RESUMEN
Previously, we generated mouse models of Rack1 deficiency to identify key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. However, other than low body weight, we did not detect an overt phenotype in mice constitutively deleted of Rack1 in intestinal epithelia ( vil-Cre: Rack1fl/fl mice), presumably because Rack1 was deleted in <10% of the total surface area of the epithelia. To assess the effect of Rack1 loss throughout the entire intestinal epithelia, we generated another mouse model of Rack1 deficiency, vil-Cre-ERT2: Rack1fl/fl. Within 5-10 days of the initial tamoxifen treatment, the mice lost over 20% of their body weight, developed severe diarrhea that for some was bloody, became critically ill, and died, if not euthanized. Necropsies revealed mildly distended, fluid-, gas-, and sometimes blood-filled loops of small and large bowel, inguinal lymphadenopathy, and thrombocytosis. Rack1 was deleted in nearly 100% of the epithelia in both the small intestine and colon when assessed by immunofluorescent or immunoblot analyses. Rack1 expression in other tissues and organs was not different than in control mice, indicating tissue specificity of the recombination. Histopathology revealed a patchy, erosive, hemorrhagic, inflammatory enterocolitis with denuded, sloughed off surface epithelium, and crypt hyperplasia. These results suggest a protective function for Rack1 in maintaining the integrity of intestinal epithelia and for survival. NEW & NOTEWORTHY Our findings reveal a novel function for Rack1 in maintaining intestinal homeostasis by protecting the epithelial barrier. Rack1 loss results in a patchy, erosive, hemorrhagic, inflammatory enterocolitis, which resembles that of inflammatory bowel diseases (IBD) in humans. Understanding mechanisms that protect barrier function in normal intestine and how loss of that protection contributes to the pathogenesis of IBD could lead to improved therapies for these and other erosive diseases of the gastrointestinal tract.
Asunto(s)
Enterocolitis/metabolismo , Células Epiteliales/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Receptores de Cinasa C Activada/metabolismo , Animales , Diarrea/genética , Diarrea/metabolismo , Diarrea/patología , Enterocolitis/genética , Enterocolitis/patología , Células Epiteliales/patología , Hemorragia Gastrointestinal/genética , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/patología , Predisposición Genética a la Enfermedad , Homeostasis , Mucosa Intestinal/patología , Ratones Noqueados , Permeabilidad , Fenotipo , Receptores de Cinasa C Activada/deficiencia , Receptores de Cinasa C Activada/genética , Transducción de Señal , Pérdida de PesoRESUMEN
Previously, we showed that receptor for activated C kinase 1 (Rack1) regulates growth of colon cells in vitro, partly by suppressing Src kinase activity at key cell cycle checkpoints, in apoptotic and cell survival pathways and at cell-cell adhesions. Here, we generated mouse models of Rack1 deficiency to assess Rack1's function in intestinal epithelia in vivo. Intestinal Rack1 deficiency resulted in proliferation of crypt cells, diminished differentiation of crypt cells into enterocyte, goblet, and enteroendocrine cell lineages, and expansion of Paneth cell populations. Following radiation injury, the morphology of Rack1-deleted small bowel was strikingly abnormal with development of large polypoid structures that contained many partly formed villi, numerous back-to-back elongated and regenerating crypts, and high-grade dysplasia in surface epithelia. These abnormalities were not observed in Rack1-expressing areas of intestine or in control mice. Following irradiation, apoptosis of enterocytes was strikingly reduced in Rack1-deleted epithelia. These novel findings reveal key functions for Rack1 in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia. NEW & NOTEWORTHY Our findings reveal novel functions for receptor for activated C kinase 1 (Rack1) in regulating growth of intestinal epithelia: suppressing crypt cell proliferation and regeneration, promoting differentiation and apoptosis, and repressing development of neoplasia.
Asunto(s)
Apoptosis , Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Receptores de Cinasa C Activada/metabolismo , Regeneración , Animales , Apoptosis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Linaje de la Célula , Proliferación Celular/efectos de la radiación , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Genotipo , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de la radiación , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Neoplasias Intestinales/prevención & control , Intestino Delgado/patología , Intestino Delgado/efectos de la radiación , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Inducidas por Radiación/metabolismo , Neoplasias Inducidas por Radiación/patología , Neoplasias Inducidas por Radiación/prevención & control , Fenotipo , Receptores de Cinasa C Activada/deficiencia , Receptores de Cinasa C Activada/genética , Regeneración/efectos de la radiación , Transducción de SeñalRESUMEN
Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Células Enteroendocrinas/metabolismo , Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Yeyuno/lesiones , Yeyuno/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Células Enteroendocrinas/patología , Regulación de la Expresión Génica , Mucosa Intestinal/patología , Yeyuno/patología , Ratones , Ratones Transgénicos , Células Madre/patologíaRESUMEN
mRNA decay is a major determinant of gene expression. In Escherichia coli, message degradation initiates with an endoribonucleolytic cleavage followed by exoribonuclease digestion to generate 5'-mononucleotides. Although the 3' to 5' processive exoribonucleases, PNPase and RNase II, have long been considered to be mediators of this digestion, we show here that another enzyme, RNase R, also participates in the process. RNase R is particularly important for removing mRNA fragments with extensive secondary structure, such as those derived from the many mRNAs that contain REP elements. In the absence of RNase R and PNPase, REP-containing fragments accumulate to high levels. RNase R is unusual among exoribonucleases in that, by itself, it can digest through extensive secondary structure provided that a single-stranded binding region, such as a poly(A) tail, is present. These data demonstrate that RNase R, which is widespread in prokaryotes and eukaryotes, is an important participant in mRNA decay.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Exorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Mensajero/metabolismo , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Poliadenilación , Estabilidad del ARN , ARN Mensajero/genéticaRESUMEN
Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42 degrees C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31 degrees C or when single mutant strains were grown at 42 degrees C. Pulse-chase analysis showed that the rRNA fragments appeared within 5 min at 42 degrees C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.
Asunto(s)
Endorribonucleasas/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Ribosómico/metabolismo , Secuencia de Bases , Northern Blotting , Cartilla de ADN , Electroforesis en Gel de Campo Pulsado , Exorribonucleasas , Calor , Control de CalidadRESUMEN
Escherichia coli RNase R, a 3' --> 5' exoribonuclease homologous to RNase II, was overexpressed and purified to near homogeneity in its native untagged form by a rapid procedure. The purified enzyme was free of nucleic acid. It migrated upon gel filtration chromatography as a monomer with an apparent molecular mass of approximately 95 kDa, in close agreement with its expected size based on the sequence of the rnr gene. RNase R was most active at pH 7.5-9.5 in the presence of 0.1-0.5 mm Mg(2+) and 50-500 mm KCl. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleotide monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3-5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ. RNase II is more active on poly(A), whereas RNase R is much more active on rRNAs. Neither RNase R nor RNase II can degrade a complete RNA-RNA or DNA-RNA hybrid or one with a 4-nucleotide 3'-RNA overhang. RNase R differs from RNase II in that it cannot digest DNA oligomers and is not inhibited by such molecules, suggesting that it does not bind DNA. Although the in vivo function of RNase R is not known, its ability to digest certain natural RNAs may explain why it is maintained in E. coli together with RNase II.