RESUMO
Axonal mRNA transport is robust in cultured neurons but there has been limited evidence for this in vivo. We have used a genetic approach to test for in vivo axonal transport of reporter mRNAs. We show that ß-actin's 3'-UTR can drive axonal localization of GFP mRNA in mature DRG neurons, but mice with γ-actin's 3'-UTR show no axonal GFP mRNA. Peripheral axotomy triggers transport of the ß-actin 3'-UTR containing transgene mRNA into axons. This GFP-3'-ß-actin mRNA accumulates in injured PNS axons before activation of the transgene promoter peaks in the DRG. Spinal cord injury also increases axonal GFP signals in mice carrying this transgene without any increase in transgene expression in the DRGs. These data show for the first time that the ß-actin 3'-UTR is sufficient for axonal localization in both PNS and CNS neurons in vivo.
Assuntos
Axônios/metabolismo , Gânglios Espinais/citologia , Neurônios/citologia , RNA Mensageiro/metabolismo , Medula Espinal/citologia , Regiões 3' não Traduzidas/genética , Actinas/genética , Actinas/metabolismo , Análise de Variância , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Dendritos/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Células de Schwann/metabolismo , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologiaRESUMO
Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.
Assuntos
Neurônios/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Actinas/genética , Actinas/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Hibridização In Situ , Glicoproteína Associada a Mielina/metabolismo , Fatores de Crescimento Neural/fisiologia , Regeneração Nervosa , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Semaforina-3A/metabolismoRESUMO
The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.