The Ccr4-Not complex has been implicated in the control of multiple steps of mRNA metabolism; however, its functions in transcription remain ambiguous. The discovery that Ccr4/Pop2 is the major cytoplasmic mRNA deadenylase and the detection of Not proteins within mRNA processing bodies have raised questions about the roles of the Ccr4-Not complex in transcription. Here we firmly establish Ccr4-Not as a positive elongation factor for RNA polymerase II (RNAPII). The Ccr4-Not complex is targeted to the coding region of genes in a transcription-dependent manner similar to RNAPII and promotes elongation in vivo. Furthermore, Ccr4-Not interacts directly with elongating RNAPII complexes and stimulates transcription elongation of arrested polymerase in vitro. Ccr4-Not can reactivate backtracked RNAPII using a mechanism different from that of the well-characterized elongation factor TFIIS. While not essential for its interaction with elongation complexes, Ccr4-Not interacts with the emerging transcript and promotes elongation in a manner dependent on transcript length, although this interaction is not required for it to bind RNAPII. Our comprehensive analysis shows that Ccr4-Not directly regulates transcription, and suggests it does so by promoting the resumption of elongation of arrested RNAPII when it encounters transcriptional blocks in vivo.

The gene encoding ribonucleotide reductase 3 (RNR3) is strongly induced in response to DNA damage. Its expression is strictly dependent upon the TAF II subunits of TFIID, which are required for the recruitment of SWI/SNF and nucleosome remodeling. However, full activation of RNR3 also requires GCN5, the catalytic subunit of the SAGA histone acetyltransferase complex. Thus, RNR3 is dependent upon both TFIID and SAGA, two complexes that deliver TATA-binding protein (TBP) to promoters. Furthermore, unlike the majority of TFIIDdominated genes, RNR3 contains a consensus TATA-box, a feature of SAGA-regulated core promoters. Although a large fraction of the genome can be characterized as either TFIID-or SAGA-dominant, it is expected that many genes utilize both. The mechanism of activation and the relative contributions of SAGA and TFIID at genes regulated by both complexes have not been examined. Here we delineated the role of SAGA in the regulation of RNR3 and contrast it to that of TFIID. We find that SAGA components fulfill distinct functions in the regulation of RNR3. The core promoter of RNR3 is SAGA-dependent, and we provide evidence that SAGA, not TAF II s within TFIID, are largely responsible for TBP recruitment. This taken together with our previous work provides evidence that SAGA recruits TBP, whereas TFIID mediates chromatin remodeling. Thus, we described an unexpected shift in the division of labor between these two complexes and provide the first characterization of a gene that requires both SAGA and TFIID.

The highly conserved Ccr4‐Not complex has been ascribed many functions, from transcription regulation to mRNA decay. Initial studies described a role of this complex in preinitiation complex formation, but a number of studies have clearly shown Ccr4‐Not mediates deadenylation of mRNAs and protein ubiquitylation in the cytoplasm. It is still not clear how Ccr4‐Not regulates gene expression, and which of these functions are controlled directly by this complex. Our study demonstrates that Ccr4‐Not complex directly regulates PolII‐dependent transcription elongation. Using purified Ccr4‐Not complex and yeast PolII in an in vitro transcription system we have studied how this complex affects transcription elongation. Our studies show the complex binds directly to the elongation complex, which is partially dependent on the emerging transcript, and crosslinking studies show it also makes contacts with the transcript. Using transcription run on assays, we demonstrate that Ccr4‐Not affects the rate of PolII transcription and stimulates elongation through transcription blocks. Our in vitro results are substantiated by in vivo studies, showing that Ccr4‐Not co‐purifies with PolII and associates with the open reading frames of transcribing genes. Together these studies support a model by which the Ccr4‐Not complex directly associates with transcribing PolII and regulates elongation. Funded by NIH (GM58672).

Many functions have been ascribed to the Ccr4‐Not complex. First described many years ago as a potential regulator of preinitiation complex (PIC) formation, more recently, a large body of evidence indicates it is predominantly cytoplasmic and regulates deadenylation of mRNA and ubiquitylation of substrates in the cytoplasm and nucleus. A direct role of this complex in transcription has not been demonstrated clearly, and the discovery of the cytoplasmic functions of this complex raises questions about how it regulates gene expression.Here we provide conclusive evidence that Ccr4‐Not directly regulates RNAPII transcription elongation. Ccr4‐Not associates with active genes in vivo, and it predominantly associates with the open reading frames of genes in a transcription and RNAPII‐dependent manner. We show that Ccr4‐Not regulates multiple elongation functions, and interestingly, mutation of different subunits of this complex results in distinct elongation phenotypes. Interestingly, Ccr4‐Not associates with RNAPII in a CTD‐independent manner in vitro and in vivo. Furthermore, using a highly purified system, we demonstrate that Ccr4‐Not associates with RNAPII elongation complexes and stimulates transcription elongation. Together, our results bring to light the functions of the Ccr4‐Not complex in the transcription cycle and provide evidence that it contributes directly to transcription by RNAPII.