List of oligonucleotides.

<p>List of oligonucleotides.</p

Treatment of <i>ods4-1</i> cells with 6-AU suppressed the exon-skipping phenotype.

<p>Serially diluted <i>ods4-1</i> and wild-type cells harboring pURA4β were spotted on MMU (+Uracil) and MM (-Uracil) plates with 160 μg/ml of 6-AU (+6-AU, lower panel) or without 6-AU (upper panel), and incubated at 26°C for 5 days. The growth of <i>ods4-1</i> and other <i>ods</i> mutants on MM (-Uracil) was impaired in the presence of 6-AU, whereas their growth on MMU (+Uracil) was not affected under the same conditions, suggesting that the exon skipping of URA4β pre-mRNA was suppressed by the treatment with 6-AU.</p

<i>S</i>. <i>pombe</i> strains used in this study.

<p><i>S</i>. <i>pombe</i> strains used in this study.</p

A transcription-coupled ordered splicing model.

<p>(A) The 5′ splice site in the nascent transcript is recognized by U1 snRNP associating with the CTD of elongating RNA polymerase II and the SF1-U2AF⁵⁹-U2AF²³/NTC- Cwf16 complex. After transcription proceeds, a branch point sequence (BP) is recognized by the U1/SF1-U2AF⁵⁹-U2AF²³/NTC-Cwf16 complex to form a pre-spliceosome. (B) The depletion of Cwf16p by the <i>ods4-1</i> mutation induces the weakened recognition of the branch and 3′ splice sites by the U1/SF1-U2AF⁵⁹-U2AF²³/NTC complex and increases an opportunity to cause exon skipping, thereby ligating to the far downstream exon.</p

Overexpression of the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> gene suppressed exon skipping in <i>ods4</i>-<i>1</i>.

<p>(A) The overexpression of the <i>cwf16</i>⁺ or <i>tif213</i>⁺ gene rescued the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with pBG1-URA4β and the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>^<i>+</i> plasmid were streaked on MMU plates and incubated at 22, 26, or 30°C to test their complementarity for the <i>cs</i> phenotype of <i>ods4-1</i>. Transformants with the <i>cwf16</i>^<i>+</i> or <i>tif213</i>^<i>+</i> plasmid grew well at 22°C, whereas <i>ods4-1</i> itself showed slow growth at the same temperature (<i>ods4</i>+URA4β+pSP1). The overexpression of Srp2p resulted in slow growth at all temperatures. (B) Total RNAs were isolated from wild type (WT), <i>ods4-1</i>, and <i>ods4-1</i> with the <i>cwf16</i>^<i>+</i>, <i>srp2</i>^<i>+</i>, or <i>tif213</i>⁺ plasmid, and subjected to RT-PCR analyses. All transformants contained pBG1-URA4β in addition to the rescued genes or pSP1 vector as indicated. Amplified cDNA products were electrophoresed on a 5% acrylamide gel, stained with ethidium bromide (upper panel), and then subjected to a Southern blot analysis using an oligonucleotide probe that specifically hybridizes to the exon-skipped product (middle panel). RT-PCR of <i>act1</i>^<i>+</i> mRNA was performed as a control (lower panel). The structures of the RT-PCR products confirmed by the sequence analysis are shown on the left. Arrows indicate the positions of the tub-3 and tub-4 primers used for RT-PCR analyses.</p

Structure of reporter plasmids for <i>ods</i> screening.

<p>(A) Structures of pURA4β (pSP1-URA4β) and pBG1-URA4β reporter plasmids. The pSP1 and pBG1 vectors have the <i>LEU2</i> and <i>his3</i> markers, respectively. The intron 1-exon 2-intron 2 region of the <i>S</i>. <i>pombe</i> β-tubulin gene (<i>nda3</i>^<i>+</i>) was amplified by PCR and inserted into the <i>Stu</i> I site in the <i>ura4</i>^<i>+</i> gene. (B) Splicing patterns of the transcripts from the chimeric <i>ura4</i> gene in the reporter plasmids. Transcripts in which the internal exon is included produce non-functional Ura4p, whereas exon-skipped transcripts produce functional Ura4p.</p

Overexpression of Tif213p promoted translation of <i>cwf16</i> mRNA containing a mutated start codon in <i>ods4-1</i>.

<p><i>ods4-1</i> was transformed with pMT-mCwf16-FLAG containing the mutated start codon (the <i>ods4-1</i> mutation). pSP1-Tif213 or pSP1 was simultaneously transformed. Five independent transformants were cultured and subjected to a western blot analysis using an anti-FLAG antibody. The intensity of each band was quantitated with a FUJIFILM LAS-1000 and graphed (lower panel).</p

Complementation analysis of <i>snh7</i> and <i>snh31</i>.

<p>The isolated <i>ura</i>^<i>+</i> mutants, <i>snh7</i> and <i>snh31</i>, were crossed with the wild-type haploid strain (<i>UR502</i>) or each of the isolated <i>ods</i> mutants to produce diploid strains. The resultant diploid strains were streaked on MMU (+Uracil) and MM (-Uracil) plates and then incubated at 26°C.</p

Homeotic Transformation Observed in the 4th Caudal Vertebra of <i>Hotair</i>^{<i>−⁄−</i>} mice.

<p>(A) Visualization of the sacral and caudal region of the mouse skeleton by microCT reveals a homeotic transformation in <i>Hotair</i>^{<i>−⁄−</i>} mice of the 4th caudal vertebra to a structure similar to that of the 3rd caudal vertebra. (B) Dorsal, lateral and ventral comparison of WT and <i>Hotair</i>^{<i>−⁄−</i>} 4th caudal vertebra reveals a structural abnormality in homozygotes indicative of a homeotic transformation.</p

Increased Expression of <i>Lincpint</i> from Postnatal Day 3 to 8 Weeks of Age.

<p><i>LacZ</i> reporter expression (blue) at 3 days, 3 weeks, and 8 weeks in F0 heterozygotes shows increasing <i>Lincpint</i> expression with age. (A) At 3 days, ßgalactosidase staining is only observed in portions of the brain, tendons and ligaments of the hind limb, and some bronchioles in the lung (arrow). (B) At 3 weeks, there is increased staining in the brain, hindlimb, atria of the heart, lung, and liver. (C) By 8 weeks of age, the whole brain, skeletal muscle of the hindlimb and chest, atria and myocardium, lung, and liver tissue all exhibit strong ß-galactosidase staining representative of increased <i>Lincpint</i> expression. Examples shown are representative of n>4 mice per group. (D) RT-PCR analysis of <i>GFP</i> in GA (gastrocnemius) muscle and kidney isolated from 8 week-old and 52 week-old wild type (WT), heterozygous (<i>Lincpint-GFP</i>^{<i>+⁄−</i>}) and homozygous (<i>Lincpint-GFP</i>^{<i>−⁄−</i>}) mice. (E) Comparison of endogenous <i>Lincpint</i> RNA level in GA (gastrocnemius) muscle and kidney of 8 week-old versus 52 week-old wild type (WT) tissues demonstrating increased in <i>Lincpint</i> gene expression. Examples shown are representative of n>4 mice per group.</p