Peyda Parham, Lin Chia-Ho, Onwuzurike Kelechi, Black Douglas L
Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA.
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA.
Genes Dev. 2025 Mar 3;39(5-6):364-383. doi: 10.1101/gad.352105.124.
The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to the large assembly of splicing regulators (LASR), a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA motifs for LASR subunits hnRNPs M, H/F, and C and Matrin3. These elements are often arranged in tandem, forming multipart modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Minigene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.
Rbfox蛋白通过与RNA元件GCAUG结合来调控前体mRNA的可变剪接。在细胞核中，大多数Rbfox与剪接调节因子大集合（LASR）结合，LASR是一种由识别其他RNA基序的RNA结合蛋白组成的复合物。然而，目前尚不清楚Rbfox/LASR复合物的不同亚基如何协同作用以结合RNA并调节剪接。我们使用核酸酶保护试验来绘制Rbfox1/LASR在新生细胞RNA上的全转录组足迹。除了GCAUG之外，Rbfox1/LASR还结合LASR亚基hnRNPs M、H/F、C和Matrin3的RNA基序。这些元件通常串联排列，形成多部分的RNA基序模块。为了区分Rbfox1与LASR亚基的接触位点，我们分析了一种突变型Rbfox1(F125A)，它失去了RNA结合能力，但仍与LASR相关联。Rbfox1(F125A)/LASR复合物不再与GCAUG相互作用，但保留了与LASR的RNA元件的结合。剪接分析表明，除了通过相邻的GCAUG元件激活外显子外，Rbfox还可以刺激LASR亚基结合位点附近的外显子。微型基因实验表明，这些不同的元件对目标外显子产生联合调节作用。这些发现阐明了一种RNA结合蛋白复合物如何通过识别串联RNA元件组来解码组合剪接调节信号。
