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Figure 4 | BMC Genomics

Figure 4

From: Evolutionarily emerged G tracts between the polypyrimidine tract and 3′ AG are splicing silencers enriched in genes involved in cancer

Figure 4

The REPA G tracts are splicing silencers and their related exons are controlled by G tract-binding splicing factors hnRNP H/F in human cells. A. (Upper left) Diagram of splicing reporter mini-genes and (Lower left) western blot of RNA interference samples of HEK293T cells using anti-nucleolin and anti-hnRNP H/F antibodies. (Right) agarose gels of RT-PCR products from transiently expressed reporters containing the G-tract-harboring 3′SS or its G- > A mutants in normal (Above) or RNA interference (Below) human HEK293T cells. Boxes: exons, horizontal lines: introns. Splicing pathways are indicated by dashed lines above or below the pre-mRNA. Heavy black bar: 3′SS with REPA G tract (WT) or its G- > A mutant (Mut). ApaI and BglII: restriction-cloning sites. *: cryptic splicing pathway through the distal 3′SS or its product. The percentages (mean ± S.D., n = 3) of molar amounts of the proximal (prox., upstream the cryptic site) 3′SS- skipped product are indicated below each lane. In the brackets above the lanes are the MaxEnt scores of the G tract or mutant 3′ splice sites of the minigenes. For PRMT5, the scores are -6.01 and -2.39, respectively. The vector DUP175 has a score of 9.50. B. (Left) western blot of RNA interference samples of HeLa cells as in A. (Right) Agarose gels of RT-PCR products of endogenous REPA G-tract-harboring genes in HeLa cells. The percentages (%, mean ± S.D., n = 3) of molar amounts of the minor isoforms (exon excluded “Excl.” except SLC44A2 as inclusion “Incl.”) are below each lane. Black dots: exon included, open circle: exon excluded. @: a shorter product consistent with a variant in frog. *: non-specific products by sequencing results.

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