Novel interaction between CCR4 and CAF1 in rice CCR4–NOT deadenylase complex

Abstract

Rice is an important crop in the world. However, little is known about rice mRNA deadenylation, which is an important regulation step of gene expression at the post-transcriptional level. The CCR4-NOT1 complex contains two key components, CCR4 and CAF1, which are the main cytoplasmic deadenylases in eukaryotic cells. In yeast and humans, CCR4 can interact with CAF1 via its N-terminal LRR domain. However, no CCR4 protein containing N-terminal LRR motifs have been found in plants. In this manuscript, we demonstrate a novel pattern of interaction between OsCCR4 and OsCAF1 in the rice CCR4-NOT complex, and that OsCAF1 acts as a bridge between OsCCR4 and OsNOT1 in this complex. Our results revealed that the Mynd-like domain at the N-terminus of rice CCR4 proteins and the PXLXP motif at the rice CAF1 N-terminus play critical roles in OsCCR4-OsCAF1 interaction. Deadenylation, also called poly(A) tail shortening, is the first rate-limiting step in general cytoplasmic mRNA degradation in eukaryotic cells. Carbon catabolite repressor (CCR)4 and CCR4-associated factor (CAF)1 in the CCR4-NOT complex function in mRNA poly(A) tail shortening. CCR4s contain N-terminal leucine-rich repeat (LRR) motifs that interact with CAF1s in yeast, fruit fly and mammals. In silico analysis has not identified any plant CCR4 proteins that contain LRR motifs. Here, two rice CCR4 homologous genes, OsCCR4a and OsCCR4b, were identified. The isolated recombinant exonuclease-endonuclease-phosphatase domain of OsCCR4a and OsCCR4b exhibited 3'-5' exonuclease activity in vitro, and point mutation of a catalytic residue in this domain disrupted the deadenylase activity. Both OsCCR4a and OsCCR4b fluorescent fusion proteins were localized in the rice cytoplasm and nucleus, and both associated with processing bodies via their N-terminus. Binding analyses showed that OsCCR4a and OsCCR4b directly interacted with three rice CAF1 family members: OsCAF1A, OsCAF1G and OsCAF1H. The zf-MYND-like domain at the N terminus of rice CCR4 and the PXLXP motif of rice CAF1 play critical roles in OsCCR4-OsCAF1 interaction. OsCAF1 proteins, but not OsCCR4 proteins, can interact with the MIG4G domain of rice OsNOT1. Our studies thus reveal a hitherto undiscovered novel interaction pattern that connects OsCCR4 and OsCAF1 in the rice CCR4-NOT complex.

RETRACTED ARTICLE:The Arabidopsis NOT4A E3 ligase promotes PGR3 expression and regulates chloroplast translation

“…Within the CCR4-NOT complex, the CAF1 subunit binds to the central NOT1 scaffold to provide deadenylase activity in humans and Drosophila 66,67 . The CAF1 family has expanded and diverged in plants, with only a proportion of members capable of binding NOT1, which are also able to bind with CCR4 to form a deadenylating module within the CCR4-NOT complex in Arabidopsis and rice 62,68 . It has been proposed that these differing binding activities of CAF1 proteins represent distinct assemblies of the CCR4-NOT complex 62 , highlighting the potential functional expansion of the complex in plants, which could be further increased via the three NOT4 homologues we have identified here.…”

Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function

“…OsCCR4a and OsCCR4b are located predominantly in visible foci, presumably PBs, that include XRN4. The EEP domain of OsCCR4s is sufficient for in vitro 39-59 exonuclease activity on poly(A), poly(U), and poly(C) substrates (Chou et al, 2017). Similarly, recombinant OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H are 39-59 exonucleases, with at least OsCAF1B associating with XRN4 in PB-like foci (Chou et al, 2014).…”

“…In yeast and mammals, CAF1 serves as a bridge between CCR4 and NOT1 via interactions between the N-terminal Leu-rich repeat of CCR4 and the MIF4G domain of NOT1. The rice CCR4-NOT complex appears to share this topology, although the N-terminal Leu-rich repeat domain of CCR4s is absent and the interaction with CAF1 is apparently replaced by an N-terminal zf-MYND-like domain of CCR4 (Chou et al, 2017).…”

DEAD-Box RNA Helicase 42 Plays a Critical Role in Pre-mRNA Splicing under Cold Stress

“…A protoplast transient expression system was used as previously described (Chou et al, 2017). Rice protoplasts were isolated from the sheaths of 10-d-old seedlings and then incubated with expression vectors and PEG for 20 min.…”