Nothwang H G, Coux O, Keith G, Silva-Pereira I, Scherrer K
Institut Jacques Monod du CNRS, Université Paris, France.
Nucleic Acids Res. 1992 Apr 25;20(8):1959-65. doi: 10.1093/nar/20.8.1959.
Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.
用RNA连接酶对HeLa细胞前体RNA进行3'末端标记后进行二维凝胶电泳，结果显示有一个明显的斑点。对3'末端部分序列的测定表明，其与兔、牛和人类的tRNA（Lys,3）3'末端的18个核苷酸完全同源。与tRNA（Lys,3）3'末端互补的寡核苷酸在Northern印迹上与HeLa细胞和鸭红细胞的前体RNA杂交。在二维聚丙烯酰胺凝胶电泳中，HeLa细胞的主要前体RNA与牛tRNA（Lys,3）迁移位置相同。用人和鸭前体的tRNA-核苷酸转移酶重建RNA的CCA 3'末端，证实了这类RNA的tRNA特性。此外，在这两个物种的前体RNA中还发现了至少一条约85个核苷酸长的额外tRNA条带。最后，为证实前体RNA的一个原始特性，我们发现体外合成的tRNA（Lys,3）能与鸭珠蛋白mRNA以及HeLa细胞的聚腺苷酸（+）和聚腺苷酸（-）RNA稳定杂交。
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