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C9orf 72与coilin相互作用并影响Cajal体动力学和剪接
C9orf 72 interacts with coilin and influences Cajal body dynamics and splicing |
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| 课程网址: |
http://videolectures.net/encals2017_gibson_cajal_body/
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| 主讲教师: |
Yolanda Gibson
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| 开课单位: |
雪菲尔大学
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| 开课时间: |
2017-07-21
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| 课程语种: |
英语
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| 中文简介: |
理由与假设：C9orf72基因中的GGCC六核苷酸重复序列扩增是肌萎缩侧索硬化症（ALS）的主要原因。虽然确切的发病机制尚不清楚，但有人提出C9orf72单倍体不足。目前对C9orf72蛋白的功能知之甚少，但一项酵母双杂交试验发现C9orf72与薏苡仁之间存在相互作用。Coilin是Cajal小体（CBs）的主要蛋白质组分，Cajal小体是作为剪接机制成熟和组装位点的核亚有机体。C9orf72 ALS患者存在与疾病严重程度相关的剪接缺陷。我们假设ALS中C9orf72蛋白的单倍体不足导致Cajal小体和下游剪接的失调。
目的：确认C9orf72与薏苡仁之间的相互作用，并研究C9orf72对Cajal小体和剪接的影响。
方法：体外GST结合试验用于确定GST-C9orf72和35S标记的薏苡仁之间是否存在直接相互作用。在Hek293细胞裂解物上进行共免疫沉淀实验，以确认myc-C9orf72和内源性薏苡仁素之间的细胞相互作用。使用原位邻近连接分析（PLA）研究相互作用的定位。在C9orf72基因敲除后，对Hek293细胞进行coilin和SMN免疫染色，以研究C9orf72对CBs的影响。最后，采用人工报告剪接试验研究C9orf72基因敲除对剪接效率的影响。
结果：GST结合试验表明C9orf72和薏苡仁素在体外存在直接相互作用。在细胞裂解物中，myc-C9orf72成功地与内源性薏苡仁素共免疫沉淀，表明这种相互作用在生理上是相关的。PLA证实了这种相互作用，并表明它发生在细胞质和细胞核中。有趣的是，在Hek293细胞中C9orf72基因敲除导致CBs数量增加，但在人工报告剪接试验中，主要和次要内含子的剪接减少。结果证实C9orf72与薏苡仁蛋白相互作用，可能调节CBs和剪接。
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| 课程简介: |
Rationale & Hypothesis: The GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the leading cause of amyotrophic lateral sclerosis (ALS). Although the precise disease mechanisms are unclear, haploinsufficiency of C9orf72 has been proposed. Little is currently known about the function of the C9orf72 protein, but a yeast two- hybrid assay found an interaction between C9orf72 and coilin. Coilin is the major protein component of Cajal bodies (CBs), nuclear suborganelles which act as sites of splicing machinery maturation and assembly. C9orf72 ALS patients have splicing defects that correlate with disease severity. We hypothesise that haploinsufficiency of the C9orf72 protein in ALS leads to dysregulation of Cajal bodies and downstream splicing.
Objectives: Confirm the interaction between C9orf72 and coilin and investigate the effect of C9orf72 on Cajal bodies and splicing.
Methodology: An in vitro GST-binding assay was used to establish if there is a direct interaction between GST-C9orf72 and 35S-labelled coilin. Co-immunoprecipitation experiments were performed on Hek293 cell lysates to confirm the cellular interaction between myc-C9orf72 and endogenous coilin. The localisation of the interaction was investigated using an in situ Proximity Ligation Assay (PLA). Immunostaining for coilin and SMN was performed on Hek293 cells following C9orf72 knockdown to investigate the effect of C9orf72 on CBs. Finally, an artificial reporter splicing assay was used to study the effect of C9orf72 knockdown on splicing efficiency.
Findings: The GST-binding assay suggests there is a direct interaction between C9orf72 and coilin in vitro. In cell lysates, myc-C9orf72 was successfully co- immunoprecipitated with endogenous coilin, suggesting the interaction is physiologically relevant. PLA confirmed the interaction and shows it occurs in both the cytoplasm and nucleus. Interestingly, C9orf72 knockdown in Hek293 cells led to an increase in the number of CBs but a decrease in the splicing of both major and minor introns in the artificial reporter splicing assay. The results confirm C9orf72 interacts with coilin and may regulate CBs and splicing.
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| 关 键 词: |
核苷酸重复序列扩增; 肌萎缩侧索硬化症; 核亚有机体
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| 课程来源: |
视频讲座网
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| 数据采集: |
2021-12-11：zkj
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| 最后编审: |
2021-12-11：zkj
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| 阅读次数: |
59
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