In vivo characterization of the Drosophila mRNA 3' end processing core cleavage complex

Area of Science:

• Molecular Biology
• RNA Processing
• Gene Expression

Background:

• The core cleavage complex (CCC), comprising CPSF73, CPSF100, and Symplekin, is crucial for the cotranscriptional 3' end processing of metazoan pre-mRNAs.
• The CCC participates in distinct complexes, including those for general cleavage/polyadenylation and the processing of nonpolyadenylated histone pre-mRNAs.
• Understanding the in vivo molecular interactions within the CCC is essential for elucidating its regulatory mechanisms.

Purpose of the Study:

• To investigate the in vivo molecular interactions and functional domains within the core cleavage complex (CCC).
• To determine the specific regions of Symplekin, CPSF73, and CPSF100 required for CCC formation and histone mRNA 3' end processing.
• To elucidate the role of Symplekin's N-terminal region in cotranscriptional processing of histone mRNAs.

Main Methods:

• Utilized Dmel-2 tissue culture cells stably expressing tagged CCC components.
• Employed RNA interference (RNAi)-depletion strategies to assess the impact of CCC factor deficiencies on mRNA processing.
• Performed deletion analyses on CCC components to map functional domains required for complex formation and processing activity.

Main Results:

• Identified specific amino acid regions in Symplekin (272-1080) and CPSF100 (C-terminal ~200 aa) as critical for efficient in vivo CCC formation.
• Demonstrated that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing, indicating dispensability of its N-terminal region.
• Showed that Symplekin deletions lacking the N-terminal region (first 271 aa) led to increased use of downstream polyadenylation sites in histone mRNAs, mimicking depletion of histone-specific factors.

Conclusions:

• CCC formation in vivo is mediated by the C-terminal regions of CPSF73, CPSF100, and Symplekin.
• The N-terminal region of Symplekin plays a facilitating role in the cotranscriptional 3' end processing of histone mRNAs.
• These findings provide a refined model for CCC assembly and function in distinct mRNA processing pathways.