List of oligonucleotides.

<p>List of oligonucleotides.</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

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

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

<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