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Upstream open reading frame in 5'-untranslated region reduces titin mRNA translational efficiency.

Cadar AG, Zhong L, Lin A, Valenzuela MO, Lim CC
Biochem Biophys Res Commun. 2014 453 (1): 185-91

PMID: 25264194 · PMCID: PMC4250383 · DOI:10.1016/j.bbrc.2014.09.085

Titin is the largest known protein and a critical determinant of myofibril elasticity and sarcomere structure in striated muscle. Accumulating evidence that mRNA transcripts are post-transcriptionally regulated by specific motifs located in the flanking untranslated regions (UTRs) led us to consider the role of titin 5'-UTR in regulating its translational efficiency. Titin 5'-UTR is highly homologous between human, mouse, and rat, and sequence analysis revealed the presence of a stem-loop and two upstream AUG codons (uAUGs) converging on a shared in frame stop codon. We generated a mouse titin 5'-UTR luciferase reporter construct and targeted the stem-loop and each uAUG for mutation. The wild-type and mutated constructs were transfected into the cardiac HL-1 cell line and primary neonatal rat ventricular myocytes (NRVM). SV40 driven 5'-UTR luciferase activity was significantly suppressed by wild-type titin 5'-UTR (∼ 70% in HL-1 cells and ∼ 60% in NRVM). Mutating both uAUGs was found to alleviate titin 5'-UTR suppression, while eliminating the stem-loop had no effect. Treatment with various growth stimuli: pacing, PMA or neuregulin had no effect on titin 5'-UTR luciferase activity. Doxorubicin stress stimuli reduced titin 5'-UTR suppression, while H2O2 had no effect. A reported single nucleotide polymorphism (SNP) rs13422986 at position -4 of the uAUG2 was introduced and found to further repress titin 5'-UTR luciferase activity. We conclude that the uAUG motifs in titin 5'-UTR serve as translational repressors in the control of titin gene expression, and that mutations/SNPs of the uAUGs or doxorubicin stress could alter titin translational efficiency.

Copyright © 2014 Elsevier Inc. All rights reserved.

MeSH Terms (21)

5' Untranslated Regions Animals Base Sequence Cell Line Connectin Doxorubicin Gene Expression Regulation Half-Life HEK293 Cells Humans Mice Molecular Sequence Data Myocytes, Cardiac Open Reading Frames Polymorphism, Single Nucleotide Protein Kinases Rats RNA, Messenger RNA Stability Sarcomeres Sequence Homology, Nucleic Acid

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