Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei
Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei
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DOI：
10.1016/j.molbiopara.2006.11.003
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发表时间：
2007-02-01
影响因子：
1.5
通讯作者：
Clayton, Christine
中科院分区：
文献类型：
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作者：
Colasante, Claudia;Robles, Ana;Clayton, Christine
在布氏锥虫中，编码磷酸甘油酸激酶的PGKB和PGKC基因共转录为多顺反子RNA的一部分。PGKB mRNA和胞质PGKB蛋白在前循环生命周期阶段比在血流形式中丰富得多，而PGKC mRNA和糖体PGKC蛋白对血流形式具有特异性。我们在这里表明，在PGKC mRNA的3 '-非翻译区的核苷酸558和779之间的序列导致低表达的氯霉素乙酰转移酶（CAT）报告基因在前环锥虫。在顺环化合物中，外泌体的RRP 45组分（3' -> 5'外切核酸酶复合物）或5' -> 3'外切核酸酶XRNA的消耗增加了CAT-PGKC mRNA的丰度，这是由于对前体和/或成熟mRNA的降解的影响。在血流形式中，反式剪接和转录的抑制导致PGKC mRNA的立即指数衰减，半衰期为46分钟。单独抑制转录产生非指数动力学，单独抑制剪接导致较长的表观半衰期。我们还发现，使用T7聚合酶生产mRNA可以影响表观半衰期，并且大量的CAT酶可能对锥虫有毒。(c)2006 Elsevier B. V.保留所有权利。
In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3'-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3' -> 5' exonuclease complex) or the 5' -> 3' exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes. (c) 2006 Elsevier B.V. All rights reserved.