TheaimofourproposalistoelucidatethemechanismsthatlinktranscriptionofspecificRNAsinthe nucleustotheirtranslation(RNAprocessing).?Wehaveidentifiedandcharacterizedanovelandhighly conservedgene,?Zfrp8/PDCD2,?andshownthatitisessentialinstemcellsinflies,mouse,andhuman,and thatitisalsorequiredforgrowthofcancercells.Wediscovered?Zfrp8/PDCD2?isrequiredforthenuclearexport ofselectmRNAsandTEtranscripts.Alsoitinteractswiththesmallribosomalsubunitandformsacomplex withmRNAbindingproteins.OurdatasuggestthatZfrp8/PDCD2controlssubcellularlocalizationofselect RNAsandtheassociationofmRNARNPswithribosomes.WealsohaveidentifiedTet/TET1asanew Zfrp8/PDCD2interactingprotein.Invertebrates,TETproteinsfunctioninDNAdemethylationconverting 5methylcytosine(5mC)into5hydroxymethylcytosine(5hmC),modificationsthatarenotdetectedin DrosophilaDNA.ArecentstudyshowsthatvertebrateTetproteinscanalsoconvert5mrCto5hmrCinRNA. Inspiredbythisdiscovery,wehaveshownthat5hmrCalsoexistsinfliesanddependsonTetactivity.We hypothesizethatTetmodifiesspecifictranscriptsandregulatestherecruitmentofZfrp8totheseRNAs,so controllingtheirprocessingandtranslation. Weproposetotestthishypothesisbyacombinationofmolecular/biochemicalandgenetic experiments.Wewillidentify5hmrCmodifiedtranscriptstranscriptomewideandstudytheirintegrity,levels, andlocalizationinwildtypeandmutanttissues.WewillmaptheTetbindingsitesonDNAandcomparetheir locationto5hmrCmodifiedtranscripts.Finally,wewilltesthow?Tet?and?Zfrp8?affectribosomaloccupancyof mRNAsandestablishhow?Tet?and5hmrCaffecttheirtranslation.Inadditiontoinvestigatingthemechanismby which?Tet?and5hmrCregulateRNAmetabolismandhowZ?frp8?affectstheprocess,wewilldeterminethe importanceofbothgenesindevelopmentandstemcelldifferentiationbystudyingthemutantphenotypesof new?Tet?allelesand?Tet?and?Zfrp8KD?tissues.BecauseoftheconservationofbothTetandZfrp8/PDCD2our resultsarelikelytoshedlightonthesameprocessinmouseandhuman.
We propose a study of the link between the transcription of the RNA in the nucleus and the mechanism that selects some of these RNAs for protein translation in the cytoplasm. We study two proteins that control these steps. Absence of these proteins results in an arrest of cell divisions in stem cells, and is also associated with infertility, neurological defects, and cancer.
| Wang, Fei; Minakhina, Svetlana; Tran, Hiep et al. (2018) Tet protein function during Drosophila development. PLoS One 13:e0190367 |
