10 research outputs found
U1C-dependent activation of an alternative 3′ splice site within U1-70K introduces a PTC: RNA-Seq analysis.
<p>(<b>A</b>) Read-density maps derived from RNA-Seq analysis of control- (blue) and U1C-siRNA-treated (ΔU1C, red) HeLa cells are shown above the exon-intron structure of the human U1-70K gene (exons 1–10 indicated as black boxes, with the narrower parts representing the untranslated regions at the 5′ and 3′ ends). Below, the conservation in vertebrates is given in green. The dashed region is shown in more detail below in panel B. (<b>B</b>) Read densities of the control- (blue) and U1C-knockdown HeLa cells (ΔU1C, red) for the U1C-dependent alternatively spliced and highly conserved exons 7–8 region. (<b>C</b>) Quantitation of the use of the alternative 3′ splice site in intron 7, as determined by specific junction-read numbers given above and below each exon-intron structure (in blue for control-, in red for U1C-knockdown). The green box indicates the potential alternative exon 7a generated by use of the alternative 3′ splice site at position +642 in intron 7, which introduces a premature termination codon (stop sign), and one of downstream cryptic 5′ splice sites (labeled A, B, C, and D).</p
U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation.
<p>(<b>A</b>) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. (<b>B</b>) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. (<b>C</b>) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. (<b>D</b>) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. <i>M</i>, DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for <i>SNHG5</i> is marked by open circles between the lanes. Lower panel: For <i>MARCH7</i> top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For <i>UFM1</i> three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.</p
Alternative 3′ splice site activation requires U1 snRNP binding to downstream cryptic 5′ splice sites.
<p>(<b>A</b>) U1-70K minigene constructs used for <i>in vivo</i> splicing analysis (see panel B), including exons 7, 7a, and 8; the arrows indicate the primers used for RT-PCR analysis. The enlargement below represents the region covered by the biotinylated transcripts used for <i>in vitro</i> binding studies (see panel C). The exact positions of the stop codon (dashed vertical line) and the three cryptic 5′ splice sites (bold vertical lines labeled with A, B, and C) are shown together with the 5′ splice site sequences, including all point mutations analyzed. The two solid lines above the enlarged exon mark the positions of the splice site blocking antisense morpholinos (see panel D); their labeling (AB and BC AMO) refers to the 5′ splice site they block. (<b>B</b>) Mutational analysis of U1-70K alternative splicing. Splicing patterns of U1-70K minigenes (as indicated) in control- (ctr) and U1C-knockdown (ΔC) HeLa cells were analyzed by RT-PCR, detecting alternative 3′ splice site activation (primers 7-7a; top panel), exon 7a inclusion and skipping (primers 7–8; middle panel), and as a loading control, exon 7 alone (bottom panel); splicing products are depicted on the right. Percentages of exon 7a inclusion are given below with standard deviations calculated from three individual experiments [n = 3]; the labels within the second panel indicate, which cryptic 5′ splice sites were used in each case for 7a inclusion, with letters in parentheses marking the less frequently used splice sites (see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3B</a></b>). (<b>C</b>) U1 snRNP binds to cryptic 5′ splice sites of U1-70K exon 7a. 3′-biotinylated RNAs spanning “exon 7a” including flanking intronic sequences (as shown enlarged in the middle of the schematic in panel A) were incubated with HeLa nuclear extracts (2% input). Bound proteins (2/3 of selected material) were analyzed by Western blotting, using antibodies against U1-70K, U1A, and U1C; bound U1 snRNA was detected by Northern blot hybridization. (<b>D</b>) Antisense morpholino (AMO) transfection in HeLa cells. In two separate assays HeLa cells were transfected with AMOs either against the cryptic 5′ splice sites of U1-70K exon 7a (AB, BC; left panel) or the 5′ end of U1 snRNA (U1; right panel), or with an unspecific control morpholino (ctr). Cells were either left untreated (−CHX) or were treated with cycloheximide (+CHX) before RT-PCR analysis. The label within the middle panel indicates which cryptic 5′ splice sites are used for exon7a inclusion, with letters in parantheses marking the less frequently used splice sites. M, DNA size markers (in bp).</p
U1-70K mRNA and protein levels are upregulated in human and zebrafish upon loss of U1C.
<p>(<b>A</b>) Exon-intron structure of U1-70K exons 7–8. The predominant splicing patterns in control and in U1C-knockdown HeLa cells are depicted by dashed lines above and below the schematic, respectively. The grey shading downstream of “exon 7a” indicates that the 7a-8 junction is not detectable in control cells without cycloheximide treatment. Arrows represent the RT-PCR primers used in (B) and (D) to detect alternative 3′ splice site activation (primers 7-7a), exon 7a inclusion, and exons 7–8 splicing. (<b>B</b>) U1-70K alternative splicing after U1C knockdown. HeLa cells were treated with two different siRNAs against U1C (ΔC or ΔC*, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#s4" target="_blank">Materials and Methods</a>), or a control siRNA (ctr), comparing untreated cells (−CHX) or cells after treatment with cycloheximide (+CHX). Alternative splicing patterns were analyzed by RT-PCR. The label within the middle panel indicates which cryptic 5′ splice sites are used for exon7a inclusion, with letters in parentheses marking the less frequently used splice sites (see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3A</a></b>). β-actin serves as an internal loading control. (<b>C</b>) U1-70K protein levels after U1C knockdown in HeLa cells. Whole cell lysates or nuclear extracts were analyzed by SDS-PAGE and Western blot, detecting U1-70K, γ-tubulin (as a loading control), and U1C. The fold change of U1-70K protein expression after U1C knockdown is given below (ΔU1C/ctr). (<b>D</b>) Add-back of U1C restores normal U1-70K splicing in HeLa and zebrafish (<i>D. rerio</i>). Left panel: Control- (ctr) and U1C-knockdown (ΔC) HeLa cells are compared with U1C-knockdown cells expressing Flag/HA-tagged U1C. Knockdown and over-expression were verified by Western blot analysis. Right panel: A wildtype zebrafish embryo (wt) is compared with two U1C-knockout mutants, without (mut) and with ZfU1C-cRNA injection (rsc = rescue). In both assays, alternative splicing patterns were analyzed by RT-PCR, and β-actin serves as an internal loading control. The rescue effect was confirmed by Western blot analysis of single-embryo lysates. M, DNA size markers (in bp).</p
U1-70K knockdown results in co-depletion of U1C protein and in U1-70K/U1C double-deficient U1 snRNPs.
