RESUMEN
The genome expression pattern is strongly modified during the heat shock response (HSR) to form an adaptive state. This may be partly achieved by modulating microRNA levels that control the expression of a great number of genes that are embedded within the gene circuitry. Here, we investigated the cross-talk between two highly conserved and universal house-keeping systems, the HSR and microRNA machinery, in Drosophila melanogaster We demonstrated that pronounced interstrain differences in the microRNA levels are alleviated after heat shock (HS) to form a uniform microRNA pattern. However, individual strains exhibit different patterns of microRNA expression during the course of recovery. Importantly, HS-regulated microRNAs may target functionally similar HS-responsive genes involved in the HSR. Despite the observed general downregulation of primary microRNA precursor expression as well as core microRNA pathway genes after HS, the levels of many mature microRNAs are upregulated. This indicates that the regulation of miRNA expression after HS occurs at transcriptional and post-transcriptional levels. It was also shown that deletion of all hsp70 genes had no significant effect on microRNA biogenesis but might influence the dynamics of microRNA expression during the HSR.
Asunto(s)
Drosophila melanogaster/genética , Respuesta al Choque Térmico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/genética , Análisis de Secuencia de ARN/métodos , Animales , Proteínas de Drosophila , Drosophila melanogaster/clasificación , Eliminación de Gen , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Interferencia de ARNRESUMEN
The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34(+) cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Síndromes Mielodisplásicos/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Células Madre/citología , Empalme Alternativo , Anemia Refractaria con Exceso de Blastos/genética , Anemia Refractaria con Exceso de Blastos/metabolismo , Antígenos CD34/metabolismo , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Exones , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Heterocigoto , Homeostasis , Humanos , Células K562 , Masculino , Mutación , Síndromes Mielodisplásicos/metabolismo , Fosfoproteínas/metabolismo , Mutación Puntual , ARN/genética , Empalme del ARN , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Análisis de Secuencia de ARNAsunto(s)
Elementos de Nucleótido Esparcido Largo/genética , Elementos de Nucleótido Esparcido Largo/fisiología , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Oligodesoxirribonucleótidos Antisentido/farmacología , ADN Polimerasa Dirigida por ARN/genética , Animales , Cinética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Modelos Animales , Ratas , Ratas WistarAsunto(s)
Elementos Transponibles de ADN , Sistemas de Lectura Abierta , Filogenia , Animales , HumanosAsunto(s)
Genes Bacterianos , Polisacáridos Bacterianos/biosíntesis , Rhizobium leguminosarum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Bases de Datos Factuales , Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Polisacáridos Bacterianos/genética , Homología de Secuencia de AminoácidoRESUMEN
Nucleotide sequences of two minireplicon fragments of the p1414 cryptic plasmid of Bacillus subtilis were determined. The fragments corresponded to the region containing ori(+) and the gene coding for Rep protein. Comparing sequences of the fragments with corresponding sequences of other ss+ plasmids suggested that ori(+) of p1414 belongs to the family of endogenous cryptic B. subtilis plasmids, which form an individual, closely related subgroup in the group of ori(+) sequences of the pC194 type. It was found that the amino acid sequence of a conservative FLTLTV motif located, together with its flanking sequences, at the N ends of Rep proteins encoded by different ss+ plasmids, is similar to those of several transmembrane proteins and signal peptides. These results, together with computer data on predicting the secondary and tertiary structure of the N-terminal domain of the p1414 Rep protein, suggest that the domain can serve as a "membrane anchor" during plasmid replication.
Asunto(s)
Bacillus subtilis/genética , ADN Helicasas , Proteínas de Unión al ADN , Plásmidos , Replicón/genética , Homología de Secuencia de Ácido Nucleico , Microbiología del Suelo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Bases de Datos Factuales , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/genética , Origen de Réplica , Homología de Secuencia de Aminoácido , Transactivadores/genéticaRESUMEN
Computer search for homology regions of retroelement ORF1 protein products and retroviral gag-encoded proteins was performed using nucleotide and protein sequences of 113 retrotransposons, conservative domains were found in amino acid sequences encoded by viral gene gag: 22 and 17 amino acid residues in capsid and nucleocapsid proteins, respectively. Similar regions were found in potential protein products of ORF1. Another conservative region was revealed by multiple alignment in amino acid sequences of potential protein products of ORF1 of mobile elements and in p19 matrix protein of some retroviruses; this region is different in mobile elements from different classes, but within each class it shows high similarity. The secondary structure of this region is an alpha-helix.
Asunto(s)
Secuencia Conservada , Genes gag , Retroelementos/genética , Retroviridae/genética , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
The Tn5 insertion into the genome of Rhizobium leguminosarum bv viciae VF39, resulting in non-mucoid growth and formation of non-N2-fixing nodule-like structures on Vicia faba plants, was mapped within a 1.4-kb EcoRV-SacI fragment. Nucleotide sequence analysis revealed an ORF (pss4) of 263 amino acids (aa). Three transcription start points (tsp) were determined. Two of them were localized upstream from the first GTG codon; the third tsp was mapped in front of the second putative start codon (GTG) corresponding to Val64 of the Pss4 aa sequence. The expression of pss4 in a T7 RNA polymerase/promoter system produced a single approx. 29-kDa protein. Pss4 reveals similarity to several proteins involved in polysaccharide biosynthesis in various Rhizobium species. A nearly complete homology was found with PssA from Rl biovar phaseoli 8002 [Borthakur et al., Mol. Gen. Genet. 213 (1988) 155-162], except that Pss4 has an additional 63 aa on its N terminus.