5S RNA Transport

核糖体在所有活细胞中蛋白质的合成中起着关键作用，无论是植物细胞、动物细胞还是细菌。在真核生物中，这些复杂的大分子由四种核糖体RNA分子和大约80种不同的核糖体蛋白组成。核糖体组装发生在细胞的核仁中，四种RRNA中的三种是在那里合成的。核糖体的所有其他成分都是在其他地方合成的，并被运输到核仁进行组装。这项研究的重点是鉴定和表征将5S核糖体RNA带到核仁组装成核糖体的运输途径和调控机制。这项研究将使用克隆的5S RNA基因及其相关蛋白的突变，试图扰乱正常的核糖体组装或功能，以辨别每个组件在正常的核糖体组装中可能如何发挥作用。突变的基因或其基因产物(5S RNAs或5S RNA结合蛋白)将被显微注射到非洲爪哇卵母细胞中，基因产物将通过放射性同位素标记和/或针对蛋白质或核蛋白成分的特异性抗体在细胞内被跟踪。体外交换研究将与分离的核仁和分离的核糖体进行，结果将通过显微注射实验在体内得到验证。蛋白质合成是所有活细胞的基本过程。蛋白质合成的机制是核糖体，这是一个由许多单独的大分子成分组成的复杂结构，既有蛋白质，也有多聚核糖核酸(RNAs)。核糖体的合成是一个复杂的过程，因为单个大分子成分的合成发生在细胞的不同部位，有时是在不同的时间，但有必要将所有不同的成分聚集在一起进行组装。核糖体的蛋白质成分本身是在原有的核糖体上合成的，这发生在细胞质中。核糖核酸是在细胞核中合成的，因为它们是核糖体基因的直接转录产物。四个RNA分子中的三个是在细胞核的一个称为核仁的特殊区域合成的，首先是一个非常长的转录本，然后被加工成三个较小的片段。所有其他成分都被带到核仁进行组装，包括第四个也是最小的rRNA分子，被称为“5S”。这种5S RNA是在细胞核中合成的，但不是在核仁中合成的。本实验室以前的研究结果表明，在非洲爪哇卵母细胞中，5S RNA先于其他rRNA组分合成，转录后与其自身的转录因子(一种名为TFIIIA的蛋白质)一起运输到细胞质并储存在那里。随后，它与TFIIIA解离，并与另一种蛋白质L5结合，L5是一种核糖体蛋白；然后，5S RNA-L5复合体被运回细胞核，特别是现在的核仁，在那里它组装成新生的核糖体。本项目将探讨这一复杂的核质运输的机制和调控。这项工作不仅对于理解核糖体的生物发生具有重要意义，它本身也是生物学中的一个非常重要的问题，而且对于理解核质运输作为一种现象是许多真核细胞调控的基础。

Ribosomes play a key role in the synthesis of proteins in all living cells, whether they be plant cells, animal cells, or bacteria. In eukaryotes, these complex giant macromolecules are composed of four kinds of ribosomal RNA molecules and approximately eighty different ribosomal proteins. Ribosome assembly occurs in the nucleolus of the cell, where three of the four RRNAS are synthesized. All other components of the ribosome are synthesized elsewhere and transported to the nucleolus for assembly. This research project is focused on identifying and characterizing the transport pathways and regulatory mechanisms that bring the 5S ribosomal RNA to the nucleolus for assembly into ribosomes. The research will use mutants of the cloned 5S RNA genes and their associated proteins to try to disrupt normal ribosome assembly or function in order to discern how each component might function in normal ribosome assembly. The mutated genes or their gene products (5S RNAs or 5S RNA-binding proteins) will be microinjected in Xenopus oocytes, and the gene products will be followed through the cell by way of the radioisotopic labelling and/or specific antibodies against the protein or nucleoprotein components. In vitro exchange studies will be carried out with isolated nucleoli and with isolated ribosomes, and the results will be verified in vivo through the microinjection experiments. %%% Protein synthesis is a fundamental process of all living cells. The machinery for protein synthesis is the ribosome, a complex structure consisting of many individual macromolecular components, both proteins and polyribonucleic acids (RNAs). The synthesis of ribosomes is a complex process, since the synthesis of the individual macromolecular components occurs in various parts of the cell, and sometimes at different times, yet it is necessary to bring all the different components together for assembly. The protein components of the ribosome are themselves synthesized on preexisting ribosomes, and this takes place in the cytoplasm. The ribonucleic acids are synthesized in the nucleus, since they are direct transcripts of the ribosomal genes. Three of the four RNA molecules are synthesized in a specialized region of the nucleus termed the nucleolus, first as a very long transcript which is processed into the three smaller pieces. It is to the nucleolus that all the other components are brought for assembly, including the fourth and smallest RRNA molecule, termed "5S." This 5S RNA is synthesized in the nucleus, but not in the nucleolus. Prior results from this laboratory have shown that, in Xenopus oocytes, the 5S RNA is synthesized in advance of the other RRNA components, and that after the 5S RNA is transcribed it is transported to the cytoplasm, together with its own transcription factor (a protein termed TFIIIA), and is stored there. Subsequently, it becomes dissociated from TFIIIA and associates with a different protein, L5, which is a ribosomal protein; the 5S RNA-L5 complex is then transported back to the nucleus, specifically now to the nucleolus, where it becomes assembled into the nascent ribosomes. This project will address the mechanism and regulation of this complicated nucleocytoplasmic trafficking. The work is of significance not only for understanding ribosomal biogenesis, which is itself a very important problem in biology, but also for understanding nucleocytoplasmic trafficking in general, which as a phenomenon is fundamental to much of eukaryotic cellular regulation.