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SMN相互作用蛋白SIP1剪接体的生物发生中起着至关重要的作用。为了明确神经元的剪接机制，我们搜索了神经剪接因子，克隆了两个新的SR蛋白，即神经突出SR蛋白（neural -salient SR protein， NSSR） 1和2，它们在大脑和睾丸中含量较高。在分化过程中，NSSR 1仅在神经元阶段检测到。纯化的重组NSSR 1和2蛋白均增强了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Flip或Flop外显子的包含，导致异常外显子跳变产物的增加。相反，NSSR 1的瞬时转染促进了Flip外显子的包含，使异常产物被剪接成成熟的剪接形式。即使在共转染NSSR 2时，也观察到NSSR 1对外显子跳变的抑制作用。结果表明，NSSR 1可能在神经元选择性剪接的调控中起重要作用。接下来，为了阐明发病机制与SIP1的相关性，除了全长SIP1-α (280AA)外，我们还鉴定了SIP1的三个新的剪接变体（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肌萎缩侧索硬化症患者肌肉样本中的表达水平。SIP1-α是主要产物，普遍表达。相比之下，SIP-β和γ的表达水平很低。在SMA和ALS患者中，与对照组相比，SIP-α显著降低（17%,19%），而SIP-β显著升高（34%,32%）。这些发现提示运动神经元疾病中发生了SIP1异常的选择性剪接事件，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.