Prediction And Identification Of Consensus Elements From Human Naturally Intronless MRNAs That Promote The Export Of MRNA

Background: mRNA nuclear export has been the focus and challenging point of RNA research.Recent study shows that mRNAs contain specific cis-acting elements that permit their efficient export to the cytoplasm.In higher organisms,mRNA nuclear export always couple with splicing process.During mRNA splicing,splicing complex can recruit the Nuclear transport complex directly and complete the living process of splicing and export together.Intron deletion can prevent the splicing process and block the mRNAs in the nucleus which can not complete the nuclear export process.Thus intron can act as cis-acting elements of export-mechanism in these mRNAs.Natural intronless mRNA only account for the 5% of the total mRNA,including important gene like IFN?histone et al.The natural intronless mRNA can not carry out the Splicing process due to deficient intron.So are there also contain the cis-acting elements for export in natural intronless mRNAs.Our lab previously study showed that the CAR-E(cytoplasmic accumulation region-element)that found in four natural intronless mRNAs(HSPB3?IFN?1?IFN?1,?c-Jun)can promote ?-globin c DNA export into cytoplasm.So,whether these genes contain the same cis-acting element in the other natural intronless mRNAs? Which protein complex is natural intronless mRNA used for the nuclear export? Up to the current research of natural intronless mRNA,no answer has been given for the above questions.Methods: 1.Use the bioinformatics method to analyze the database of natural intronless mRNA,and try to find their potential common fragments.2.Recombine the common fragments and ?-globin c DNA,and search for the fragments which can promote the process of nuclear export.3.Use the Bioinformatics software FIMO to anti-Retrieval the database of natural intronless mRNA.Find the natural intronless mRNA with the cis-acting elements which can promote c DNA nuclear export.Modify it at the gene level to test if it can lead to the promotion of nuclear export in the natural intronless mRNA.4.Construct stable cell line by Flp-In system.And through the MS2-MBP protein purification system and mass spectrometry method determine the role of cis-acting element binding proteins.Results: We have found that natural intronless mRNAs contain several common motifs by comparing and analyzing 679 natural intronless mRNAs data using bioinformatics software MEME.The functional experiment indicated that 4 motifs can promote c DNA nuclear export.We named motif 1-AS?motif 1v-AS?motif 4-AS?motif 5.There are 9 motif 1v-AS in the KRN1 gene,which is a natural intronless mRNA.And we show the 6 closely elements as a whole cis-acting element and design CAR-C.Molecular cloning method is carried out for deleting and modifying the motifs.The results of FISH indicated that,after deletion and mutation,the KRN1 mRNA blocked in the nucleus.After completion of construction of stable cell line,we achieve the capture of RNP by the MS2-MBP combined method,and observe the existence of distinct stripes by silver stain.Conclusion: The experimental data indicated that the predicted motifs can not only promote c DNA nuclear export process,but also influence the nuclear transport process in the natural intronless gene.