In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei
In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei
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DOI：
10.1261/rna.5258504
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发表时间：
2004-04-01
期刊：
影响因子：
4.5
通讯作者：
Carmo-Fonseca, M
中科院分区：
文献类型：
--
作者：
Custódio, N;Carvalho, C;Carmo-Fonseca, M
近年来的研究表明，mRNA的核输出和前体mRNA的加工之间存在广泛的耦合。在这里，我们可视化的外显子连接复合物（EJC）蛋白和RNA输出因子相对于丰富的前mRNA合成的网站在细胞核中的分布。我们分析了用腺病毒感染的HeLa细胞和用人β-珠蛋白基因稳定转染的鼠红白血病（MEL）细胞。使用原位杂交和共聚焦显微镜，我们观察EJC蛋白（REF/Aly，Y14，SRm 160，UAP 56，RNPS 1和Magoh）和核心剪接体成分（U snRNP）在转录位点的积累。这表明EJC蛋白稳定地与前mRNA共转录结合。没有浓度的出口因子NXF 1/TAP，p15和Dbp 5检测新生的成绩单，认为在哺乳动物细胞中，这些蛋白质结合的mRNA之前或之后不久从网站的转录释放。这些结果还表明，EJC蛋白与mRNA的结合不足以募集TAP-p15，这与最近的研究结果一致，该研究结果表明EJC在mRNA输出中不起关键作用。与在表达正常人β-珠蛋白转录物的MEL细胞中获得的结果相反，剪接和3 '末端加工缺陷的突变前mRNA不与SRm 160、REF、UAP 56或Sm蛋白共定位。这表明EJC蛋白在转录位点的积累需要新生前mRNA的有效加工，认为转录本身不足以稳定组装EJC。
Studies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human beta-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human beta-globin transcripts, mutant pre-mRNAs defective in splicing and 3'end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.