第16回千葉カルシウム代謝研究会

Gene ontology term enrichments for RNA-Seq data from differentiated TSC2 deletion cell lines and microarray data of patient SEGAs (related to Fig. 2f). (XLSX 27.7 kb

Comparison of alternative mRNA isoforms across 25 human tissues

<p><b>Copyright information:</b></p><p>Taken from "Variation in alternative splicing across human tissues"</p><p>Genome Biology 2004;5(10):R74-R74.</p><p>Published online 13 Sep 2004</p><p>PMCID:PMC545594.</p><p>Copyright © 2004 Yeo et al.; licensee BioMed Central Ltd.</p> Color-coded representation of values between pairs of tissues (see Figure 4 and Materials and methods for definition of SJD). Hierarchical clustering of SJD values using average-linkage clustering. Groups of tissues in clusters with short branch lengths (for example, thyroid/ovary, B-cell/bone) have highly similar patterns of AS. Mean SJD values (versus other 24 tissues) for each tissue

RT-PCR Confirmation of Exon Skipping Patterns in Human Tissues

<p>Eleven orthologous exons (≤250 nucleotides in length) were selected from the analysis of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030158#pbio-0030158-g005" target="_blank">Figure 5</a> for RT-PCR analysis in a panel of eight human tissues. These exons are derived from the intersection dataset, in which conserved TAGG and GGGG motifs are present in combination in the human and mouse orthologous exons. Additional cDNA and EST evidence for these skipping events are summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030158#pbio-0030158-t101" target="_blank">Table 1</a>. Specific primer pairs were designed for each test exon to amplify the exon-included (double arrowhead) and exon-skipped (single arrowhead) products by RT-PCR. Each gel panel shows the products of reactions for a single test exon resolved on agarose gels in the arrangement given in the inset. Gene name, exon number, and Ensembl number (in parentheses) are provided above each gel panel. The far left and far right lanes of each gel panel contain DNA molecular weight markers. </p

Computational Analysis of UAGG and GGGG Motif Patterns Reveals Association with Exon Skipping Genome-Wide

<p>At the top is a flow chart for the computational analysis used to illustrate the procedure used to identify human exons with and without the CI cassette silencer motif pattern (≥1 exonic UAGG and a 5′-splice-site-proximal GGGG), followed by the determination of the percentage of confirmed skipped exons in each group. The reciprocal pattern (≥1 exonic GGGG and a 5′ splice site UAGG) and related variants were analyzed for comparison as indicated in the graph and table. The graph (middle) shows exons with the motif pattern on the left and the remaining exons without the pattern (w/o) on the right; x-axis, 5′ splice site motif; y-axis, percent confirmed skipped exons; z-axis, exonic motif. Confirmed skipped exons were defined as those skipping events supported by 20 or more individual cDNA and/or EST entries. Exonic motifs were allowed at any position within the exon, but not overlapping the splice sites, and the 5′ splice site motif was restricted to bases 3–10 of the intron. Only exons of 250 nucleotides or fewer were considered. The table (bottom) shows, for each motif pattern, the percentage of confirmed skipped exons within that group (as shown in the graph) and the number of exons in the group (in parentheses). The CI cassette silencer motif pattern is boxed. </p

Genome-Wide Identification of Exons with UAGG and GGGG Silencing Motifs

<p>A database of 96,089 orthologous human and mouse exon pairs was searched for TAGG located anywhere in the exon and GGGG in bases 3–10 of the intron. Venn diagrams indicate the number of exons containing either or both sequence motifs in the human subset and the mouse subset of the database. The number of exons (19) in which UAGG and GGGG silencer motifs are conserved in orthologous human and mouse exons is also shown (intersection). The motif patterns are shown in the context of the exon (uppercase) and 5′ splice site region (lowercase) for 12 examples from the intersection dataset (human sequences are shown). Colon indicates 5′ splice site. The conserved TAGG and GGGG motifs are highlighted in red to illustrate variations in their positions. Gene name (HUGO ID) and exon number within the gene are indicated at far right. For one uncharacterized transcript, the GenBank accession is given instead (NM_018469_8). </p

Model for Differential Regulation of the CI Cassette Exon by the Interplay of hnRNP A1 and H and a Ternary Motif Pattern

