title: Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.
abstract: Since DNA and histone levels must be closely balanced for cell survival, histone expressions are highly regulated. The regulation of replication-dependent histone expression is mainly achieved at the mRNA level, as the mRNAs are rapidly removed when DNA replication is inhibited during S-phase. Histone mRNA degradation initiates with addition of multiple uridines (oligouridylation) following the 3' stem-loop (SL) catalyzed by terminal uridyltransferase (TUTase). Previous studies showed that histone mRNA degradation occurs through both 5' → 3' and 3' → 5' processes, but the relative contributions are difficult to dissect due to lack of established protocols. The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by structural modifications at the both 5' and 3' termini. In this chapter, we present methods of utilizing modified cap dinucleotide analogs to block 5' → 3' degradation of a reporter mRNA containing canonical histone mRNA 3' SL and monitoring how oligouridylation and 3' → 5' degradation occur. Protocols are presented for synthesis of reporter mRNA containing the histone 3' SL and modified cap analogs, monitoring mRNA stability and unidirectional degradation either from 5' or 3' termini, and detection of oligo(U) tracts from degradation products by either traditional or deep sequencing.
authors.affiliation: Department of Neurology, University of Washington, Seattle, WA, 98195, USA. wsu2@uw.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Su
authors.affiliation: Good Start Genetics, 237 Putnam Ave, Cambridge, MA, 02139, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Slevin
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: William F
authors.initials: WF
authors.lastname: Marzluff
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Rhoads
mesh_terms: D006367:HeLa Cells; D006657:Histones / Q000235:genetics*; D006801:Humans; D009843:Oligoribonucleotides / Q000378:metabolism; D014176:Protein Biosynthesis; D012314:RNA Cap Analogs / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D058615:Synthetic Biology; D014500:Uracil Nucleotides / Q000378:metabolism
chemical_list: D006657:Histones; D009843:Oligoribonucleotides; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D014500:Uracil Nucleotides; C027916:oligo(U); D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase
keywords: Cap analogs; Deep sequencing; Nucleoporation; Oligourdiylation; mRNA stability