Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus.
Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus.
复制标题
布氏锥虫中多顺反子前体 mRNA 的成熟：分析 hsp70 基因座新生 RNA 转录物的反式剪接和 Poly(A) 添加。
DOI：
10.1128/mcb.11.6.3180-3190.1991
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发表时间：
1991
影响因子：
5.3
通讯作者：
vanderPloeg,LH
中科院分区：
文献类型：
--
作者：
Huang,J;vanderPloeg,LH
原生动物布鲁氏锥虫的许多蛋白质编码基因排列在串联阵列中，并以多链式转录。前mrna转录本通过转剪接处理，导致添加一个带帽的39个核苷酸（nt）的小外显子，并通过poly(a)添加。我们希望确定hsp70位点上RNA加工事件的顺序，并解决可能发生的共转录RNA加工。我们测定了离体细胞核中hsp70位点的转录延伸率，其测量值在20到40 nt/min之间。这种低的RNA链延伸率使我们能够用一个短的（约180-nt）32P尾巴标记hsp70新生RNA的3 '端。标记的nascenthsp70RNA的结构可以通过RNase T和RNase Tj/RNase A作图分析。我们发现hsp70pre- mrna的转剪接并没有在3 ‘剪接受体位点合成后立即发生，并且在3 ’剪接受体位点以外含有约550 nt RNA的新生RNA分子仍然没有获得小外显子。相比之下，可以检测到具有5 '端映射到hsp70基因聚腺苷化位点的新生RNA，这表明锥虫中pre-mRNA的成熟涉及到对新生sp70rna的快速切割（在合成该位点后的几秒钟内）以添加poly(a)。我们的数据表明，多顺反子前mrna不太可能全部合成，而似乎是通过切割多聚(A)添加进行共转录处理。
Numerous protein-coding genes of the protozoanTrypanosoma bruceiare arranged in tandem arrays that are transcribed poly cist ronically. The pre-mRNA transcripts are processed bytranssplicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at thehsp70locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at thehsp70locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3′ end ofhsp70nascent RNA with a short (about 180-nt)32P tail. The structure of the labeled nascenthsp70RNA could then be analyzed by RNase T, and RNase Tj/RNase A mapping. We show that thetranssplicing ofhsp70pre-mRNA did not occur immediately after the synthesis of the 3′ splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3′ splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5′ end that mapped to the polyadenylation site of thehsp70genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascenthsp70RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly (A) addition.