<p>At the top is a schematic of intron/exon structure and prominent splicing patterns observed in the forebrain (top) and hindbrain (bottom) of rat brain. Below is a summary of splicing regulatory motifs functionally defined in this study depicted on an expanded version of the <i>GRIN1</i> CI cassette exon (yellow). ESEs are indicated above the exon. Nucleotides complementary to U1 small nuclear RNA and the interaction of the positive regulator NAPOR/CUGBP2 with the downstream intron are indicated (‡; as determined in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030158#pbio-0030158-b26" target="_blank">26</a>]). UAGG and GGGG splicing silencing motifs defined in this study are highlighted in red. The working model for splicing silencing, based on the results shown here, proposes that the CI cassette is a strong exon silenced by a combination of two exonic UAGG motifs and a 5′-splice-site-proximal GGGG. HnRNP A1 mediates silencing and hnRNP H mediates anti-silencing via these RNA signals. </p

Computational Analysis of Exonic UAGG Motifs and Exon Skipping Patterns Genome-Wide

<p>Computational searches were performed to identify exons with two or more UAGGs and to determine the association of confirmed exon skipping events with this group. Exons with a single UAGG were analyzed for comparison. The following constraints were applied: (1) exon lengths of 250 bases or fewer and (2) both UAGG motifs conserved in sequence and position in the orthologous mouse exons. The graph illustrates the percentage of confirmed exon skipping events associated with one UAGG or two or more UAGGs (blue bars), or with the remaining exons lacking these motifs (red bars). The list of 16 human exons identified with two or more UAGGs is shown with the Ensembl ID, exon number, 5′ splice site sequence, and gene name. It is not unexpected to find exon 19 of the glutamate NMDA receptor <i>GRIN1</i> and exon 13 of <i>NCOA2,</i> which have a 5′-splice-site-proximal GGGG, since the sequence of the 5′ splice site was not specified in the search. </p

Effect of Number and Position of CI Cassette Exon Splicing Silencer Motifs

<p>Splicing reporters were constructed with variations in the number and position of UAGG and/or GGGG motifs. Three sets of schematics (boxed at center) illustrate the CI cassette exon and adjacent 5′ splice site region with positions of exonic UAGG (black vertical bars) and 5′ splice site GGGG (grey vertical stripe) motifs. Splicing reporter names are indicated at left. Vertical arrowhead indicates 5′ splice site. Each splicing reporter was generated by site-directed mutagenesis from parent plasmid wt0. Natural UAGG positions 51 and 93 represent the starting position of the motif relative to the first base of the exon. Engineered UAGG positions 11, 76, and 100 are also indicated (see schematic in center box at top). Sequence changes of the mutations are underscored: 11, GUGG→<u>UA</u>GG; 51, UAGG→<u>AU</u>GG; 76, CCAG→<u>UAG</u>G; 93, UAGG→<u>GU</u>GG; 100, UCCAA→U<u>AGGC</u>. Representative splicing patterns in PC12 cells (left gel panels) and C2C12 cells (right gel panels) are shown together with average percent exon inclusion values. The correlation between motif pattern and strength of splicing silencing is summarized (bottom). Exon-included (double arrowheads) and exon-skipped (single arrowheads) products are indicated. </p

Identification and Functional Roles of Protein Factors That Bind to GGGG and UAGG Motifs

