Perry K L, Watkins K P, Agabian N
School of Public Health, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1987 Dec;84(23):8190-4. doi: 10.1073/pnas.84.23.8190.
A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.
用核糖核酸酶T2消化布氏锥虫的多聚腺苷酸（poly(A)+）RNA后，会释放出一个单帽寡核苷酸。这一观察结果支持了所有布氏锥虫信使核糖核酸（mRNA）共享一个共同前导序列的假说。用核苷酸焦磷酸酶消化对T2有抗性的物种表明，帽核苷酸是7-甲基鸟苷5'-单磷酸（pm7G）。对T2抗性片段的进一步表征表明，在前四个转录核苷酸上存在修饰；因此，布氏锥虫mRNA的5'末端可被描述为“帽4”结构。在布氏锥虫剪接前导序列（SL）RNA上发现了相同的5'-帽结构；这一观察结果与小SL RNA作为mRNA的SL供体的假说相符。然而，我们发现，在纯化的SL RNA群体中，存在一些被加帽但修饰不完全的物种。这些未修饰和部分修饰物种的存在使我们能够分析SL RNA转录本的5'序列。结果表明，转录从报道的5'端上游四个核苷酸处开始。因此，布氏锥虫SL转录本实际上长39个核苷酸，而不是35个核苷酸。我们还分析了一种远缘锥虫——巨大细滴虫（Leptomonas collosoma）的加帽寡核苷酸，发现它与布氏锥虫的相同。根据锥虫基因表达的观察结果，讨论了这些结果的潜在意义。
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