The Role And Mechanism Of Phase Separation In SKIP-mediated RNA Splicing

Liquid-liquid phase separation（LLPS）is the main mechanism to promote the formation of membrane-less organelles.The main feature of LLPS is the local high concentration of macromolecules,which can concentrate enzymes and substrates within specialized subcellular compartments,promoting more efficient enzymatic reactions.Phase separation play critical roles in multiple biological processes,including transcription,signal transduction,and RNA processing^[1-3].Whether phase separation is involved in regulation of RNA splicing remains unclear.SKIP has been identified as a key factor regulating pre-m RNA splicing^[4].It was found by NMR that SKIP contains an intrinsic disordered region^[5],suggesting SKIP may undergoes LLPS.This study takes SKIP as the starting point to explore the relationship between the phase separation and RNA splicing,which may help us to better understand the process of splicing.In order to investigate whether SKIP can undergo phase separation and its functions,we purified the SKIP（SKIP-IDR,residues 1-129）in vitro.The droplet formation assay showed that SKIP-IDR can form a spherical droplet structure,suggesting that SKIP-IDR may undergo phase separation.Phase separation is driven by the interactions between multivalent macromolecules,such as hydrophobic interactions,electrostatic interactions^[6].After 1,6-hexanediol treatment,which was reported to destroy hydrophobic interactions^[7],the size of SKIP-IDR droplets became smaller.Through FRAP experiment,we found that the droplets formed by SKIP-IDR phase separation showed better fluidity.Next,we investigated SKIP phase separation in vivo.Through cellular immunofluorescence staining,1,6-hexanediol treatment and FRAP assay,we found that the puncta containing SKIP have liquid-like properties in vivo.Collectively,these data indicate that SKIP can undergo phase separation in vitro and in vivo.Besides,we found that the tyrosines in IDR are important for phase separation of SKIP.We found that SKIP is localized in nuclear speckle,where concentrates large amounts of splicing factors,but the SKIP mutant（contain tyrosine mutations in IDR）show diffuse staining patterns,which suggests that the function of SKIP in splicing may be depend on its phase separation.To investigate whether the role of SKIP in pre-m RNA splicing depends on its phase separation,we constructed SKIP phase separation defect mutant and studied its effect on RNA splicing of SKIP target genes（sororin and APC2）^[8].Through the q PCR assay,we found that SKIP expression can restore target gene splicing defect caused by SKIP depletion,while overexpression of SKIP mutant showed similar effects as SKIP depletion.Taken together,these results indicated that SKIP phase separation plays a key role in pre-m RNA splicing.U1 and U4 are released during activation of spliceosome,and proteins such as PRP19 complex and PRP19-related proteins are recruited.SKIP is a PRP19-related protein^[9],we found that SKIP can recruit PRP19 and CDC5L to the nuclear speckle,while the SKIP mutant disturbed the recruitment of PRP19 and CDC5L to nuclear speckle,suggesting that SKIP phase separation may play an important role in the activation of spliceosome.One study reported that SKIP regulates sister chromatid cohesion by splicing sororin and APC2pre-m RNA^[8].To find out whether phase separation of SKIP is essential for sister chromatid cohesion,we performed karyotype staining assay and found that overexpression of SKIP rescued the cohesion defect caused by SKIP RNAi,while the expression of SKIP mutant exhibited similar effects as SKIP depletion,suggesting that SKIP can regulate sister chromatid cohesion by phase separation.This study reveals the connection between phase separation and pre-m RNA splicing.The data suggest that SKIP phase separation may offer a binding site for the recruitment of related proteins to nuclear speckles,where the spliceosome resides,thereby promoting the spliceosome activation.