<p>(<b>A–B</b>) 96 hours after siRNA transfection, whole-cell lysates were prepared from control- (ctr) and U1-70K-knockdown- (Δ70K) cells. (<b>A</b>) U1-70K knockdown in HeLa cells results in co-depletion of U1C. Whole-cell lysates were analyzed by SDS-PAGE and Western blot, detecting U1-70K, γ-tubulin, and U1C. (<b>B</b>) U1-70K/U1C double-deficient U1 snRNPs in U1-70K-knockdown HeLa cells. U1 snRNPs were affinity-purified from whole-cell lysates (5% of input, lanes 1 and 3; 2/3 of purified material, lanes 2 and 4) and analyzed by SDS-PAGE and Western blot, detecting U1-70K, U1A, and U1C. U1 snRNA was detected by Northern blotting, analyzing 1/3 of the purified material. Asterisks mark unspecific bands detected by the anti-U1-70K antibody. (<b>C</b>) Exon 7a skipping upon U1-70K knockdown. 72 hours after siRNA transfection, the U1-70K wildtype minigene construct (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen-1003856-g004" target="_blank"><b>Figure 4A</b></a>) was transfected into HeLa cells, and 24 hours later minigene splicing patterns were analyzed by RT-PCR, comparing control- (ctr) and U1-70K- (Δ70K) knockdown samples. Specific primer sets were used to detect alternative 3′ splice site activation (product 7-7a), exon 7a inclusion (product 7-7a-8; letters indicate which cryptic 5′ splice sites are used, with parentheses marking the less frequently used splice site; see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3A</a></b>), exon 7a skipping (product 7–8), and as a loading control, exon 7 alone (product 7). The identities of the splicing products are depicted on the right. (<b>D</b>) U1C mRNA is stable after U1-70K knockdown. After four days of U1-70K knockdown in HeLa cells, total RNA was isolated from control- (ctr) and U1-70K- (Δ70K) knockdown cells. Endogenous mRNA levels of U1-70K, U1C, and β-actin (as indicated on the right) were analyzed by RT-PCR. The numbers given in parentheses refer to the amplified exons. M, DNA size markers (in bp).</p
Model of intra-U1 snRNP U1-70K/U1C cross-regulation.
<p>Binding of intact U1 snRNPs to three cryptic 5′ splice sites (labeled with A, B, C) within intron 7 of the U1-70K pre-mRNA (middle) activates an alternative 3′ splice site. Inclusion of the alternative exon 7a introduces a premature termination codon (stop sign) into the mature U1-70K mRNA, which is degraded by nonsense-mediated decay (NMD; following the pathway upwards). Reduced U1-70K mRNA and protein levels result in a co-depletion of U1C protein; thus, U1 snRNPs are assembled inefficiently. U1C/U1-70K-deficient U1 snRNPs are unable to activate the alternative 3′ splice site, therefore, constitutive U1-70K splicing is enhanced, and more functional U1-70K mRNA and protein are produced (following the pathway downwards). Normal U1 snRNP assembly is restored and alternative 3′ splice site activation can occur again to close the regulatory circle (for a detailed description, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#s3" target="_blank"><i>Discussion</i></a>).</p
MOESM2 of Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure
Additional file 2: Table S1. Transcription level of the genes from the studied region (CPM). Only genes with CPM > 1 in at least 2 replicates are presented. CPM values were averaged over replicates. Log2-fold change and FDR of difference between experiments are presented (see “Methods”). For genes that have not passed filtering procedure, only CPM are present
MOESM4 of Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure
Additional file 4: Table S2. Sequences and characteristics of the 5C primers used in this study. 5C primers were designed using the alternating scheme in my5C.primers and chicken reference genome assembly galGal4
Additional file 1: Figure S1. of Between Lake Baikal and the Baltic Sea: genomic history of the gateway to Europe
Quality control process. (PDF 25Â kb
Additional file 3: Table S1a. of Between Lake Baikal and the Baltic Sea: genomic history of the gateway to Europe
List of samples included in “Extended” dataset. Table S1b List of samples included in “Core” dataset. Table S1c List of samples included in “Ancient” dataset. Table S2 Results of ADMIXTURE for K = 9. Table S3 Results of ADMIXTURE for K = 6, 7, 8. Table S4 Results of f3 test. Table S5 Results of IBD sharing analysis in 1–3 cM and 4–10 cM bins. Table S6 Total amount of shared IBD between populations. Table S7 Standard residue of linear regression analysis of distance-IBD sharing. Table S8 Distance and shared IBD between pairs of populations. Table S9: Results of f3 outgroup test with ancient samples. (XLSX 482 kb