| Background: Genetic material is transcribed by DNA in the nucleus to produce mRNA,mRNA translate into proteins and play its biological function after transfer to the cytoplasm.In this process,the transfer of mRNA from the nucleus to the cytoplasm is an indispensable step.As a consequence,the study of mRNA export has always been a hot and difficult point in the field of biology.mRNA export requires the recruitment of splicing complex to remove intron connection exons and then recruit the nuclear transport complex to complete the process of mRNA splicing and export.After the intron of the gene was artificially deleted,the transcribed mRNA could not be successfully recruited and transported out of the nuclear complex.After all,it could not complete the splicing event,resulting in its transcripts being blocked in the nucleus.In recent years,it has been found that there are some cis-acting element and trans-acting factors in mRNA that can facilitate the smooth transport of its transcripts out of the nucleus.However,there are two special types of mRNA,in RNA,one is naturally intronless mRNA,which accounts for 5% of all mRNA.It contains genes that have important functions for organisms,such as interferon,histone and so on.The second type is the circular RNA,formed by special splicing on the basis of mRNA.In recent years,it has also been reported that it is closely related to a variety of diseases.The former does not have a splicing process because it does not contain introns.In the meanwhile,the latter is formed by a special splicing process on the basis of mRNA.Do these two special forms of mRNA also have cis-acting element elements and trans-acting factors that can affect their export?The current research still does not solve these problems.In this paper,we planned to study two special forms of mRNA export mechanism in order to improve it.In the previous study of our laboratory,the bioinformatics analysis of 679 coding regions of the first type of naturally intronless genes was verified by reporter gene level experiments.The element motif1V-AS,which can promote the export of globin c DNA,was found in its common sequence.In the previous experiment,a series of naturally intronless genes containing motif1V-AS elements were found by FIMO inverse search.Apart from the KRN1 gene without introns known in the previous work,are there any cis-acting element original genes that inhibit the export of motif1V-AS elements in other genes containing motif1V-AS elements? In the study of the second kind of circular RNA,the mechanism of its localization and distribution is not clear.Whether the export and nuclear arrest of circular RNA are similar to mRNA,and whether there are specific proteins? In order to solve these problems,34 naturally intronless genes containing motif1V-AS and 5 circular RNA with different lengths were selected as the research objects so as to improve the export mechanism of special mRNA.Methods: We found that the element that can promote the export of globin c DNA was named motif1V-AS,in the previous study of naturally intronless mRNA,356 naturally intronless mRNA were found after FIMO inverse search.In this paper,we intend to further study on this basis: 1)Sequencing the number of motif1V-AS elements in 356 naturally intronless genes containing motif1V-AS elements,selecting34 genes that contain a large number of elements and currently available in our laboratory,and cloning their full length into p CDNA3 vectors.2)RNA fluorescence in situ hybridization(RNA-FISH)was used to detect the location of transient transfection of genes containing motif1V-AS elements in Hela cells.3)The full-length sequences of these genes were truncated and then transfected into Hela cells to detect their localization.Observe whether the location after truncation is different from the full length.For the second kind of circular RNA,we find a large number of literatures and selected five kinds of circular RNA of different length for follow-up experiments,including three kinds of circ PVT1(410nt),circ HIPK3(1099nt),circ CDR1as(1500nt)mainly located in the cytoplasm,and two kinds of circ EIF3J(647nt)and circ PAIP2(985nt)mainly located in the nucleus.The main results are as follows: 1)Firstly,the endogenous expression and localization of the above five circular RNA in Hela,293 T,TJ905,A549,MDA-MB-231,MCF7 and MRC5 cell lines were verified after nucleocytoplasmic separation and RT-PCR technique.And Hela cells were selected for follow-up experiments,Because of the large size and obvious nuclear and cytoplasmic distribution.2)Five kinds of circular RNA were transfected into Hela cells by cationic liposome,and the exogenous expression and localization of the above five kinds of circular RNA in Hela were verified by nuclear and cytoplasmic separation and RT-PCR technique.3)By designing circular RNA transfection experiments at different time points in order to find out the time point of circ RNA export.Results: For the first type of naturally intronless mRNA,we first analyzed the356 naturally intronless genes obtained from previous work,and selected 34 genes according to the number of motif1V-AS elements for follow-up experiments.The full length of these genes and truncated operations were cloned into p CDNA3 vectors,combined with RNA-FISH screening.As a result,it was found that the mRNA transcribed from the full-length sequences of the naturally intronless genes SLC18A3,HPS6 and GPR148 was located in the cytoplasm.However,the mRNA transcribed after deletion of the sequence 1 to 3 was mainly located in the nucleus.It was preliminarily concluded that there were cis-acting elements in the genes containing motif1V-AS elements that could promote their export.For the second kind of circular RNA,the transfection experiments at different time periods were designed to find out the approximate time point of export after the detection of endogenous and exogenous content and location.However,no positive results were obtained.Conclusion: It suggests that the cis-acting element of intronless mRNA can regulate the export. |
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