Structural assembly of the nucleic-acid-binding Thp3-Csn12-Sem1 complex functioning in mRNA splicing

Area of Science:

• Molecular Biology
• Structural Biology
• Gene Regulation

Background:

• PCI domain proteins are vital for post-transcriptional gene regulation.
• The Thp3-Csn12-Sem1 complex is structurally related to the TREX-2 complex and mediates pre-mRNA splicing.

Purpose of the Study:

• To determine the high-resolution structure of the yeast Thp3186-470-Csn12-Sem1 ternary complex.
• To elucidate the structural basis of nucleic acid binding and its role in pre-mRNA splicing.

Main Methods:

• X-ray crystallography at 2.9 Å resolution.
• Biochemical assays to assess nucleic acid binding affinity.
• Site-directed mutagenesis to investigate functional roles of specific residues.

Main Results:

• The Thp3-Csn12-Sem1 complex adopts an inverted V-shape, with Sem1 stabilizing Csn12.
• The winged-helix domains of Thp3 and Csn12 form a continuous surface for nucleic acid binding (micromolar affinity).
• Mutations in these domains impair nucleic acid binding and pre-mRNA splicing in vivo.

Conclusions:

• The determined structure provides insights into the molecular architecture of the Thp3-Csn12-Sem1 complex.
• The winged-helix domains are key for nucleic acid interaction and spliceosome recruitment.
• This structural information is foundational for understanding pre-mRNA splicing mechanisms.