<div><p>(A) Detection of protein binding to the 5′ splice site GGGG motif by UV crosslinking in HeLa nuclear extract. Wild-type (cs1 and 3h1) and mutant (cs3 and 3h3) RNA substrates were internally labeled at guanosine nucleotides; mutations are underscored. Pattern of UV crosslinking is shown following RNase digestion and SDS-PAGE (lanes 1–4). Immunoprecipitation reactions (lanes 5–11) contained the 3h1 substrate together with antibody specific for hnRNP F or H/H′; control samples contained preimmune rabbit serum. Gel panel shows the pellet (P), supernatant (S), and input (I) of the immunoprecipitation reactions following SDS-PAGE. The positions of hnRNP H/H′ and F (arrowheads) and protein molecular weight standards (in kilodaltons) are indicated. The hnRNP F and H/H′ antibodies were a gift of C. Milcarek. </p> <p>(B) UV crosslinking of exonic position 93 UAGG motif in HeLa nuclear extract. RNA substrates were prepared with a single radiolabeled nucleotide as indicated by the asterisk; sequences are shown (bottom). The wild-type (wt3) and mutant (mt3) substrates are identical except for the underscored mutation. The A1winner substrate corresponds to the high-affinity hnRNP A1 binding sequence previously identified by SELEX. The position of hnRNP A1 is indicated (arrowhead). Monoclonal antibody 9H10 was a gift of G. Dreyfuss.</p> <p>(C) Exon inclusion is enhanced by co-expression of hnRNP F or H. Gel panel shows splicing pattern resulting from co-transfection of wild-type (wt) or mutant (5m2) splicing reporter with hnRNP F or H expression plasmid; splicing reporters are identical to those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030158#pbio-0030158-g001" target="_blank">Figure 1</a>A. Control samples were transfected with empty vector; grey wedge indicates two levels (4 and 6 μg) of protein expression plasmid. Arrowhead indicates 5′ splice site. For immunoblot verification of transfected protein expression (bottom), nuclear extracts from transfected cells were separated by SDS-PAGE, transferred to nylon membrane, and developed with an antibody specific for the Xpress tag at the N-terminus of each pcDNA–protein sample. Raw percent exon inclusion values are shown below gel image.</p> <p>(D) Silencing effect of hnRNP A1 requires the intact 5′ splice site GGGG motif and full-length downstream intron. Structures of chimeric splicing reporters are shown in which the CI cassette exon and intron flanks were introduced into an unrelated splicing reporter containing sequences from the GABA_A receptor γ2 transcript: rGγCI-wt0 (both introns truncated), -up (full-length upstream intron, truncated downstream intron), and -dn (truncated upstream intron, full-length downstream intron). Numbers above indicate length of each intron segment in nucleotides. Arrowhead indicates 5′ splice site. The splicing reporters rGγCI-dn5m2 and -dn5m4 contain the full-length downstream intron with 5′ splice site mutations of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030158#pbio-0030158-g001" target="_blank">Figure 1</a>A. Gel panel shows splicing pattern resulting from co-transfection of splicing reporter with hnRNP A1 expression plasmid or vector control. Immunoblot verification of transfected protein expression (bottom) is as described in (C). </p></div

Exonic UAGG and 5′ Splice Site GGGG Motifs Are Required in Combination for Silencing of the CI Cassette Exon

<div><p>(A) A GGGG splicing silencer motif at the 5′ splice site. Top: Sequence of the 5′ splice site region (5′ to 3′) with exonic (uppercase) and intronic (lowercase) nucleotides. Numbering is relative to the first nucleotide of the intron. Arrowhead indicates 5′ splice site. A predicted SRp40 motif overlying the last seven bases of the exon is indicated. Engineered mutations and names of splicing reporters are indicated immediately below the affected nucleotides. Effect of mutations on the pattern of splicing is shown in a 5′ to 3′ arrangement (gel panel and graph). All splicing reporter plasmids have a three-exon structure in which CI is the middle exon (in the schematic, vertical bars indicate exons and horizontal lines indicate introns). Splicing reporter plasmids were expressed in vivo in mouse C2C12 cells, and splicing patterns assayed by radiolabeled RT-PCR of cellular RNA harvested from the cells. PCR primers are specific for the flanking exons. Results of multiple experiments are shown graphically as the average percent of exon included in product (y-axis) for each splicing reporter construct (x-axis). </p> <p>(B) Analysis of ESE motifs. An exonic UAGG splicing silencer motif overlaps an ASF/SF2 motif. Sequence of the CI exon (5′ to 3′) is shown, with engineered mutations (underscored) and names of splicing reporters indicated immediately below the affected nucleotides (bold). Numbering is relative to the first nucleotide of the exon. Predicted ESE motifs for ASF/SF2 and SC35 are highlighted above the exonic sequence as indicated in brackets. The UAGG motif required for silencing (boxed) is indicated below the overlapping ASF/SF2 motif (asterisk). Effect of mutations on the in vivo pattern of splicing is shown in a 5′ to 3′ arrangement (gel panel and graph).</p> <p>Error bars in (A) and (B) represent standard deviations.</p></div