title: Translation attenuation via 3' terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk.
abstract: The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5' and 3' UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5' and 3' UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3' terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.
authors.affiliation: a Animal and Poultry Science; University of Guelph ; Guelph , Ontario , Canada.
authors.forename: Julie J
authors.initials: JJ
authors.lastname: Kim
authors.forename: Jaeju
authors.initials: J
authors.lastname: Yu
authors.forename: Jnanankur
authors.initials: J
authors.lastname: Bag
authors.forename: Marica
authors.initials: M
authors.lastname: Bakovic
authors.forename: John P
authors.initials: JP
authors.lastname: Cant
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000096:biosynthesis / Q000235:genetics*; D002417:Cattle; D002474:Cell-Free System / Q000378:metabolism; D003062:Codon / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D008892:Milk / Q000737:chemistry*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011132:Polyribosomes / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D002364:Caseins; D003062:Codon; D020033:Protein Isoforms
keywords: 40S, small ribosomal subunit; 60S, large ribosomal subunit; AA, amino acid; ARE, AU-rich element; Apaf-1, apoptosis protease activating factor 1; DLG1, disc large 1 ncosuppressor; FMR1, fragile X mental retardation 1; HRP, horseradish eroxidase; IE, inhibitory element; IRE, iron-responsive element; IRES, nternal ribosome entry site; IRP, iron-regulatory protein; MACT, bovine mammary epithelial cell; PABP, poly(A) binding protein; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PVDF, polyvinylidene fluoride; RACE, rapid amplification of cDNA ends; RBP, RNA-binding protein; RRL, rabbit reticulocyte lysate; RT, reverse transcription; SDS, sodium dodecyl sulfate; SE, standard error; STR, single-stranded nucleic acid binding protein; TBS-T, Tris-buffered saline containing 0.5%; TfR, transferrin receptor; Tween 20; UTR, untranslated region; aa-tRNA, aminoacyl-tRNA; aaRS, aminoacyl-tRNA synthetase; bovine casein; cDNA, complementary DNA; cell-free translation; coding region; codon usage; eEF, eukaryotic elongation factor; eIF, eukaryotic initiation factor; eRF, eukaryotic termination factor; m7G, 7-methylated uanidine; mRNA, messenger RNA; qPCR, real-time polymerase chain reaction; sAUG, start codon; tRNA, transfer RNA; translational efficiency; uAUG, upstream start codon; uORF, open reading frame; untranslated region; ΔG, free energy
references.citation: RNA. 2006 May;12(5):851-61
references.pmid: 16540693
references.citation: Trends Genet. 2012 Nov;28(11):574-81
references.pmid: 22921354
references.citation: Reprod Nutr Dev. 2006 Sep-Oct;46(5):567-78
references.pmid: 17107646
references.citation: Nat Genet. 2007 Mar;39(3):415-21
references.pmid: 17277776
references.citation: J Chromatogr A. 2007 Mar 30;1146(1):110-7
references.pmid: 17320885
references.citation: Curr Opin Biotechnol. 2007 Apr;18(2):163-9
references.pmid: 17292602
references.citation: J Dairy Res. 2007 Aug;74(3):356-66
references.pmid: 17655779
references.citation: Mol Biol Cell. 2007 Dec;18(12):5004-13
references.pmid: 17928403
references.citation: Bioinformatics. 2008 Feb 1;24(3):367-73
references.pmid: 18056736
references.citation: Nucleic Acids Res. 2008 Feb;36(3):861-71
references.pmid: 18086709
references.citation: Protein Expr Purif. 2008 May;59(1):94-102
references.pmid: 18289875
references.citation: J Dairy Sci. 2009 Apr;92(4):1338-53
references.pmid: 19307615
references.citation: Cell. 2010 Apr 16;141(2):227-9
references.pmid: 20403320
