HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs.

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5'-3' messenger RNA decay.

XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5'-3' mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4-NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the 'C-terminal interacting region' or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4-NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4-NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5'-3' exonucleolytic decay.

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex.

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions.

A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin.

Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex.

Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos.

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4-NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4-NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4-NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1-3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4-NOT to assemble analogous repressive complexes.

miRISC and the CCR4-NOT complex silence mRNA targets independently of 43S ribosomal scanning.

miRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4-NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4-NOT complex in Drosophila melanogaster Here, we show that miRNAs, AGOs, GW182, the CCR4-NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning-independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.

Ubiquitination-mediated interaction among domains is responsible for inhibition of RNA endonuclease activity of mRNA cycling sequence binding protein from L. donovani (LdCSBP).

In nearly complete absence of transcriptional regulation, messenger RNA (mRNA) turnover mediated through specific cis-elements plays a predominant role in the control of differential gene expression for the disease causing trypanosomatid parasites. In these organisms, the periodic accumulation of S-phase messages during cell cycle is determined by the presence of one or more copies of a conserved CAUAGAAG octanucleotide motif in the untranslated regions of mRNAs. In our previous studies, a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani was characterized, which binds specifically to the octamer-containing RNAs via its uniquely arranged CCCH-type Zn fingers and degrades them through its small MutS-related (Smr) endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Interestingly, the protein is modified by the incorporation of a monoubiquitin residue, and the posttranslational modification inhibits its riboendonuclease activity. However, the mechanism of such inhibition was previously unknown. Here, we establish that the CCCH-type Zn finger domain is the site of ubiquitination in LdCSBP and the interaction of CUE domain of the protein with the ubiquitinated Zn finger domain is responsible for inhibition of its riboendonuclease activity. The findings elucidate an inhibitory mechanism of RNA cleavage through ubiquitination-mediated intramolecular interaction among domains of the enzyme. Furthermore, the riboendonuclease activity is inhibited by anti-leishmanial drug paromomycin suggesting that the regulation of RNA metabolism could be a target of the drug.

An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to mediate mRNA deadenylation and decay.

The PAN2-PAN3 complex functions in general and microRNA-mediated mRNA deadenylation. However, mechanistic insight into PAN2 and its complex with the asymmetric PAN3 dimer is lacking. Here, we describe crystal structures that show that Neurospora crassa PAN2 comprises two independent structural units: a C-terminal catalytic unit and an N-terminal assembly unit that engages in a bipartite interaction with PAN3 dimers. The catalytic unit contains the exonuclease domain in an intimate complex with a potentially modulatory ubiquitin-protease-like domain. The assembly unit contains a WD40 propeller connected to an adaptable linker. The propeller contacts the PAN3 C-terminal domain, whereas the linker reinforces the asymmetry of the PAN3 dimer and prevents the recruitment of a second PAN2 molecule. Functional data indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay, which lead to complete mRNA degradation.

Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression.

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Ubiquitination of mRNA cycling sequence binding protein from Leishmania donovani (LdCSBP) modulates the RNA endonuclease activity of its Smr domain.

In trypanosomatid parasites, an octanucleotide sequence (C/A)AUAGAA(G/A) in the UTRs primarily determines the stability of S-phase specific mRNAs. A multi-domain protein LdCSBP from Leishmania donovani interacts with the UTR of an S-phase RNA containing the octanucleotide sequence through its unique CCCH-type Zn-finger motifs. Interestingly, the RNA binding protein contains a previously characterized DNA endonuclease domain - Smr. It has been demonstrated here that the LdCSBP Smr domain independently possesses both DNA and RNA endonuclease activities, but the full-length LdCSBP exhibits only riboendonuclease activity. Moreover, LdCSBP protein has been shown to be ubiquitinated, resulting in the down-regulation of its riboendonuclease activity. In conclusion, the results described here suggest a novel regulatory mechanism of mRNA degradation through ubiquitination in eukaryotes.

mRNA cycling sequence binding protein from Leishmania donovani (LdCSBP) is covalently modified by ubiquitination.

The lack of transcriptional regulation in trypanosomatids suggests the presence of distinct posttranscriptional mechanisms to control differential gene expression. In fact, the stability of S-phase specific mRNAs in these parasites is determined primarily by the presence of the octanucleotide sequence (C/A)AUAGAA(G/A) in the UTRs of the transcripts. Here, the characterization of LdCSBP is reported, which specifically binds to the octanucleotide containing RNA. The LdCSBP protein contains multiple putative functional domains, including two types of ubiquitin binding domains (UBA and CUE), two CCCH-type Zn-finger motifs probably responsible for specific RNA binding activity and a speculative endonuclease domain SMR. Interestingly, the protein is covalently modified through ubiquitination. This observation and the occurrence of multiple ubiquitin binding domains in the protein raise the possibility of regulation of the activity of LdCSBP by ubiquitination.