The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation

Abstract

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

Translational Capacity of a Cell Is Determined during Transcription Elongation via the Ccr4-Not Complex

“…Previous studies have shown that Not5 is required for association of Not1 to specific mRNAs (Villanyi et al, 2014) and that in the absence of Not5 the expression of nuclear encoded mitochondrial protein mRNAs was upregulated (Azzouz et al, 2009b; Cui et al, 2008). In not5Δ Not1 interaction with most other Ccr4-Not subunits is reduced (Fig.…”

“…Our recent finding that Not5 is necessary for cytoplasmic functions of Rpb4 suggested that the multisubunit Ccr4-Not complex might also play a key role in regulating the gene expression circuitry (Villanyi et al, 2014). Ccr4-Not is a conserved multifunctional eukaryotic regulator composed of nine subunits in the yeast S.cerevisiae .…”

“…Several evidences point towards the Ccr4-Not complex mediating this cross-talk. Recently, we have shown that Not5 plays a role in translatability and assembly of the RNAPII complex (Villanyi et al, 2014). In addition, components of the Ccr4-Not complex, particularly Not4 and Not5, are important for transcription elongation (Kruk et al, 2011) and protein quality control (Dimitrova et al, 2009; Halter et al, 2014; Preissler et al, 2015).…”

Next-generation analysis of gene expression regulation – comparing the roles of synthesis and degradation

“…In many of these cases, buffering is likely mediated by proteins that function at multiple levels of gene expression. For example, the yeast RNA binding protein Rpb4 appear to affect both transcription and translation, as do the CCR4/Not complex and Not5 ref 75. .…”

Interaction of CCR4–NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

“…Recently, the CCR4-NOT complex has also been shown to regulate transcription elongation (Kruk et al 2011;Dutta et al 2015) and cotranslational quality control by its associated ubiquitin E3 ligase activity (for review, see Panasenko 2014). The CCR4-NOT complex has been implicated in the promoter-mediated coordination of transcription and mRNA decay by interacting with the Rpb4/7 subunits of RNA polymerase II (Lotan et al 2005;Villanyi et al 2014). The CCR4-NOT complex is assembled around the CNOT1 subunit, which serves as a scaffold and interacts with various modules.…”