RESUMO
The bacterial pathogen Listeria monocytogenes encodes seven homologous small regulatory RNAs, named the LhrC family of sRNAs. The LhrCs are highly induced under infection-relevant conditions and are known to inhibit the expression of multiple target mRNAs encoding virulence-associated surface proteins. In all cases studied so far, the LhrCs use their CU-rich regions for base pairing to complementary AG-rich sequences of the ribosomal binding site (RBS) of specific target mRNAs. Consequently, LhrC-mRNA interaction results in inhibition of translation followed by mRNA degradation, corresponding to the canonical model for sRNA-mediated gene regulation in bacteria. Here, we demonstrate that the LhrC sRNAs employ a different regulatory mechanism when acting to down-regulate the expression of tcsA, encoding a T cell-stimulating antigen. In this case, LhrC base pairs to an AG-rich site located well upstream of the RBS in tcsA mRNA. Using an in vitro translation assay, we found that LhrC could not prevent the ribosome from translating the tcsA messenger. Rather, the LhrC sRNAs act to decrease the half-life of tcsA mRNA in vivo. Importantly, LhrC-mediated destabilization of tcsA mRNA relies on an intact LhrC binding site near the 5´-end of the tcsA mRNA and occurs independently of translation. Based on these findings, we propose an alternative mechanism for LhrC-mediated control in L. monocytogenes that relies solely on sRNA-induced degradation of a target mRNA.
Assuntos
Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Interferência de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Regiões 5' não Traduzidas , Sequência de Bases , Sítios de Ligação , Listeriose/imunologia , Modelos Biológicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/química , Pequeno RNA não Traduzido/químicaRESUMO
All organisms contain RNA modifications in their ribosomal RNA (rRNA), but the importance, positions and exact function of these are still not fully elucidated. Various functions such as stabilizing structures, controlling ribosome assembly and facilitating interactions have been suggested and in some cases substantiated. Bacterial rRNA contains much fewer modifications than eukaryotic rRNA. The rRNA modification patterns in bacteria differ from each other, but too few organisms have been mapped to draw general conclusions. This study maps 23S ribosomal RNA modifications in Clostridium sporogenes that can be characterized as a non-toxin producing Clostridium botulinum. Clostridia are able to sporulate and thereby survive harsh conditions, and are in general considered to be resilient to antibiotics. Selected regions of the 23S rRNA were investigated by mass spectrometry and by primer extension analysis to pinpoint modified sites and the nature of the modifications. Apparently, C. sporogenes 23S rRNA contains few modifications compared to other investigated bacteria. No modifications were identified in domain II and III of 23S rRNA. Three modifications were identified in domain IV, all of which have also been found in other organisms. Two unusual modifications were identified in domain V, methylated dihydrouridine at position U2449 and dihydrouridine at position U2500 (Escherichia coli numbering), in addition to four previously known modified positions. The enzymes responsible for the modifications were searched for in the C. sporogenes genome using BLAST with characterized enzymes as query. The search identified genes potentially coding for RNA modifying enzymes responsible for most of the found modifications.
