RESUMO
Upstream of N-ras (UNR) is a conserved RNA-binding protein that regulates mRNA translation and stability by binding to sites generally located in untranslated regions (UTRs). In Drosophila, sex-specific binding of UNR to msl2 mRNA and the noncoding RNA roX is believed to play key roles in the control of X-chromosome dosage compensation in both sexes. To investigate broader sex-specific functions of UNR, we have identified its RNA targets in adult male and female flies by high-throughput RNA binding and transcriptome analysis. Here we show that UNR binds to a large set of protein-coding transcripts and to a smaller set of noncoding RNAs in a sex-specific fashion. The analyses also reveal a strong correlation between sex-specific binding of UNR and sex-specific differential expression of UTRs in target genes. Validation experiments indicate that UNR indeed recognizes sex-specifically processed transcripts. These results suggest that UNR exploits the transcript diversity generated by alternative processing and alternative promoter usage to bind and regulate target genes in a sex-specific manner.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Regiões não Traduzidas , Animais , Drosophila melanogaster/genética , Feminino , Masculino , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fatores Sexuais , Transcrição GênicaRESUMO
Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that SXL recruits the protein upstream of N-ras (UNR) to the 3' UTR of msl-2 mRNA. UNR is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). Here, we dissect the domains of UNR required for translational repression and complex formation with SXL and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with SXL and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human UNR can also form a complex with SXL and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that UNR binding to SXL and msl-2 can be distinguished from translation inhibition. Repression by tethered UNR requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of UNR resides within the first 397 amino acids of the protein. Our results identify domains and residues required for UNR function in translational control.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/metabolismo , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RNA-binding proteins regulate every aspect of RNA metabolism, including pre-mRNA splicing, mRNA trafficking, stability, and translation. This review summarizes the available information on molecular mechanisms of translational repression by RNA-binding proteins. By using a specific set of well-defined examples, we also describe how regulation can be reversed.
Assuntos
Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Inativação Gênica , Modelos Biológicos , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (SXL) is an essential regulatory step for X-chromosome dosage compensation in Drosophila melanogaster. The mammalian upstream of N-ras (UNR) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. Here we have identified the Drosophila homolog of mammalian UNR as a cofactor required for SXL-mediated repression of msl-2 translation. UNR interacts with SXL, a female-specific protein. Although UNR is present in both male and female flies, binding of SXL to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits UNR to adjacent regulatory sequences, thereby conferring a sex-specific function to UNR. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with SXL in translational control.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila/genética , Proteínas Nucleares/fisiologia , Biossíntese de Proteínas/fisiologia , Fatores de Transcrição/fisiologia , Cromossomo X/metabolismo , Regiões 3' não Traduzidas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Mecanismo Genético de Compensação de Dose/fisiologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Masculino , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismoRESUMO
MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (SXL), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Here, we purify translationally silenced msl-2 mRNPs and identify UNR (upstream of N-ras) as a protein recruited to the 3' UTR by SXL. We demonstrate that SXL requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. Our results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a "fail-safe" dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Feminino , Humanos , Masculino , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Homologia de Sequência , Fatores de Transcrição/genética , TransfecçãoRESUMO
BACKGROUND: Factor V Leiden has been described as a common genetic risk factor for venous thromboembolism. The geographic distribution of this abnormality varies greatly, being high in Europe and almost absent in Asia and Africa. Particularly high prevalence is observed in some Mediterranean countries, which suggests the Mediterranean origin of this mutation. Similarly, prevalence of silent mutation 1311 of the G6PD gene seems to be higher among Mediterranean populations. Since the Dalmatian population (of south Croatia) geographically belongs to the Mediterranean populations we analyzed the prevalence of FV-Leiden and silent mutation 1311 in this region. Furthermore, because the coincidence of G6PD deficiency and venous thromboembolism was described earlier, we tested a possible association of FV-Leiden and G6PD deficiency. METHODS: One hundred sixty-eight healthy blood donors and 55 G6PD deficient individuals originating from the Dalmatian region were tested for the presence of FV-Leiden mutation and silent mutation 1311. RESULTS: Prevalence of FV-Leiden among blood donors was 2.4%, while among G6PD deficient individuals it was significantly higher, 11% (p=0.011). Prevalence of silent mutation 1311 among blood donors and G6PD deficient individuals was 21 and 15%, respectively. CONCLUSIONS: Observed allele frequencies among individuals originating from the Dalmatian region is similar to the neighboring European and Mediterranean populations. Interestingly, our results indicate the association of the FV-Leiden and G6PD deficiency and warrant further studies.
