Ethanol destabilizes liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase, mRNA by depleting a 3'-untranslated region-specific binding protein.
Asialoconjugates are viable biomarkers for alcohol abuse. We previously showed that chronic ethanol feeding down-regulated liver Gal beta l, 4GlcNAc alpha2,6-sialyltransferase (ST6Gal l) 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 l mRNA. Using (32)P-labeled RNA probes generated from a 2.7-kb 3'-UTR of ST6Gal l 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 l 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 nucleotides, AG and TC, among the consensus sequences were critical for the RNA-protein interaction. Therefore, 5'-CAGCCTCCTCCCT-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 l mRNA may account for its destabilization and consequent appearance of asialoconjugates as alcohol biomarkers.
Garige
Mamatha
M
Department of Biochemistry, George Washington University, Washington, DC, USA.
Gong
Maokai
M
Lakshman
M Raj
MR
D020413:3' Untranslated Regions / Q000737:chemistry*; D000818:Animals; D051152:Clusterin / Q000097:blood; D000431:Ethanol / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D019158:N-Acetylneuraminic Acid / Q000097:blood; D020871:RNA Stability / Q000187:drug effects*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012799:Sialyltransferases / Q000235:genetics*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D020413:3' Untranslated Regions; C498500:Clu protein, rat; D051152:Clusterin; D016601:RNA-Binding Proteins; D000431:Ethanol; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase