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Updated: Jun 23, 2026
Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies
Published on: November 28, 2017
A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A)
Kelly D Sullivan1, Mindy Steiniger, William F Marzluff
1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract:
Metazoan histone mRNAs are unique: their pre-mRNAs contain no introns, and the mRNAs are not polyadenylated, ending instead in a conserved stem-loop structure. In Drosophila, canonical poly(A) signals are located downstream of the normal cleavage site of each histone gene and are utilized when histone 3' end formation is inhibited. Here we define a subcomplex of poly(A) factors that are required for histone pre-mRNA processing. We demonstrate that Symplekin, CPSF73, and CPSF100 are present in a stable complex and interact with histone-specific processing factors. We use chromatin immunoprecipitation to show that Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes. Depletion of SLBP recruits CstF50 to histone genes. Knockdown of CPSF160 or CstF64 downregulates Symplekin but does not affect histone pre-mRNA processing or association of Symplekin with the histone locus. These results suggest that a common core cleavage factor is required for processing of histone and polyadenylated pre-mRNAs.
Insights
This study identifies a core complex of polyadenylation factors, including Symplekin and CPSF73, essential for processing unique histone pre-mRNAs in Drosophila. These factors associate with histone genes during transcription, revealing a shared mechanism for mRNA processing.
Area of Science:
- Molecular Biology
- RNA Processing
- Gene Regulation
Background:
- Metazoan histone mRNAs possess a unique structure, lacking introns and polyadenylation, terminating instead in a conserved stem-loop.
- Canonical polyadenylation signals in Drosophila histone genes are activated upon inhibition of normal 3' end formation.
Purpose of the Study:
- To define the subcomplex of polyadenylation factors involved in histone pre-mRNA processing.
- To investigate the interaction and association of these factors with histone genes.
Main Methods:
- Biochemical complex isolation and interaction studies.
- Chromatin immunoprecipitation (ChIP) to assess cotranscriptional association with histone genes.
- RNA interference (RNAi) for gene knockdown and functional analysis.
Main Results:
- Symplekin, CPSF73, and CPSF100 form a stable complex interacting with histone-specific processing factors.
- Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes; SLBP depletion recruits CstF50.
- Knockdown of CPSF160 or CstF64 affects Symplekin levels but not histone pre-mRNA processing or Symplekin's histone locus association.
Conclusions:
- A common core cleavage factor complex is implicated in the processing of both histone and polyadenylated pre-mRNAs.
- Symplekin and CPSF73 are key components of the machinery governing histone mRNA 3' end formation.
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