Research On The Molecular Mechanism Of Aberrant RNA Splicing Associated With SF3B1 Mutations

RNA splicing is a fundamental biological process in eukaryotic cells,which involves the removal of introns and the ligation of exons.Pre-mRNA can be spliced in multiple patterns,resulting in various mature mRNA subtypes.This phenomenon is called alternative splicing.Although alternative splicing can improve gene expression diversity and the flexibility of post-transcriptional regulation,aberrant alternative splicing might cause various diseases.About 50% of disease-related mutations affect alternative splicing.The products of aberrant alternative splicing are involved in many cancer-related pathways.Therefore,aberrant alternative splicing is a promising target for cancer therapy.RNA splicing is undertaken by spliceosome,which is a highly dynamic complex composed of numerous ribonucleic acids and proteins.The proteins are called RNA splicing factors.SF3B1 is a RNA splicing factor and the mutations of SF3B1 are found in many cancers,such as chronic lymphocytic leukemia,myelodysplastic syndrome,and uveal melanoma.SF3B1 is an important component of U2 sn RNP.SF3B1 is responsible for recognizing and stabilizing the interaction between branch sites and U2 sn RNP during RNA splicing.SF3B1 mutations lead to aberrant alternative splicing.Some of these aberrantly spliced products participate in signaling pathway leading to tumorigenesis,such as the DVL2 gene in the Wnt signaling pathway and the MAP3K7 gene in the Notch signaling pathway.Therefore,inhibiting of aberrant spliced RNA is a very promising target for the treatment of cancers with SF3B1 mutations.The RNA splicing process is mediated by the interaction of cis-acting elements on pre-mRNA and trans-acting factors of spliceosome.Therefore,the mechanisms under which SF3B1 mutations promote aberrant splicing of RNA can be investigated from two ways.On one hand,previous researches showed that the aberrant splicing associated with SF3B1 mutation requires a new branch site,new 3' splice site and the normal polypyrimidine tract.On the other hand,cancer hotspot SF3B1 mutations are located in the 5th to 8th HEAT domains of its C-terminal region,which is a protein interaction domain.Therefore,mutations may affect the aberrant splicing by influencing its interaction with other proteins.We have explored methods to inhibit the SF3B1 associated aberrant splicing from these two directions.We found that the amount of aberrantly spliced MAP3K7 was increased in cancer cells containing SF3B1-K700E(the highest mutation rate in blood cancer,about 50%).In this study,we verified that SF3B1-K700 E promotes the aberrant splicing of MAP3K7,and found that the aberrantly spliced product was degraded by NMD pathway.We demonstrated that the disruption of 3' ss,the nucleic acid upstream of3'ss,aberrant branch site,and aberrant polypyrimidine tract can eliminate the aberrant splicing of MAP3K7.In addition to the SF3B1-K700 E mutation,we also constructed nine other mutations that are frequently found in cancer or previously investigated in yeast.All the mutations can cause aberrant splicing of MAP3K7,and also rely on the same cis-acting elements as those required by the SF3B1-K700 E mutation to cause the aberrant splicing of MAP3K7.Therefore,aberrant splicing associated with SF3B1 mutation can be inhibited via the disruption of cis-acting elements on pre-mRNA.In Chapter 3,we used mass spectrometry to identify splicing factors with different binding abilities to wild type SF3B1 and K700 E mutated SF3B1,and found that DDX42 showed decreased binding ability to SF3B1-K700 E.DDX42 is a member of the DExH/D family of RNA helicases,which have the ability to hydrolyze ATP.The DExH/D family members influence the recognition of branch site by using the energy provided from the hydrolyzation of ATP,and therefore regulate RNA splicing.In this study,we overexpressed DDX42 in HEK293 T cells and found that overexpression of DDX42 increased the interaction between DDX42 and SF3B1-K700 E and inhibited aberrant splicing;then we generated Q280 R and F254 A mutated DDX42,which disrupted the ATP hydrolysis ability of DDX42,then the two mutations abolished the ability of DDX42 to inhibit aberrant splicing,demonstrating the requirement of energy from ATP hydrolysis in the inhibition of aberrant splicing.In addition,other SF3B1 mutations also weakened the interactions between SF3B1 and DDX42,and overexpression of DDX42 can inhibit the aberrant splicing caused by these SF3B1 mutations.Therefore,aberrant splicing associated with SF3B1 mutation can be inhibited via the disruption of trans-acting factors of spliceosome.However,DDX42 knockdown did' t cause aberrant splicing.We speculate that DDX42 might be one of many RNA helicases that interact with SF3B1,and the effects of DDX42 knockdown might be resaved by other RNA helicases.While our project is in progress.professor James Manley's team from Columbia University found that SF3B1 mutations disrupted the interaction between SF3B1 and the splicing factor SUGP1,and they speculated that SUGP1 mediates the aberrant splicing of RNA through some unidentified RNA helicases.Since DDX42 is an RNA helicase,we explored the role of DDX42 in aberrant splicing associated with SUGP1 in chapter 4.We first verified that knockdown of SUGP1 or overexpression of mutant SUGP1 could indeed lead to aberrant splicing similar as the K700 E mutated SF3B1.However,overexpression of DDX42 couldn't inhibit the aberrant splicing associated with SUGP1 knockdown.Immunoprecipitation showed that there was no interaction between SUGP1 and DDX42.Therefore,our results demonstrated that SUGP1 might not affect aberrant splicing through DDX42.In summary,we found ways to inhibit aberrant splicing associated with SF3B1 mutations through both the cis-acting element,the aberrantly spliced RNA and the trans-acting factors with changed binding ability to the mutated SF3B1.Our study reveals that inhibiting the aberrant splicing associated with SF3B1 mutation might be potential target of cancer therapies.