title: Upstream open reading frame in 5'-untranslated region reduces titin mRNA translational efficiency.
abstract: 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.
authors.affiliation: Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN, United States; Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN, United States.
authors.forename: Adrian G
authors.initials: AG
authors.lastname: Cadar
authors.affiliation: Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN, United States.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhong
authors.affiliation: Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN, United States.
authors.forename: Angel
authors.initials: A
authors.lastname: Lin
authors.affiliation: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, United States.
authors.forename: Mauricio O
authors.initials: MO
authors.lastname: Valenzuela
authors.affiliation: Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN, United States; Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN, United States. Electronic address: chee.lim@vanderbilt.edu.
authors.forename: Chee C
authors.initials: CC
authors.lastname: Lim
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D064211:Connectin / Q000096:biosynthesis / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D016366:Open Reading Frames; D020641:Polymorphism, Single Nucleotide; D011494:Protein Kinases / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
chemical_list: D020121:5' Untranslated Regions; D064211:Connectin; D012333:RNA, Messenger; C577285:TTN protein, human; C577295:Ttn protein, rat; D004317:Doxorubicin; D011494:Protein Kinases; C579730:titin protein, mouse
keywords: 5′-UTR; Cardiomyocytes; Titin; Translation; Ttn; uORF