RESUMEN
Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.
Asunto(s)
Codón Iniciador/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Transducción de Señal/genética , Virus Sindbis/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Línea Celular Tumoral , Codón Iniciador/metabolismo , Factor 2 Eucariótico de Iniciación/deficiencia , Factor 2 Eucariótico de Iniciación/genética , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Haploidia , Interacciones Huésped-Patógeno/genética , Humanos , Secuencias Invertidas Repetidas , Leucina/genética , Leucina/metabolismo , Metionina/genética , Metionina/metabolismo , Conformación de Ácido Nucleico , ARN Mensajero/metabolismo , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Valina/genética , ARN de Transferencia de Valina/metabolismo , ARN Viral/metabolismo , Replicón , Virus Sindbis/metabolismo , Valina/genética , Valina/metabolismoRESUMEN
PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.
Asunto(s)
Regiones no Traducidas 5' , Regulación de la Expresión Génica , Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , Receptores de Superficie Celular/genética , Secuencia de Bases , Hipoxia de la Célula/genética , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Receptores Patched , Receptor Patched-1 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Transducción de SeñalRESUMEN
It is widely known that an optimal nucleotide sequence context immediately upstream of the AUG start codon greatly improves the efficiency of translation initiation of mRNA in mammalian and plant somatic cells, which in turn increases protein levels. However, it is still unclear whether a similar regulatory mechanism is also present in highly differentiated cells. Here, we surveyed this issue in Arabidopsis thaliana sperm cells and found that the sequence context-mediated regulation of translation initiation in sperm cells is generally similar to that in somatic cells. A simple motif of four adenine nucleotides at positions - 1 to - 4 greatly improved the efficiency of translation initiation, and when the motif was present there, translation was even initiated at some non-AUG codons in sperm cells. However, unlike that in mammalian cells, a mainly effective nucleotide site to regulate the efficiency of translation initiation was not present at positions - 1 to - 4 in sperm cells. Meanwhile, different from somatic cells, sperm cells did not use eukaryotic translation initiation factor 1 to regulate the efficiency in a poor context consisting of the lowest frequency nucleotides. All these results contribute to our understanding of the cytoplasmic event of translation initiation in highly differentiated sperm cells.