Alternative splicing of CNOT7 diversifies CCR4-NOT functions

Nucleic Acids Res. 2017 Aug 21;45(14):8508-8523. doi: 10.1093/nar/gkx506.

Abstract

The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions.

MeSH terms

  • Alternative Splicing*
  • Arginine / genetics
  • Arginine / metabolism
  • Blotting, Western
  • Cell Line, Tumor
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cell Proliferation / genetics
  • Cytoplasm / genetics*
  • Cytoplasm / metabolism
  • Exoribonucleases
  • Gene Expression Profiling / methods*
  • HEK293 Cells
  • Humans
  • MCF-7 Cells
  • Methylation
  • Microscopy, Confocal
  • Protein Binding
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • RNA Interference
  • Repressor Proteins
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription Factors / chemistry
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism

Substances

  • Protein Isoforms
  • Repressor Proteins
  • Transcription Factors
  • Arginine
  • CNOT7 protein, human
  • Exoribonucleases