Function of the interacting protein of SMN, the product of the spinal muscular atrophy gene, and neural specific splicing.

SMN蛋白是SMA基因的产物，被认为在与SMN相互作用蛋白SIP 1的剪接体的生物发生中起关键作用。为了阐明神经元中的剪接机制，我们寻找神经剪接因子，并克隆了两个新的SR蛋白，神经突起SR蛋白（NSSR）1和2，这两个蛋白在脑和睾丸中存在较高水平。在分化过程中，NSSR 1仅在神经元阶段被检测到。纯化的重组NSSR 1和2蛋白均增强核提取物的体外剪接活性。此外，NSSR 2的过表达阻止了GluR-B基因剪接中包含Flip或Flop外显子，导致异常外显子跳跃产物的增加。相反，用NSSR 1瞬时转染促进包含Flip外显子，使得异常产物剪接成成熟剪接形式。甚至在共转染NSSR 2的情况下也观察到NSSR 1对外显子跳跃的这种抑制。接下来，为了阐明SIP 1与神经元选择性剪接的相关性，我们在SIP 1-α（280 AA）的基础上，进一步鉴定了SIP 1的三种新的剪接变体（SIP-β、γ和δ）。我们检测了这些剪接变体在各种正常人体组织和SMA和ALS患者肌肉样本中的表达水平。SIP 1-α是一种主要的产物，并且广泛表达。相比之下，SIP-β和γ以非常低的表达水平检测到。在SMA和ALS患者中，与对照组相比，SIP-α显著降低（17%，19%），而SIP-β在两种疾病中均显著升高（34%，32%）。这些结果表明，SIP 1的异常选择性剪接事件发生在运动神经元疾病中，并参与了SMA和ALS的病理过程。

SMN protein, the product of the SMA gene, is considered to play a crucial role in biogenesis of spliceosomes with the SMN-interacting protein, SIP1. To clarify splicing mechanism in neuron, we searched neural splicing factors and cloned two new SR proteins, Neural-salient SR proteins (NSSR) 1 and 2, which are present at higher levels in brain and testis. During the differentiation, NSSR 1 is detected only in the neuronal stage. Both the purified recombinant NSSR 1 and 2 proteins enhance the in vitro splicing activity of nuclear extract. Moreover, overexpression of NSSR 2 prevents the inclusion of either the Flip or Flop exons in the splicing of the GluR-B gene, resulting in an increase in the abnormal exon-skipping product. In contrast, transient transfection with NSSR 1 promotes the inclusion of the Flip exon so that the abnormal product is spliced to the mature spliced form. This suppression of exon skipping by NSSR 1 is observed even with cotransfection of NSSR 2. Results indicate that NSSR 1 may play a crucial role in the regulation of alternative splicing in neurons.Next, to clarify relevancy of pathogenesis and SIP1, we identified three novel splicing variants of the SIP1 (SIP-β、γ and δ), in addition to the full-length SIP1-α (280AA). We examined the expression levels of these splicing variants in various normal human tissues and in muscle samples from patients with SMA and ALS. The SIP1-α was a major product and ubiquitiously expressed. In contrast, SIP-β and γ were detected at very low expression level. In patients with SMA and ALS, the SIP-α was dramatically decreased(17%, 19%) compared to the controls, while the SIP-β was significantly increased(34%, 32%) in both diseases. These findings suggest that aberrant alternative splicing event in SIP1 occur in the motor neuron disease and contribute the pathological process of SMA and ALS.