Müller S, Wolpensinger B, Angenitzki M, Engel A, Sperling J, Sperling R
M. E. Müller-Institute for Microscopy, Biozentrum Universität Basel, Klingelbergstrasse 70, Basel, CH-4065, Switzerland.
J Mol Biol. 1998 Oct 23;283(2):383-94. doi: 10.1006/jmbi.1998.2078.
Pre-mRNA splicing is an important regulatory step in the expression of most eukaryotic genes. In vitro studies have shown splicing to occur within 50-60 S multi-component ribonucleoprotein (RNP) complexes termed spliceosomes. Studies of mammalian cell nuclei have revealed larger complexes that sediment at 200 S in sucrose gradients, termed large nuclear RNP (lnRNP) particles. These particles contain all factors required for pre-mRNA splicing, including the spliceosomal U snRNPs and protein splicing factors. Electron microscopy has shown them to consist of four apparently similar substructures. In this study, mass measurements by scanning transmission electron microscopy of freeze-dried mammalian lnRNP preparations, both confirm the similarity between the lnRNP particles and reveal the mass uniformity of their subunits. Thus, the tetrameric lnRNP particle has a mass of 21.1(+/-1.6) MDa, while each repeating subunit has a mass of 4.8(+/-0.5) MDa, which is close to the estimated mass of the fully assembled 60 S spliceosome. The 1.9 MDa discrepancy between the lnRNP particle's mass and the cumulative masses of its four subunits may be attributed to an additional domain frequently observed in the micrographs. Notably, strands and loops of RNA were often seen emanating from lnRNP particles positively stained with uranyl formate. Our results support the idea that the nuclear splicing machine is a supraspliceosome complex. For clarity, we define spliceosomes devoid of pre-mRNA as spliceosome cores, and propose that the supraspliceosome is constructed from one pre-mRNA, four spliceosome cores, each composed mainly of U snRNPs, and additional proteins. In this way a frame is provided to juxtapose exons about to be spliced.
前体mRNA剪接是大多数真核基因表达过程中的一个重要调控步骤。体外研究表明，剪接发生在称为剪接体的50 - 60 S多组分核糖核蛋白（RNP）复合物中。对哺乳动物细胞核的研究揭示了在蔗糖梯度中以200 S沉降的更大复合物，称为大核RNP（lnRNP）颗粒。这些颗粒包含前体mRNA剪接所需的所有因子，包括剪接体U snRNP和蛋白质剪接因子。电子显微镜显示它们由四个明显相似的亚结构组成。在本研究中，通过对冻干的哺乳动物lnRNP制剂进行扫描透射电子显微镜质量测量，既证实了lnRNP颗粒之间的相似性，又揭示了其亚基的质量均匀性。因此，四聚体lnRNP颗粒的质量为21.1（±1.6）MDa，而每个重复亚基的质量为4.8（±0.5）MDa，这接近完全组装的60 S剪接体的估计质量。lnRNP颗粒质量与其四个亚基累积质量之间1.9 MDa的差异可能归因于在显微照片中经常观察到的一个额外结构域。值得注意的是，在用甲酸双氧铀阳性染色的lnRNP颗粒中经常可以看到RNA链和环。我们的结果支持核剪接机器是一种超级剪接体复合物的观点。为了清晰起见，我们将不含前体mRNA的剪接体定义为剪接体核心，并提出超级剪接体由一个前体mRNA、四个主要由U snRNP组成的剪接体核心和其他蛋白质构建而成。通过这种方式，提供了一个框架来并列即将被剪接的外显子。
