Identification Of Export-promoting Proteins Recruited To The Cis Element Of Naturally Intronless Gene KRN1

Background: Splicing-dependent TREX recruitment is vital for the export of spliced m RNAs,however,mechanism on export of naturally intronless m RNAs is still elusive.In the process of splicing,splicing complexes will be recruited to cut introns and connect exons.At the same time,nuclear transport complexes will be recruited directly,so that the nuclear transport of m RNA can be completed.When the intron of the gene is deleted artificially,the transcription products of the gene can not recruit nuclear complexes because of the termination of splicing events,and eventually be blocked in the nucleus Naturally intronless genes account for 5% of all human genes,including some genes which encode histones,interferons and other proteins that play important roles in the life process.It is of great significance to study the export mechanism of naturally intronless m RNAs.In previous studies,through bioinformatics analysis on the coding regions of 679 naturally intronless m RNAs followed by predictive sequence deletion and mutation verification at reporter gene level,we found that the conserved sequence CAR-C(cytoplasmic accumulation region-cluster)in the coding region of naturally intronless gene KRN1 acted as cis-acting element.It is essential for the nuclear transport of KRN1 m RNA transcripts However,the key proteins bind to the cis-element to facilitate this export process are still unknown.In this paper,we will identify the functional trans-factors associated to CAR-C region and its fundamental role in the export of global intronless m RNAs.Methods: 1.In vitro transcription technique will be utilized to obtain the biotin-labeled CAR-C RNA probe,which will then be incubated with the nuclear extract of He La cell.The RNP purification products will be sent for mass spectrometry analysis to get the candidate proteins.2.Carry out si RNA screen among the candidates followed by transient transfection of reporter gene containing KRN1 coding sequence.Apply fluorescence in situ hybridization(FISH)to show the intracellular localization of its m RNA transcripts.3.Construct inducible hn RNPR overexpression stable cell line,and obtain other proteins or complexes associated with CAR-C by immunoprecipitation.4.In He La cells,si RNA was used to knock down hn RNPR,followed by nucleocytoplasmic separation.The RNAs were extracted respectively followed by RNA-seq analysis and RT-PCR.Results: Using KRN1 reporter,we identified hn RNPR as key factor governing the export process of KRN1 m RNA transcripts with RNP purification followed by mass spectrometry and si RNA screen.The results of fluorescence in situ hybridization(FISH)showed that after knockdown the protein hn RNPR,KRN1 transcripts were significantly blocked in the nucleus.Then we constructed inducible hn RNPR overexpression stable cell line.Immunoprecipitation further revealed that hn RNPR interacts with NPC component NUP93 to promote the export.Depletion of hn RNPR or NUP93 resulted in severe export defect for intronless reporter transcripts as well as endogenous intronless m RNAs.Moreover,si RNA screen of all the reported 31 nuclear pore components one by one,revealed that knockdown of 10 components(NUP107,NUP205,NUP62,NUP214,TPR,GLE1,NUP88,NUP98,Ran BP2,RAE1)resulted in export defect of the KRN1 transcripts.In addition,through RNA-seq and RT-PCR results we found that the knockdown of hn RNPR affected the distribution of endogenous expression of naturally intronless transcripts in He La cell line.Conclusion: Our data provide direct evidence to support a fundamental role of sequence-dependent recruitment of hn RNPR in the export of naturally intronless m RNAs.