title: Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH.
abstract: Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-β CH(2) group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the β-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the β-phosphate with BH(3) or Se, or substituted at either the α-β or β-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m(2)(7,2'-O)Gpp(S)pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m(7)Gpp(BH3)pm(7)G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t(1/2) ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.
authors: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
authors: Wei
authors: W
authors: Su
authors: Sergey
authors: S
authors: Slepenkov
authors: Ewa
authors: E
authors: Grudzien-Nogalska
authors: Joanna
authors: J
authors: Kowalska
authors: Marta
authors: M
authors: Kulis
authors: Joanna
authors: J
authors: Zuberek
authors: Maciej
authors: M
authors: Lukaszewicz
authors: Edward
authors: E
authors: Darzynkiewicz
authors: Jacek
authors: J
authors: Jemielity
authors: Robert E
authors: RE
authors: Rhoads
mesh_terms: D000818:Animals; D001888:Boric Acids / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D017672:Nitrogen Compounds / Q000378:metabolism*; D011122:Polyphosphates / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D012315:RNA Caps; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry; D012643:Selenium / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D001888:Boric Acids; D017672:Nitrogen Compounds; D011122:Polyphosphates; D012315:RNA Caps; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004722:Endoribonucleases; C467723:DCP2 protein, human; D012643:Selenium; C005692:triphosphoric acid; C032688:boric acid