Kunkel G R, Pederson T
Mol Cell Biol. 1985 Sep;5(9):2332-40. doi: 10.1128/mcb.5.9.2332-2340.1985.
Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.
通过对分离的HeLa细胞核中新生链进行³²P标记，研究了U1小核RNA生物合成的转录近端阶段。将标记的RNA与固定在硝酸纤维素上的单链M13 DNA克隆杂交，这些克隆对应于人U1 RNA基因内部或侧翼区域。1微克/毫升的α-鹅膏蕈碱可使U1 RNA的转录受到超过95%的抑制，这与之前关于它由RNA聚合酶II合成的证据一致。未检测到与成熟U1 RNA 5'端紧邻区域（-6至-105个核苷酸）的DNA发生杂交，这表明与所有已研究的聚合酶II起始情况一样，U1 RNA的转录起始于帽位点或非常接近帽位点。然而，与之前描述的mRNA转录单位不同，在mRNA转录单位中，成熟mRNA 3'端之后的数百或数千个核苷酸会进行等摩尔转录，而标记的U1 RNA杂交在成熟U1 RNA 3'端下游的一个非常短的区域（约60个核苷酸）内急剧下降。同样与前体mRNA不同，前体mRNA在仍是新生RNA链时就组装成核糖核蛋白（RNP）颗粒，而根据与小核RNP Sm蛋白的单克隆抗体反应的标准，在分离细胞核中转录的U1 RNA并未形成RNP复合物。这表明，与前体mRNA - RNP颗粒形成不同，U1小核RNP组装直到转录完成后才发生。这些结果表明，尽管mRNA和U1小核RNA由RNA聚合酶II共同合成，但它们在3'加工程度和RNP组装的时间模式上都存在显著差异。
