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Cancers (Basel) ; 13(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375322


Understanding the molecular signatures of colorectal cancer progression under chemotherapeutic treatment will be crucial for the success of future therapy improvements. Here, we used a xenograft-based mouse model to investigate, how whole transcriptome signatures change during metastatic colorectal cancer progression and how such signatures are affected by LDM chemotherapy using RNA sequencing. We characterized mRNAs as well as non-coding RNAs such as microRNAs, long non-coding RNAs and circular RNAs in colorectal-cancer bearing mice with or without LDM chemotherapy. Furthermore, we found that circZNF609 functions as oncogene, since over-expression studies lead to an increased tumor growth while specific knock down results in smaller tumors. Our data represent novel insights into the relevance of non-coding and circRNAs in colorectal cancer and provide a comprehensive resource of gene expression changes in primary tumors and metastases. In addition, we present candidate genes that could be important modulators for successful LDM chemotherapy.

Nucleic Acids Res ; 48(18): 10368-10382, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32955563


Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.

Sítios Internos de Entrada Ribossomal/genética , Metiltransferases/genética , Biossíntese de Proteínas , RNA Circular/genética , Sequência de Bases/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sítios de Splice de RNA/genética , RNA Circular/classificação , Proteínas de Ligação a RNA/genética
Cell Death Differ ; 27(9): 2586-2604, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152556


Angiotensin-(1-9) is a peptide from the noncanonical renin-angiotensin system with anti-hypertrophic effects in cardiomyocytes via an unknown mechanism. In the present study we aimed to elucidate it, basing us initially on previous work from our group and colleagues who proved a relationship between disturbances in mitochondrial morphology and calcium handling, associated with the setting of cardiac hypertrophy. Our first finding was that angiotensin-(1-9) can induce mitochondrial fusion through DRP1 phosphorylation. Secondly, angiotensin-(1-9) blocked mitochondrial fission and intracellular calcium dysregulation in a model of norepinephrine-induced cardiomyocyte hypertrophy, preventing the activation of the calcineurin/NFAT signaling pathway. To further investigate angiotensin-(1-9) anti-hypertrophic mechanism, we performed RNA-seq studies, identifying the upregulation of miR-129 under angiotensin-(1-9) treatment. miR-129 decreased the transcript levels of the protein kinase A inhibitor (PKIA), resulting in the activation of the protein kinase A (PKA) signaling pathway. Finally, we showed that PKA activity is necessary for the effects of angiotensin-(1-9) over mitochondrial dynamics, calcium handling and its anti-hypertrophic effects.

Angiotensina I/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Dinaminas/metabolismo , Hipertrofia , MicroRNAs/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Miócitos Cardíacos/ultraestrutura , Fatores de Transcrição NFATC/metabolismo , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
Proc Natl Acad Sci U S A ; 117(6): 2894-2905, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988137


The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association of Mediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an "activation helix" close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allow us to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

Quinase 8 Dependente de Ciclina/metabolismo , Complexo Mediador/metabolismo , Domínio Catalítico , Ciclina C/genética , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/química , Quinase 8 Dependente de Ciclina/genética , Ativação Enzimática , Humanos , Complexo Mediador/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
Nat Microbiol ; 4(4): 578-586, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30692667


Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.

Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , beta Carioferinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Ribonucleoproteínas/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Replicação Viral