西亞試劑：Far upstream element-binding protein 1 and RNA secondary st

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression

Huang Lia, Zhijia Wanga, Xuexia Zhoua, Yuanming Chenga, Zhiqin Xiea, James L. Manleyb,1, and Ying Fenga,c,1

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3′splice site, and the other involves an exonic splicing silencer (ESS) situated 3′ to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.

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