Beltrame M, Tollervey D
EMBL, Heidelberg, Germany.
EMBO J. 1992 Apr;11(4):1531-42. doi: 10.1002/j.1460-2075.1992.tb05198.x.
It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.
长期以来，人们都知道U3可以通过氢键与核糖体前体RNA分离，但相互作用位点的特征却鲜为人知。在这里，我们表明酵母U3在脱蛋白提取物和活细胞中都可以与35S核糖体前体RNA交联。交联位点定位于5'外部转录间隔区（ETS），然后在核苷酸水平上进行鉴定。U3靠近5'端的两个区域在体内和体外都与核糖体前体RNA交联；进化上保守的A框区域和一个与ETS序列具有完美互补性的10个核苷酸（nt）序列。在ETS中检测到两个体内交联，一个在与U3互补区域内的+470处，另一个在+655处，靠近18S rRNA 5'端的切割位点。使用标记的rDNA构建体来追踪体内ETS突变的影响。ETS中+470交联位点周围的一个小缺失会阻止18S rRNA的合成。该区域与脊椎动物ETS切割位点同源。我们提出，该位点可能在进化上是保守的，以指导产生18S rRNA的切割所需的核糖体前体RNA加工复合物的组装。
