Das Biswadip, Das Satarupa, Sherman Fred
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Proc Natl Acad Sci U S A. 2006 Jul 18;103(29):10871-6. doi: 10.1073/pnas.0604562103. Epub 2006 Jul 10.
We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Delta or cbc2-Delta mutants lacking components of the cap-binding complex and in rrp6-Delta mutants lacking Rrp6p, a 3' to 5' nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Delta and rrp6-Delta. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Delta and rrp6-Delta. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this "retention signal" causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.
我们之前证明，保留在酿酒酵母细胞核中的mRNA会受到一种名为DRN（细胞核中mRNA降解）的降解系统的作用，该系统在缺乏帽结合复合体成分的cbc1-Δ或cbc2-Δ突变体以及缺乏3'至5'核外切核酸酶Rrp6p的rrp6-Δ突变体中会减弱。通过筛选众多lys2突变体以寻找cbc1-Δ和rrp6-Δ的抑制作用，发现了两个突变体lys2-187和lys2-121。这两个突变体是相同的，都包含两个碱基变化，其中一个形成了TGA无义密码子。来自lys2-187及相关突变体的LYS2 mRNA会迅速降解，并且这种降解会被cbc1-Δ和rrp6-Δ抑制。使用U1A-GFP成像程序表明lys2-187 mRNA部分保留在细胞核中，这解释了其对DRN的敏感性。通过定点诱变创建lys2-187的几个衍生物表明，框内TGA本身并不是导致对DRN敏感性增加的原因。因此，对DRN敏感的mRNA可以由2个碱基的变化形成。此外，通过改变其中一个碱基对会丢失这种导致对DRN敏感的“保留信号”，这表明对DRN敏感和不敏感的mRNA可归因于适当背景下的单个核苷酸。
