RESUMEN
Human inherited disorders of interferon-gamma (IFN-γ) immunity underlie severe mycobacterial diseases. We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to interleukin-23 (IL-23) and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN-γ by innate-like adaptive mucosal-associated invariant T cells (MAIT) and γδ T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-γ.
Asunto(s)
Interferón gamma , Janus Quinasa 2 , Infecciones por Mycobacterium , Humanos , Masculino , Proteínas de Ciclo Celular/metabolismo , Interferón gamma/inmunología , Interleucina-12 , Interleucina-23 , Janus Quinasa 2/metabolismo , Mycobacterium/fisiología , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/metabolismo , Proteínas Oncogénicas/metabolismoRESUMEN
Roughly half of animal mRNAs contain upstream open reading frames (uORFs). These uORFs can represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5' end and then scan for ORFs in a 5'-to-3' fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important instance of post-transcriptional regulation that affects gene expression. Few molecular factors regulating or facilitating this process are known. Here we show that the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C impact translation initiation. We find that they bind eukaryotic translation initiation factors and preinitiation complexes, and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote leaky scanning past translation start codons, thereby promoting translation of mRNAs containing uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.
Asunto(s)
Iniciación de la Cadena Peptídica Traduccional , Ribosomas , Animales , Codón Iniciador/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Codón/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sistemas de Lectura Abierta/genética , Biosíntesis de ProteínasRESUMEN
Familial Dysautonomia (FD) is an autosomal recessive disorder caused by a splice site mutation in the gene ELP1, which disproportionally affects neurons. While classically characterized by deficits in sensory and autonomic neurons, neuronal defects in the central nervous system have also been described. Although ELP1 expression remains high in the normal developing and adult cerebellum, its role in cerebellar development is unknown. To explore the role of Elp1 in the cerebellum, we knocked out Elp1 in cerebellar granule cell progenitors (GCPs) and examined the outcome on animal behavior and cellular composition. We found that GCP-specific conditional knockout of Elp1 (Elp1cKO) resulted in ataxia by 8 weeks of age. Cellular characterization showed that the animals had smaller cerebella with fewer granule cells. This defect was already apparent as early as 7 days after birth, when Elp1cKO animals also had fewer mitotic GCPs and shorter Purkinje dendrites. Through molecular characterization, we found that loss of Elp1 was associated with an increase in apoptotic cell death and cell stress pathways in GCPs. Our study demonstrates the importance of ELP1 in the developing cerebellum, and suggests that loss of Elp1 in the GC lineage may also play a role in the progressive ataxia phenotypes of FD patients.
Asunto(s)
Cerebelo , Disautonomía Familiar , Ratones Noqueados , Fenotipo , Animales , Disautonomía Familiar/genética , Disautonomía Familiar/patología , Cerebelo/metabolismo , Cerebelo/patología , Ratones , Modelos Animales de Enfermedad , Ataxia/genética , Ataxia/patología , Ataxia/metabolismo , Células-Madre Neurales/metabolismo , Apoptosis/fisiología , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5' TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.
Asunto(s)
Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína S6 Ribosómica/genética , Animales , Células Cultivadas , Células HeLa , Humanos , Immunoblotting , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Mutación , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , RNA-Seq/métodos , Proteína S6 Ribosómica/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
From the cellular perspective, organ growth is determined by production and growth of cells. Uncovering how these two processes are coordinated is essential for understanding organogenesis and regulation of organ growth. We utilized phenotypic and genetic variation of 252 natural accessions of Arabidopsis thaliana to conduct genome-wide association studies (GWAS) for identifying genes underlying root growth variation; using a T-DNA line candidate approach, we identified one gene involved in root growth control and characterized its function using microscopy, root growth kinematics, G2/M phase cell count, ploidy levels and ribosome polysome profiles. We identified a factor contributing to root growth control: Arabidopsis Adenylate Kinase 6 (AAK6). AAK6 is required for normal cell production and normal cell elongation, and its natural genetic variation is involved in determining root growth differences between Arabidopsis accessions. A lack of AAK6 reduces cell production in the aak6 root apex, but this is partially compensated for by longer mature root cells. Thereby, aak6 mutants exhibit compensatory cell enlargement, a phenomenon unexpected in roots. Moreover, aak6 plants accumulate 80S ribosomes while the polysome profile remains unchanged, consistent with a phenotype of perturbed ribosome biogenesis. In conclusion, AAK6 impacts ribosome abundance, cell production and thereby root growth.
Asunto(s)
Adenilato Quinasa , Proteínas de Arabidopsis , Raíces de Plantas/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proliferación Celular , Tamaño de la Célula , Regulación de la Expresión Génica de las Plantas , Estudio de Asociación del Genoma Completo , Homeostasis , Ribosomas/metabolismoRESUMEN
Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Factor 4E Eucariótico de Iniciación , Biosíntesis de Proteínas , ARN Mensajero , Ribosomas , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ribosomas/metabolismo , Unión Proteica , Caperuzas de ARN/metabolismo , Células HEK293 , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Ciclo Celular , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Mechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.
Asunto(s)
Complejos Multiproteicos , Transducción de Señal , Animales , Encéfalo/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.