Savkur R S, Olson M O
Department of Biochemistry, The University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.
Nucleic Acids Res. 1998 Oct 1;26(19):4508-15. doi: 10.1093/nar/26.19.4508.
Protein B23 is an abundant nucleolar protein and a putative ribosome assembly factor which possesses an intrinsic ribonuclease activity. In the current work, the effects of RNA sequence and secondary structure on the cleavage preference by protein B23 were studied. Protein B23 ribonuclease preferentially cleaved the single-stranded homopolymers poly(A), poly(U) and poly(C). However, double-stranded co-polymers and poly(G) were resistant to cleavage. No base specificity was observed with an oligoribonucleotide substrate. The action of protein B23 ribonuclease on different regions of pre-rRNA was studied using transcripts synthesized in vitro from cloned rDNA segments. Although no specific cleavages were detected in transcripts containing sequences from the 5' external transcribed spacer or the first internal transcribed spacer, the enzyme preferentially cleaved the second internal transcribed spacer (ITS2) approximately 250 nt downstream from the 3'-end of 5.8S rRNA. Preferential cleavage was retained when the transcript was extended by 100 nt at the 3'-end, but abolished in a transcript lacking this cleavage site. Furthermore, this site was not susceptible to cleavage by RNase A or RNase T1. These results, in conjunction with the sub-nucleolar localization of the protein with elements of the processing machinery, suggest that the protein B23 endoribonuclease could play a role in pre-rRNA processing in ITS2.
蛋白质B23是一种丰富的核仁蛋白，也是一种假定的核糖体组装因子，具有内在的核糖核酸酶活性。在当前的研究中，研究了RNA序列和二级结构对蛋白质B23切割偏好性的影响。蛋白质B23核糖核酸酶优先切割单链均聚物聚腺苷酸（poly(A)）、聚尿苷酸（poly(U)）和聚胞苷酸（poly(C)）。然而，双链共聚物和聚鸟苷酸（poly(G)）对切割具有抗性。对于寡核糖核苷酸底物，未观察到碱基特异性。使用从克隆的rDNA片段体外合成的转录本，研究了蛋白质B23核糖核酸酶对前体rRNA不同区域的作用。尽管在包含5'外部转录间隔区或第一个内部转录间隔区序列的转录本中未检测到特异性切割，但该酶优先切割位于5.8S rRNA 3'端下游约250 nt处的第二个内部转录间隔区（ITS2）。当转录本在3'端延长100 nt时，仍保留优先切割，但在缺乏该切割位点的转录本中则被消除。此外，该位点不易被核糖核酸酶A或核糖核酸酶T1切割。这些结果，连同该蛋白在核仁亚区与加工机制元件的定位，表明蛋白质B23内切核糖核酸酶可能在ITS2的前体rRNA加工中发挥作用。
