Deadenylation-dependent and -independent decay pathways for α1-tubulin mRNA in Chlamydomonas reinhardtii
Deadenylation-dependent and -independent decay pathways for α1-tubulin mRNA in Chlamydomonas reinhardtii
复制标题
DOI：
10.1128/mcb.18.3.1498
复制
发表时间：
1998-03-01
影响因子：
5.3
通讯作者：
Baker, EJ
中科院分区：
文献类型：
--
作者：
Gera, JF;Baker, EJ
莱茵衣藻α-微管蛋白和β-微管蛋白mRNAs在不同条件下呈现不同的半衰期：在组成性表达时，半衰期约为1h，而在偏转诱导下，半衰期仅为10~15min。为了研究在这两种情况下使用的衰变途径(S)，将一个在3‘非翻译区插入30个胍基残基的α1-微管蛋白基因构建到细胞中。该转基因在稳定转化的细胞中得到了高效表达，其表达的半衰期与α1-微管蛋白的半衰期相似。Northern印迹分析显示，从含聚(G)的α1-微管蛋白转录本中出现了一个3‘RNA片段。在放线菌素D处理的细胞中，3‘片段随着全长mRNA的消失而积聚，这表明了前体-产物关系，在第一个多聚(G)区上游插入第二个聚(G)区导致只有一个较长的3’片段积聚，这表明衰变中间产物是由5‘-到-3’外切消化产生的。在环己亚胺存在下细胞被放缩的实验证明了产生3‘片段需要翻译。对片段Poly(A)长度的分析表明，这些片段在未脱胶的细胞中至多是寡腺化的，但在脱胶的细胞中有一条长的Poly(A)尾巴。这些发现表明，寡腺化的片段是依赖于去烯化的结构性降解途径中的衰退中间体，在脱胶的细胞中绕过了去烯化的要求，这是第一个被证明在不同的细胞条件下针对不同的衰退途径的单一转录本的例子。
The alpha- and beta-tubulin mRNAs of Chlamydomonas reinhardtii exhibit different half-lives under different conditions: when expressed constitutively, they degrade with half-lives of about 1 h, whereas when induced by deflagellation, they degrade with half-lives of only 10 to 15 min. To investigate the decay pathway(s) used under these two conditions, an alpha 1-tubulin gene construct which included an insert of 30 guanidylate residues within the 3' untranslated region was introduced into cells. This transgene was efficiently expressed in stably transformed cells, and the mRNA exhibited constitutive and postinduction half-lives like those of the alpha 1-tubulin mRNA. Northern blot analysis revealed the occurrence of a 3' RNA fragment derived from the poly(G)-containing alpha 1-tubulin transcripts. The 3' fragment was shown to accumulate as full-length mRNA disappeared in actinomycin D-treated cells, indicating a precursor-product relationship, Insertion of a second poly(G) tract upstream of the first resulted in accumulation of only a longer 3' fragment, suggesting that the decay intermediate is generated by 5'-to-3' exonucleolytic digestion, A translational requirement for generation of the 3' fragment was demonstrated by experiments in which cells were deflagellated in the presence of cycloheximide. Analysis of fragment poly(A) length revealed that the fragments were, at most, oligoadenylated in nondeflagellated cells but had a long poly(A) tail in deflagellated cells, These findings suggest that the oligoadenylated fragment is a decay intermediate in a deadenylation-dependent, constitutive degradation pathway and that the requirement for deadenylation is bypassed in deflagellated cells, This represents the first example in which a single transcript has been shown to be targeted to different decay pathways under different cellular conditions.