Insight Into The Structure And Function Of The Saccharomyces Cerevisiae Thp3-Csn12-Sem1 Complex Functioning In MRNA Splicing

The eukaryotic post-transcriptinal processing is a necessary part of gene expresstion,and it’s also an important prerequisite to guarantee the normal cellular function of cells.The studies focusing on eukaryotic mRNA post-transcriptional process will be of great significance for helping to understand the mechanism of gene expression regulation.In this study,we conducted structural and functional studies on Thp3-Csn12-Seml complex in Saccharomyces cerevisiae that involved in regulating RNA processing.Csn 12 was initially described as a subunit of Saccharomyces cerevisiae COP9 signalosome complex(CSN)and played vital roles in guiding the nuclear localization of Csn5 functional subunit directing arrangement of the complex subunits and maintaining the integrity of CSN structure.However,deletion of Csn12 failed to accumulate the neddylated Cdc53 as observed for other subunits of CSN,suggesting the distinctive role of Csn12 beyond regulating CRL activity.Only Csn12,but not other subunits of CSN,could physically interact with a number of proteins(SMB1,SMX2 and SMX3)involved in mRNA splicing and Thp3(also called Ypr045c).It was then confirmed that Csn12 and Thp3 could interact specifically to form a complex independent of the rest subunits of CSN.The Thp3-Csn12 complex was cotranscriptionally recruited to actively-transcribing gene and functionally linked to spliceosome.Deletion of either Csn12 or Thp3 had a splicing defect,causing accumulation of intron-containing pre-mRNA,which was similar to isyl Δ strain and several other splicing factor mutants.It is intriguing to understand how Thp3-Csn12 complex is associated with spliceosome for mRNA splicing.Additionally,the specific participation mechanisms and functional mechanisms of Thp3-Csn12 in the pre-mRNA splicing process are still unclear.At the same time,the possible molecular mechanism underlying the model that Csn12 could separate from COP9 and function in transcription and transcription-related pre-mRNA splicing process with Thp3 remains elusive.In addition,the versatile small Sem1 protein was also found to co-purified with Thp3 and Csn12 in vivo.However,the precise binding mode and molecular details of interaction among Csn12,Thp3 and Sem1 as well as the exact role played by Sem1 in Thp3-Csn12 complex are unknown.In this study,we first confirmed that Thp3,Csn12 and Sem1 could form a stable complex in vivo by His pull-down results and then solved the structures of yeast Thp3(186-470)-Csn12-Sem1 ternary complex in P3221 and P212121 space group by x-ray crystallography at 2.85 ? and 2.75 ? resolution,respectively,with one complex molecule in per asymmetric unit.The overall structure of Thp3(186-470)-Csn12-Sem1 complex has an inverted V-shape with Thp3 and Csn12 forming the two sides,both Thp3 and Csn12 adopt a typical PCI structural fold characterized by a stack of α-helices capped by a C-terminal winged-helix(WH)domain.A fishhook-shaped Sem1 makes extensive contacts on Csn12 to stabilize its conformation.The unique structural properties and patterns of interaction among those three proteins explain the structural mechanism by which Csn12 can be independent of other subunits of COP9.To further investigate the effector mechanism of this complex,we demonstrated that the Thp3-Csn12-Sem1 complex has direct nucleic acid binding activity through fluorescence anisotropy experiments for the first time.Both the single component of complex and the key basic residues located on the continuous nucleic acid binding surface at the C-terminus of Thp3-Csn12 heterodimer are critical for the nucleic acid binding ability of the complex.The C-terminal WH domains of Thp3 and Csn12 form a continuous surface to bind different forms of nucleic acids(ssDNA,dsDNA and ssRNA)with micromolar affinity.Single subunit deficiency or structure-based charge reversal mutation of the key basic residues in the WH domains of Thp3 and Csn12 affects nucleic acid binding in vitro and mRNA splicing in vivo.The overall structure of Thp3(186-470)-Csn12-Sem1 complex resembles the previously reported Sac3(N+M)-Thp1-Sem1 module of TREX-2 complex,but also has significant structural differences.The differential nucleic acid binding affinities(μmol/L vs.nmol/L)may hint an ordered RNA processing mechanism from pre-mRNA splicing to mRNA nuclear export across these two complexes,and their significant structural differences(The Ca RMSD of main chain is 5.8 ?)allow the selective interactions of Thp3-Csn12-Seml and TREX-2 with spliceosome and nuclear pore complexes by different structural surfaces,respectively,which may account for their distinctive functions in mRNA splicing vs.mRNA nuclear export,two different but related processes of post-transcriptional regulation.Together,the detailed structural information,interaction studies between Thp3Csn12-Seml complex and nucleic acids as well as pre-mRNA splicing level studies of single component deletion or key basic residues mutation in vivo will help to understand in depth the molecular basis of mRNA splicing mediated by the Thp3Csn12-Seml ternary complex,and provide new ideas for further exploring transcription-coupled pre-mRNA splicing.