RESUMO
Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.
Assuntos
Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Proteínas Nucleares/biossíntese , Biossíntese de Proteínas , RNA/metabolismo , Ribossomos/metabolismo , Animais , Linhagem Celular , Códon de Iniciação , Códon de Terminação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição Forkhead/metabolismo , Genótipo , Cabeça , Espectrometria de Massas , Camundongos , Mutação , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Estado Nutricional , Fenótipo , RNA/química , RNA/genética , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Circular , Ratos , Ribossomos/química , Ribossomos/genética , Inanição/genética , Inanição/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.
Assuntos
Células-Tronco Adultas/fisiologia , Planárias/genética , Planárias/fisiologia , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Regeneração/genética , Regeneração/fisiologia , Células-Tronco Adultas/citologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Modelos Biológicos , Conformação de Ácido Nucleico , Planárias/citologia , Interferência de RNA , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA de Helmintos/química , Homologia de Sequência do Ácido NucleicoRESUMO
Introduction: Controlled randomized trials, molecular analytics, and guideline recommendations have so far been irreplaceable tools to ensure appropriate treatment and decision-making for physicians and patients. Individual patient models are increasingly complementing these methods, particularly in the case of advanced cancers, rare cancers, and cancers of unknown primary (CUP), as in these cases comprehensive clinical evidence is unavailable, often resulting in poor treatment success, even after stratification. Case Presentation: Here we report a 53-year-old patient with CUP with axillary lymph node metastases for whom patient-derived 3D (PD3D®) tumor organoids successfully guided personalized treatment. PD3D tumor models were used to screen drugs that are effective at the suspected primary tumor site. The screen revealed sensitivity to doxorubicin, which is not indicated for CUP treatment but hinted toward breast cancer that was subsequently confirmed as triple-negative breast cancer (TNBC). The patient showed partial remission to first-line doxorubicin and cyclophosphamide, which were followed by docetaxel. Subsequent radiotherapy eventually led to a complete remission, which is still ongoing. Conclusion: We conclude that pre-therapeutic drug sensitivity screening with PD3D tumor models can be essential in guiding and enabling an effective personalized treatment for patients with hard-to-treat cancers, like CUP or TNBC.