Mechanistic Studies Of PQBP1 In Nuclear Import Of The Splicing Factor And MRNA Alternative Polyadenylation

The evolutionarily conserved PQBP1 is an intrinsically disordered protein ubiquitously expressed in various tissues,especially with a high level in the nervous system.PQBP1 plays important roles in neurodevelopment for multiple mutations in this gene are associated with a type of X-linked intellectual disability(XLID),named Renpenning syndrome.Its molecular functions remain to be elucidated.PQBP1 is dominantly located in the nucleus and associates with various splicing and transcription factors through its different domains,indicating its involvement in mRNA metabolic regulation.This dissertation investigated the molecular functions of PQBP1 through its interactions with the associated proteins.The effects of different mutations of PQBP1 on its interaction with splicing factor TXNL4 A were first carefully examined.Then a novel PQBP1 binding partner CPSF6 was identified by mass spectrometry analysis.Further investigation using in combination of genetics,molecular biology,biochemistry,and cell biology approaches elucidates the novel molecular function of PQBP1 in mRNA alternative polyadenylation(APA).PQBP1 binds to the splicing factor TXNL4 A through its C-terminal Yxx Pxx VL motif,but the biological function of this interaction has not been elucidated.Our study found that the interaction of PQBP1-TXNL4 A can promote the nuclear import of TXNL4 A,and the mutations of PQBP1 in the C-terminal domain can distrupt this interaction and cause the abnormal subcellular localization of TXNL4 A.Further analysis demonstrated that PQBP1 is recognized by the nuclear import receptor karyopherin β2 through its nuclear localization signal and facilitates the nuclear import of TXNL4 A via a piggyback mechanism.In order to investigate the unknown molecular function of PQBP1,we used mass spectrometry analysis and protein binding assays to identify new associating proteins.We discovered the interaction of PQBP1 with the 3’ end processing core factor CPSF6 and demonstrated that PQBP1 is a component of the 3’ end processing complex.Next,we detected the effects of knock-out or knock-down of PQBP1 on the selection of mRNA poly(A)sites in neural cells using high-throughput sequencing.The results showed that downregulation of PQBP1 is correlated with an increasing usage of distal poly(A)sites.Further biochemical analysis demonstrated that PQBP1 does not affect the protein expression or complex assembly of CFIm,but competes with CPSF5 for the UGUA sites in the pre-mRNA substrate,which changes the selection of mRNA poly(A)sites.In summary,this dissertation reveals new molecular functions of PQBP1 both in the nuclear transport of splicing factors and in the regulation of mRNA alternative polyadenylation.These findings expand our understanding of the molecular functions of PQBP1 and provide novel insights into the pathogenesis of Renpenning syndrome.