Mauchi Naoko, Ohtake Yoshiaki, Irie Kenji
Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tennoudai, Tsukuba, Japan.
Cell Struct Funct. 2010;35(2):95-105. doi: 10.1247/csf.10011. Epub 2010 Oct 13.
Khd1p (KH-domain protein 1) is a yeast RNA-binding protein highly homologous to mammalian hnRNP K. Khd1p associates with hundreds of potential mRNA targets including a bud-localized ASH1 mRNA and mRNAs encoding membrane-associated proteins such as Mid2p and Mtl1p. While Khd1p negatively regulates gene expression of Ash1p by translational repression, Khd1p positively regulates gene expression of Mtl1p by mRNA stabilization. To investigate how Khd1p regulates the stability of MTL1 mRNA, we searched for cis-acting elements and trans-acting factors controlling MTL1 mRNA stability. Regional analysis revealed that partial deletion of the coding sequences of MTL1 mRNA restored the decreased MTL1 mRNA and protein levels in khd1Δ mutants. This region, encompassing nucleotides 532 to 1032 of the Mtl1p coding sequence, contains CNN repeats that direct Khd1p-binding. Insertion of this sequence into other mRNAs conferred mRNA instability in khd1Δ mutants. We further searched for factors involved in the destabilization of MTL1 mRNA. Mutations in CCR4 and CAF1/POP2, encoding major cytoplasmic deadenylases, or of SKI genes, which code for components of a complex involved in 3' to 5' degradation, did not restore the decreased MTL1 mRNA levels caused by khd1Δ mutation. However, mutations in DCP1 and DCP2, encoding a decapping enzyme complex, and XRN1, encoding a 5'-3' exonuclease, restored the decreased MTL1 mRNA levels. Furthermore, Khd1p colocalized with Dcp1p in processing bodies, cytoplasmic sites for mRNA degradation. Our results suggest that MTL1 mRNA bears a cis-acting element involved in destabilization by the decapping enzyme and the 5'-3' exonuclease, and Khd1p stabilizes MTL1 mRNA through binding to this element.
Khd1p（KH结构域蛋白1）是一种酵母RNA结合蛋白，与哺乳动物的hnRNP K高度同源。Khd1p与数百个潜在的mRNA靶标相关联，包括芽定位的ASH1 mRNA以及编码膜相关蛋白（如Mid2p和Mtl1p）的mRNA。虽然Khd1p通过翻译抑制对Ash1p的基因表达起负调控作用，但Khd1p通过mRNA稳定对Mtl1p的基因表达起正调控作用。为了研究Khd1p如何调节MTL1 mRNA的稳定性，我们寻找了控制MTL1 mRNA稳定性的顺式作用元件和反式作用因子。区域分析表明，MTL1 mRNA编码序列的部分缺失恢复了khd1Δ突变体中降低的MTL1 mRNA和蛋白质水平。该区域包含Mtl1p编码序列的第532至1032个核苷酸，含有指导Khd1p结合的CNN重复序列。将该序列插入其他mRNA会导致khd1Δ突变体中的mRNA不稳定。我们进一步寻找参与MTL1 mRNA去稳定化的因子。编码主要细胞质脱腺苷酸酶的CCR4和CAF1/POP2或编码参与3'至5'降解的复合物组分的SKI基因中的突变，并未恢复由khd1Δ突变引起的降低的MTL1 mRNA水平。然而，编码去帽酶复合物的DCP1和DCP2以及编码5'-3'核酸外切酶的XRN1中的突变恢复了降低的MTL1 mRNA水平。此外，Khd1p与Dcp1p在加工体（mRNA降解的细胞质位点）中共定位。我们的结果表明，MTL1 mRNA带有一个顺式作用元件，参与由去帽酶和5'-3'核酸外切酶介导的去稳定化，并且Khd1p通过与该元件结合来稳定MTL1 mRNA。
