A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4
A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4
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DOI：
10.1261/rna.1212008
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发表时间：
2008-10-01
期刊：
影响因子：
4.5
通讯作者：
Lynch, Kristen W.
中科院分区：
文献类型：
--
作者：
Topp, Justin D.;Jackson, Jason;Lynch, Kristen W.
人CD45基因编码一种蛋白酪氨酸磷酸酶，由于三个可变外显子的选择性剪接，该蛋白酪氨酸磷酸酶在静息和活化的T细胞中表现出不同的同种型表达。在此之前，我们已经使用生物化学方法来确定两个调节蛋白，hnRNP L和PSF，这有助于激活诱导的跳跃CD45通过可变外显子4中的ESS 1调控元件。在这里，我们报告的第三个CD45调节因子，hnRNP L样（hnRNP LL）的鉴定，通过基于细胞的筛选克隆变体，表现出激活样表型的CD45剪接，即使在静息条件下。两个剪接改变克隆的微阵列分析显示，相对于野生型细胞，hnRNP LL的表达增加。我们进一步证明，hnRNP LL蛋白的表达及其与ESS 1的结合在野生型细胞激活后上调。在野生型细胞中hnRNP LL的强制过表达导致外显子抑制的增加，而hnRNP LL的敲低消除了激活诱导的外显子跳跃。有趣的是，hnRNP L和hnRNP LL与ESS 1突变体的结合分析表明，这些蛋白质具有重叠但不同的结合要求。总之，这些数据确立了hnRNP LL在信号诱导的CD45调节中起着关键和独特的作用，并证明了基于细胞的筛选用于鉴定新型剪接调节因子的实用性。
The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knockdown of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.