references.citation: Cell. 2010 Apr 16;141(2):344-54
references.pmid: 20403328
references.citation: Cell. 2010 Apr 16;141(2):355-67
references.pmid: 20403329
references.citation: Genes Dev. 2010 Oct 1;24(19):2146-56
references.pmid: 20837656
references.citation: Nat Rev Genet. 2011 Jan;12(1):32-42
references.pmid: 21102527
references.citation: Mol Biol Evol. 2011 Jul;28(7):2053-61
references.pmid: 21245413
references.citation: Nature. 2011 Jul 7;475(7354):118-21
references.pmid: 21734708
references.citation: J Control Release. 2011 Aug 10;153(3):206-16
references.pmid: 21338636
references.citation: Genes Dev. 2000 May 15;14(10):1236-48
references.pmid: 10817758
references.citation: J Nutr. 2000 Jul;130(7):1865S-7S
references.pmid: 10867064
references.citation: Science. 2001 Sep 7;293(5536):1832-6
references.pmid: 11546873
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Genome Biol. 2002;3(3):REVIEWS0004
references.pmid: 11897027
references.citation: Genet Sel Evol. 2002 Jul-Aug;34(4):509-19
references.pmid: 12270107
references.citation: Reprod Nutr Dev. 2002 Sep-Oct;42(5):433-59
references.pmid: 12537255
references.citation: J Mammary Gland Biol Neoplasia. 2002 Jul;7(3):327-45
references.pmid: 12751895
references.citation: Curr Pharm Des. 2003;9(16):1309-23
references.pmid: 12769739
references.citation: Nucleic Acids Res. 2003 Jun 15;31(12):3174-84
references.pmid: 12799445
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: Nucleic Acids Res. 2013 Feb 1;41(4):2073-94
references.pmid: 23293005
references.citation: Biochim Biophys Acta. 2006 Mar-Apr;1759(3-4):132-40
references.pmid: 16714065
references.citation: Trends Biotechnol. 2004 Jul;22(7):346-53
references.pmid: 15245907
references.citation: J Biol Chem. 1972 Nov 10;247(21):6770-80
references.pmid: 5082126
references.citation: Nucleic Acids Res. 1984 Jan 25;12(2):857-72
references.pmid: 6694911
references.citation: Nucleic Acids Res. 1987 Oct 26;15(20):8125-48
references.pmid: 3313277
references.citation: Mol Biol Evol. 1987 May;4(3):231-41
references.pmid: 2833669
references.citation: J Dairy Sci. 1988 Jan;71(1):24-8
references.pmid: 3372804
references.citation: FASEB J. 1989 Oct;3(12):2360-70
references.pmid: 2676679
references.citation: Am J Clin Nutr. 1990 Jan;51(1):37-46
references.pmid: 1688683
references.citation: J Dairy Sci. 1993 Jun;76(6):1645-53
references.pmid: 8326033
references.citation: Am J Physiol. 1994 Jul;267(1 Pt 1):C115-26
references.pmid: 8048474
references.citation: Gene. 1994 Sep 30;147(2):179-87
references.pmid: 7926797
references.citation: Mol Cell Biol. 1994 Nov;14(11):7322-30
references.pmid: 7935446
references.citation: EMBO J. 1995 Jan 3;14(1):151-8
references.pmid: 7828587
references.citation: Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5431-5
references.pmid: 7777525
references.citation: J Dairy Sci. 1996 Oct;79(10):1781-91
references.pmid: 8923249
references.citation: EMBO J. 1996 Dec 16;15(24):7147-55
references.pmid: 9003790
references.citation: J Dairy Res. 1997 May;64(2):207-19
references.pmid: 9161914
references.citation: Physiol Rev. 1997 Jul;77(3):643-69
references.pmid: 9234961
references.citation: Gene. 1997 Dec 31;205(1-2):95-102
references.pmid: 9461382
references.citation: Nucleic Acids Res. 2011 Aug;39(14):6201-12
references.pmid: 21486747
references.citation: Genome Biol. 2011;12(2):R12
references.pmid: 21284851
references.citation: J Dairy Sci. 2011 Nov;94(11):5691-4
references.pmid: 22032393
references.citation: Nutr Rev. 2012 Apr;70(4):241-55
references.pmid: 22458697
references.citation: PLoS Genet. 2012;8(3):e1002603
references.pmid: 22479199
references.citation: Mol Biol Evol. 2013 Oct;30(10):2263-7
references.pmid: 23883522