RESUMO
Experimental observations suggested that the length of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA 3' end has a role in regulating rates of translation in the parasitic protists Trypanosoma brucei, Leishmania donovani, and Trichomonas vaginalis. Using a PCR assay for poly(A) tail length, we measured the size of the RNA 3' end under different growth conditions in all three species. Our results showed that the combined 3' untranslated region and poly(A) tail of GAPDH mRNA do not vary with different rates of translation.
Assuntos
Regiões 3' não Traduzidas/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Leishmania donovani/genética , Biossíntese de Proteínas , Trichomonas vaginalis/genética , Trypanosoma brucei brucei/genética , Animais , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Leishmania donovani/enzimologia , Poli A/genética , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA de Protozoário/genética , Trichomonas vaginalis/enzimologia , Trypanosoma brucei brucei/enzimologiaRESUMO
The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA.
Assuntos
Poli A/análise , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/química , Northern Blotting , Primers do DNA , DNA ComplementarRESUMO
During early development in mouse and Xenopus, translational activation of stored maternal mRNAs by cytoplasmic polyadenylation requires both the nuclear polyadenylation signal AAUAAA and U-rich cis-acting adenylation control elements (ACEs), also termed cytoplasmic polyadenylation elements, located in the 3' UTR. Using an ACE-based PCR strategy (Sallés et al., 1992) we have isolated two novel cDNAs from mouse oocytes: OM2a and OM2b (for Oocyte Maturation). Each message contains an ACE consensus sequence upstream of AAUAAA, is specifically transcribed in the growing oocyte, and is cytoplasmically polyadenylated upon oocyte maturation. Comparison of the mouse and rat homologs reveals considerable nucleotide sequence homology and conservation of overall gene organization. However, the predicted open reading frames are far less conserved, suggesting that these genes may not be functioning as proteins. The tissue specificity and tight temporal regulation of the RNAs suggest a role for these genes during early development.
Assuntos
Citoplasma/metabolismo , Oogênese/genética , Poli A/biossíntese , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Ratos , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência , Caracteres Sexuais , Especificidade da Espécie , Distribuição TecidualRESUMO
A rapid and sensitive technique is described that measures the length of the poly(A) tail on a specific mRNA within subnanogram quantities of total cellular RNA [the Poly(A) test (PAT)]. In a single-tube reaction, a poly(dT) primer is synthesized in situ on the poly(A) tail of mRNAs using oligo(dT) and DNA ligase. By modulating the annealing temperature and primer concentrations, a GC-rich adapter sequence is targeted to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor is then used to prime reverse transcription of the mRNA, yielding a library of PAT cDNAs. The length of a poly(A) tail is determined by PCR amplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quantitative determination of changes in polyadenylation of a given mRNA. This technique overcomes many of the pitfalls associated with conventional poly(A) tail length assessments and should prove useful in studying a variety of processes relating to polyadenylation.
Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/química , Animais , Sequência de Bases , Primers do DNA , Feminino , Indicadores e Reagentes , Camundongos , Dados de Sequência Molecular , Oócitos/química , Oócitos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Sensibilidade e Especificidade , Especificidade da Espécie , Fatores de Tempo , XenopusRESUMO
Pattern formation in Drosophila depends initially on the translational activation of maternal messenger RNAs (mRNAs) whose protein products determine cell fate. Three mRNAs that dictate anterior, dorsoventral, and terminal specification--bicoid, Toll, and torso, respectively--showed increases in polyadenylate [poly(A)] tail length concomitant with translation. In contrast, posteriorly localized nanos mRNA, although also translationally activated, was not regulated by poly(A) status. These results implicate at least two mechanisms of mRNA activation in flies. Studies with bicoid mRNA showed that cytoplasmic polyadenylation is necessary for translation, establishing this pathway as essential for embryogenesis. Combined, these experiments identify a regulatory pathway that can coordinate initiation of maternal pattern formation systems in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Homeodomínio , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Receptores Proteína Tirosina Quinases , Receptores de Superfície Celular , Transativadores , Animais , Sequência de Bases , Citoplasma/metabolismo , Drosophila/genética , Desenvolvimento Embrionário , Feminino , Hormônios de Inseto/genética , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Morfogênese , Ovário/metabolismo , Proteínas Tirosina Quinases/genética , RNA Mensageiro/genética , Receptores Toll-LikeAssuntos
Oligonucleotídeos Antissenso/farmacologia , Oócitos/metabolismo , RNA/biossíntese , Animais , Separação Celular/métodos , Células Cultivadas , Técnicas de Cultura/métodos , Feminino , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Microinjeções/métodos , Oócitos/citologia , Ovário/citologia , Transcrição Gênica , XenopusRESUMO
The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.
Assuntos
Citoplasma/metabolismo , Poli A/metabolismo , RNA Mensageiro/isolamento & purificação , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The use of purified piscine plasminogen in a chromogenic solution assay enabled us to detect plasminogen activator (PA) activity in crude homogenates of goldfish optic nerve following nerve injury. In contrast, no activity was detected in the homogenates of uninjured nerve. Under conditions allowing regeneration of the optic axons (optic nerve crush), PA activity peaked 8 days after crush, and decreased to undetectable levels by 60 days. Under conditions allowing only degeneration of the axons (enucleation), the activity peaked at 8 days but decreased more rapidly. Casein zymography of samples after fractionation in SDS-PAGE showed that PA activity migrated as a doublet at Mr = 60-65 kd. Using this assay, activity was also observed in uninjured control nerves. This plasminogen-dependent activity migrated as three bands of higher molecular weight (Mr = 75, 95 and 120 kd) and was undetectable in solution assays of unfractionated extracts, suggesting complex formation with an inhibitor(s). Fibrin overlay assay of retinal explants and isolated primary cells in culture suggest that the goldfish PA is associated with the glial cells of the goldfish visual pathway.
