RESUMEN
miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.
Asunto(s)
Hipertrofia/genética , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Biosíntesis de Proteínas/genética , Animales , Regulación de la Expresión Génica , Humanos , Ratones , MicroARNs/metabolismo , Miocitos Cardíacos/patología , Poliadenilación/genética , Procesamiento Postranscripcional del ARNRESUMEN
Transcription-induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription-associated recombination, as they both affect transcription elongation and hyper-recombination in a concerted manner. Using a yeast strain that has the wild-type THO2 gene replaced by one encoding a His(6)-HA-tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti-Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Delta and tho2Delta cells, mft1Delta and thp2Delta cells show mitotic hyper- recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper-recombination conferred by mft1Delta and thp2Delta is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/genética , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos , Sustancias Macromoleculares , Mitosis , Mutación , Proteínas Nucleares , Fenotipo , Recombinación Genética , Saccharomyces cerevisiae/citología , Fracciones Subcelulares/metabolismo , Temperatura , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae , Sitios de Unión , Transporte Biológico , Citoplasma/metabolismo , Mitocondrias/genética , Mutación , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/fisiología , Secuencia de Aminoácidos , Animales , División Celular/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Meiosis , Microscopía Electrónica , Datos de Secuencia Molecular , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esporas Fúngicas , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/ultraestructuraRESUMEN
When overexpressed in Saccharomyces cerevisiae, beta-galactosidase fusion proteins directed to the mitochondria are toxic, preventing growth of yeast cells on non-fermentable carbon sources (Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L., Takeda, M., and Douglas, M. G. (1986) J. Cell Biol. 102, 523-533). We show that such fusion proteins interfere with the assembly of respiratory complexes in the mitochondrial inner membrane, without blocking protein translocation. The gene YME1, encoding an ATP-dependent metalloprotease of the mitochondrial inner membrane, acts as a suppressor of this defect; a 3-fold overexpression of Yme1p is sufficient to restore respiratory complex assembly and mitochondrial function. Detailed knowledge of the topology and effect of the toxic beta-galactosidase fusion proteins will permit the identification and characterization of components that control protein sorting and protein assembly within the mitochondrial inner membrane.
Asunto(s)
Membranas Intracelulares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Proteínas de Saccharomyces cerevisiae , beta-Galactosidasa/toxicidad , Proteasas ATP-Dependientes , Adenosina Trifosfatasas , Transporte Biológico , Proteínas Fúngicas/biosíntesis , Sustancias Macromoleculares , Consumo de Oxígeno/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae , Supresión Genética , beta-Galactosidasa/genéticaRESUMEN
Yeast cells harboring mft1 mutations are compromised in mitochondrial protein targeting, and Mft1p has previously been identified as a ribosomal protein. However, two genes, PLC2 and YML062C, are present in the MFT1 locus, and we show that mft1 mutant cells are compromised in the function of the cytosolic protein encoded by YML062C. The ribosomal protein (YS3a) is actually encoded by the tightly linked PLC2 gene, and does not play a role in targeting proteins to the mitochondria.
Asunto(s)
Compartimento Celular , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Genes Fúngicos , Mitocondrias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Levaduras/genética , Secuencia de Aminoácidos , Ligamiento Genético , Datos de Secuencia Molecular , Mutagénesis Insercional , Unión Proteica , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Homología de Secuencia de AminoácidoRESUMEN
We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.