Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation

The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were ass...

Full description

Saved in:
Bibliographic Details
Published in:RNA (Cambridge) 2010-07, Vol.16 (7), p.1356-1370
Main Authors: Temme, Claudia, Zhang, Lianbing, Kremmer, Elisabeth, Ihling, Christian, Chartier, Aymeric, Sinz, Andrea, Simonelig, Martine, Wahle, Elmar
Format: Article
Language:English
Subjects:
Adenosine Monophosphate - metabolism
Animals
Cell Line
Drosophila melanogaster - enzymology
Drosophila melanogaster - metabolism
Drosophila Proteins - analysis
Drosophila Proteins - metabolism
Genetics
Life Sciences
Proteome - analysis
Retinoblastoma-Binding Protein 4 - metabolism
Ribonucleases - chemistry
Ribonucleases - metabolism
RNA, Messenger - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.2145110