Ethanol Destabilizes Liver Gal beta 1,4GlcNAc alpha 2,6-Sialyltransferase,mRNA by Depleting a 3'-Untranslated Region-Specific Binding Protein

摘要

Asialoconjugates are viable biomarkers for alcohol abuse.We previously showed that chronic ethanol feeding down-regulated liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase(ST6Gal 1)mRNA by destabilizing it.Since RNA-binding proteins are known to stabilize many eukaryotic mRNAs by interacting with the 3'-untranslated region(UTR),we have delineated the possible mechanism by which ethanol destabilizes ST6Gal 1 mRNA.Using ~(32)P-labeled RNA probes generated from a 2.7-kb 3'-UTR of ST6Gal 1 mRNA,we identified a liver cytosolic 41-kDa specific binding protein that interacts with its 3'-UTR domain and protects it from degradation in normal rat liver but disappears after chronic ethanol treatment.Mapping of the binding region revealed that four RNA probes of 80-base pair(bp)length spanning the 304 bp of the 3'-UTR of ST6Gal 1 mRNA showed equal binding intensity.The corresponding cDNA sequences for the four 80-bp RNA probes share the 13-bp consensus sequence.Mutagenesis analysis identified that four nucleo-tides,AG and TC,among the consensus sequences were critical for the RNA-protein interaction.Therefore,5'-CAGCCTC-CTCCCT-3' serves as a cis-element critically involved in this interaction.The RNA-protein complex formation progressively decreased with increasing dietary ethanol,resulting in its virtual disappearance with 36% of the dietary calories as ethanol.Concomitantly,the same ethanol diet decreased sialic acid index of plasma apolipoprotein J by 45%(p < 0.05).Thus,depletion of a binding protein that specifically interacts with its 3'-UTR region of ST6Gal 1 mRNA may account for its destabi-lization and consequent appearance of asialoconjugates as alcohol biomarkers.