The Species-specific Processing,subcellular Localization And Function Of LncRNAs

lncRNAs(long noncoding RNAs)are pervasively transcribed in eukaryotic cells,which are longer than 200 nucleotides and have no protein coding ability.Although a number of lncRNAs have been proved with important regulatory functions,enormous lncRNAs' function is unknown.It is still essential to reveal novel regulatory lncRNAs.lncRNA different subcellular localizations determine their diverse functions.While lncRNAs are rapidly evolved and with less sequence conservation,which make it changellege to study or predict their function.Whether conserved lncRNAs have conserved processing,localization and function is largely unknown.In this dissertation,I used molecular and cellular biological method to study the conservation of conserved lncRNAs processing,localization and function of in human and mouse embryonic stem cells(ESCs),as well as to study novel regulatory lncRNAs in stem cells.Firstly,I fractionated cytoplasmic and nuclear RNA form human and mouse ESCs for RNA-sequencing.We found that lncRNA subcellular localization is distinct between h ESCs and m ESCs.A significantly higher fraction of lncRNAs is localized in the cytoplasm of h ESCs than in m ESCs,while both m RNAs are cytoplasmic localized.Analysis the subcellular localization of both sequence conserved lncRNAs and positionally conserved lncRNAs reveales their distinct localization patterns,while the localization of conserved m RNAs is similar.Secondly,we found that knockdown of the cytoplasmic localized conserved lncRNAs usually alertes h ESC pluripotency,while knockout of nuclear localized conserved lncRNAs has no effect on m ESC pluripotency.Further,I studied the function and mechanism of a novel lncRNA named FAST(FOXD3 Antisense transcript 1)which is specifically expressed in ESCs.Human FAST(h FAST)is localized at cytoplasm and binds E3 ubiquitin ligase b-Tr CP to protecte b-catenin from degradation,which result in activating of WNT signaling and maintaining ESC pluripotency.Nuclear localized mouse Fast(m Fast)neither binds b-Tr CP nor regulates m ESC pluripotency.Cytoplasmic localized cem FAST(crab-eating macaque FAST)also required for monkey ESC pluripotency.In this part,we revealed the mechanism of h FAST regulating h ESC pluripotency and confirmed the importance of lncRNA subcellular localization for their function.We also revealed the nonconserved function and localization of conserved lncRNAs between h ESCs and m ESCs.What contributes to lncRNA distinct localization in h ESCs and m ESCs?We found that the splicing factor PPIE(peptidylprolyl isomerase E)can regulate RNA processing and localization by bioinformatics analysis and experimental screening.Knockdown of PPIE can promote lncRNA processing and export in h ESCs and m ESCs.While the expression of PPIE is distinct between h ESCs and m ESCs.Highly expressed Ppie in m ESCs inhibits lncRNA processing and result in lncRNAs retention in nucleus.We also found that the expression level of PPIE is negatively corelated with RNA processing among human,monkey and mouse ESCs.The expression of PPIE is decreased while the RNA processing is enhanced during the species evolution.Together,we revealed that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.