RESUMEN
It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4-NOT deadenylase complex involved in posttranscriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4-NOT deadenylase complex and might act in posttranscriptional mRNA regulation. The rapid gene evolution in the ancient posttranscriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.
Asunto(s)
Proteínas de Drosophila , Inactivación del Cromosoma X , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolución Molecular , Genes Ligados a X , MasculinoRESUMEN
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.
Asunto(s)
Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores CCR4/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Línea Celular , Secuencia Conservada , Cristalización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Cuaternaria de Proteína , Estabilidad del ARN/genética , Receptores CCR4/química , Factores de Transcripción/genéticaRESUMEN
Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. Here, we investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, we found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an α-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. We further show that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40.
Asunto(s)
Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Estabilidad del ARN , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular , ADN Helicasas/química , ADN Helicasas/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Proteínas de Unión al ARN , Ribonucleasas/química , Ribonucleasas/genética , Alineación de Secuencia , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases.
Asunto(s)
Colitis/genética , Ciclooxigenasa 2/genética , Silenciador del Gen , Técnicas de Transferencia de Gen , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Colitis/terapia , Modelos Animales de Enfermedad , Regulación hacia Abajo , Escherichia coli/genética , Escherichia coli/metabolismo , Microbioma Gastrointestinal , Expresión Génica , Terapia Genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR-101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR-101 re-expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR-101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR-101 expression and important CRC pro-malignant features, such as inflammation, activation of the Wnt/ß-catenin signalling pathway and epithelial-mesenchymal transition (EMT). We then propose that up-regulated miR-101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR-101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis.