hypothetical RNA structure generated by M-fold is illustrated along with the position of the proximal TAF6 5′ splice site (green box). Selected mutation are indicated with arrows (red text) (B) The names of mutations within alternative exon 2 (2a) of the TAF6 minigene constructs are indicated at the right. HeLa cell transfection and splice product analysis was carried out as in Figure 3. The percentage of exogenous TAF6δ mRNA is graphically shown (x-axis). Mutations (red nucleotides) are shown to the right in hypothetical RNA secondary structures generated using the M-Fold algorithm. (C) As in panel A except that mutations (red nucleotides) are shown to the left. (P<0.05 = *; P<0.01 = **; P<0.001 = ***).
Figure 8.
A hypothetical model for TAF6δ alternative splicing.
(A) The TAF6 minigene construct is schematically shown with putative exonic splicing enhancer (ESE), exonic splicing silencer (ESS) and intronic splicing enhancer (ISE) motifs indicated with boxes. Enhancer or silencer definitions are given with respect to the pro-apoptotic TAF6δ isoform but likely act simultaneously to repress one 5′ SS while enhancing the other because of the small distance (30 nucleotides) between them. (B) As in panel A, except that mutations were focused on poly G motifs found within intron 2.