Differential response to RNA trans-splicing signals within the phosphoglycerate kinase gene cluster in Trypanosoma brucei.
Differential response to RNA trans-splicing signals within the phosphoglycerate kinase gene cluster in Trypanosoma brucei.
复制标题
布氏锥虫磷酸甘油酸激酶基因簇内对 RNA 转拼信号的差异反应。
DOI：
10.1093/nar/21.17.4067
复制
发表时间：
1993
影响因子：
14.9
通讯作者：
Bellofatto,V
中科院分区：
文献类型：
--
作者：
Kapotas,N;Bellofatto,V
在锥虫中，核前体mRNA剪接仅为反式剪接反应，其中加帽的39 nt外显子（迷你外显子）位于开放阅读框的5'端。RNA的差异剪接可能反映了小外显子和3'剪接位点的相互作用。为了检验这一假设，我们比较了小外显子添加到位于单个前mRNA转录物内的三个天然3'剪接受体位点（SAS）的效率。在布氏锥虫中，磷酸甘油酸激酶A、B和C基因（PGKA、B和C）共表达为多顺反子前mRNA上的三个连续序列。该前mRNA产生不等量的PGK A、B和C mRNA。当将来自每个基因的SAS置于荧光素酶开放阅读框的上游，并将所得构建体瞬时转染入T.在布鲁氏原环细胞中，荧光素酶活性水平指示差异的SAS利用。当来自A基因的SAS存在时，酶活性低。当比较B和C SAS时，水平无法区分。在测试构建体中用氯霉素乙酰转移酶替换荧光素酶后，酶活性显示与mRNA量直接相关。因此，差的剪接效率解释了PGK前mRNA成熟期间PGK A mRNA的差异表达。该反应似乎反映了3'剪接受体位点内的聚嘧啶模式。
In trypanosomatlds, nuclear pre-mRNA splicing is exclusively atrans-spliclng reaction in which a capped, 39 nt exon, the mini-exon, is positioned 5' to an open reading frame. Differential RNA splicing might reflect specific mini-exon and 3' splice site Interactions. To test this hypothesis, we compared the efficiency of mini-exon addition to three natural 3' splice acceptor sites (SASs) located within a single pre-mRNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive sequences on a polycistronic pre-mRNA. This pre–mRNA gives rise to unequal amounts of PGK A, B and C mRNAs. When the SAS from each gene was placed upstream of the luciferase open reading frame and the resultant constructs transiently transfected Into T. brucei procyclic cells, luciferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels were indistinguishable when the B and C SASs were compared. After replacing luciferase with chloramphenicol acetyl transferase in the test constructs, enzyme activities were shown to directly correlate with mRNA amounts. Thus, poor splicing efficiency accounts for the differential expression of the PGK A mRNA during PGK pre-mRNA maturation. This reaction appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.