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.
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