Mechanism Of Trf4-1 In MRNA And MiRNA Transcriptional Control In Drosophila Melanogaster

The reported Trfs that varied from yeast to Drosophila and mammals were defined as poly(A)polymerase or terminal nucleotide transferase(PAP/TNTase).They could modify the terminus of RNAs,of which basely were uridylation or adenylation.Those PAP/TNTases mainly modified the mRNA,pre-miRNA,miRNA and nuclearlized snRNA?The modification of poly-oligonucleotion would caused the modified RNA decay by recruiting the associated 3'-5' exonucleases,like SDN1 in plant,Dis3l2 in mammals and RRP6 in Drosophila.The mononuleodytion of specified RNAs was indicated to modify the special structural RNA that may own 1nt loss at its 3' terminus,like pre-miRNA and miRNA.Their 3' termimus shared 2 nt overhang which was satisfied for their normal functions.Trf was firstly founded in yeast.In addition to the reported PAP/TNTase activity,other functions were uncovered in previous researches.Firstly,Trf4 worked in the mitotic events of chromosome condensation,spindle elongation,and nuclear segregation.Besides,Trf4 and its redundant homologue Trf5 were reported to associate with the chromosomes,by which to possess DNA polymerase activity(DNA polymerase ?)in vitro.Furthermore,yeast Trf4 mutant showed a transcriptiondependent hyperrecombination phenotype mediated by R-loops,but transcription seemed not to be affected.In Drosophila melanogaster,Trf4-1 was isolated based on the homologues comparison with Trf4 in yeast and named as DmTrf4-1.Trf4-1 which hold the PAP/TNTase domain exhibited the activity in vitro,while its homolog DmTrf4-2 did not.DmTrf4-1 was firstly identified in the nucleus and involved in the polyadenylation-mediated degradation of snRNAs in vivo by which to assist the DmRRP6.Interstingly,another study deduced that DmTrf4-1 located in the cytoplasm rather than the nucleus,which could promote the mRNA decay in the cytoplasm.Taken these two researches togother,it was concluded that Trf4-1 could localize in both cytoplasm and nuleus.To ignore their location,the function of Trf4-1 related to its PAP/TNT catalytic activity.In our work,knock down of Trf4-1 caused the mRNA level downregulated.The positive control of Trf4-1 on mRNA indicated that it could protect or help mRNA from downgrade with different mechanisms.With the indication of GRO(global running on)assay,Trf4-1 regualted the nascent mRNA positively.We also got a conclusion that SAIA mutant worked similarly as wildtype Trf4-1 with the rescue assay.It is indicated that PAP/TNT active domain was not necessary for mRNA positive regulation.The overexpressed SAIA mutant in S2 cells could also get the mRNA upregulation.Taken together,the positive control of Trf4-1 on mRNA did not depend on its PAP/TNTase activity,Trf4-1 may affect mRNA transcrion.Primary miRNA transcripts(pri-miRNAs)contained similar structures as mRNA and were also transcribed by pol ?.In our experiment,Trf4-1 worked upstream of Drosha clevage and positively regulated pri-miRNA expression level as well.Knocked down of Trf4-1 resulted in decreasing of primiRNA biogenesis and downregution of mature miRNA.With the analysis of trancriptom sequencing and small RNA sequencing,Trf4-1 could worked globally in mRNA and miRNA transcription.Trf4-1 affected the translational core protein pol ? indirectly and by which caused pol ? stall at any region of chromatin and abnormal accumulation.The abnormal accumulation of pol ? provoked the hijack and cut down the turn over of pol ?.Otherwise,H3K4me3 show lower bound at the promoter region when Trf4-1 was knocked down.However,western blot did not indicat the effect of Trf4-1 on the stability of these protein or their modifications.There was not any direct binding with the chromatin dectected in the HA-Trf4-1-ChIP(Chromatin immunoprecipitation)assay.The co-immunoprecipitation-mass spectrum(I.P.-M.S.)assay had help us find considerable transcriptional associated proteins,nucleic localizied proteins and ribosomal proteins,especially the H2 A.V protein in the varying nucleosome.The homolog in mammals,PAPD5,had been found in our last work that it could also mediate mRNA transcription and I.P.-M.S.assay got similar result with DmTrf4-1.Above all,we concluded that Trf4-1 could control mRNA/miRNA transcription via affecting the function of pol ? and related histones.