Since their serendipitous discovery in nematodes, microRNAs (miRNAs) have emerged as key regulators of biological processes in animals. These small RNAs form complex networks that regulate cell ...differentiation, development and homeostasis. Deregulation of miRNA function is associated with an increasing number of human diseases, particularly cancer. Recent discoveries have expanded our understanding of the control of miRNA function. Here, we review the mechanisms that modulate miRNA activity, stability and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, viral factors, transport from the cytoplasm and regulation of miRNA-target interactions. We conclude by discussing intriguing, unresolved research questions.
microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host ...miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5' end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.
Vertebrate corpse decomposition provides an important stage in nutrient cycling in most terrestrial habitats, yet microbially mediated processes are poorly understood. Here we combine deep microbial ...community characterization, community-level metabolic reconstruction, and soil biogeochemical assessment to understand the principles governing microbial community assembly during decomposition of mouse and human corpses on different soil substrates. We find a suite of bacterial and fungal groups that contribute to nitrogen cycling and a reproducible network of decomposers that emerge on predictable time scales. Our results show that this decomposer community is derived primarily from bulk soil, but key decomposers are ubiquitous in low abundance. Soil type was not a dominant factor driving community development, and the process of decomposition is sufficiently reproducible to offer new opportunities for forensic investigations.
MicroRNAs (miRNAs) are short noncoding RNAs, which bind to messenger RNAs and regulate protein expression. The biosynthesis of miRNAs includes two precursors, a primary miRNA transcript (pri-miRNA) ...and a shorter pre-miRNA, both of which carry a common stem-loop bearing the mature miRNA. MiR-122 is a liver-specific miRNA with an important role in the life cycle of hepatitis C virus (HCV). It is the target of miravirsen (SPC3649), an antimiR drug candidate currently in clinical testing for treatment of HCV infections. Miravirsen is composed of locked nucleic acid (LNAs) ribonucleotides interspaced throughout a DNA phosphorothioate sequence complementary to mature miR-122. The LNA modifications endow the drug with high affinity for its target and provide resistance to nuclease degradation. While miravirsen is thought to work mainly by hybridizing to mature miR-122 and blocking its interaction with HCV RNA, its target sequence is also present in pri- and pre-miR-122. Using new in vitro and cellular assays specifically developed to discover ligands that suppress biogenesis of miR-122, we show that miravirsen binds to the stem-loop structure of pri- and pre-miR-122 with nanomolar affinity, and inhibits both Dicer- and Drosha-mediated processing of miR-122 precursors. This inhibition may contribute to the pharmacological activity of the drug in man.
Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with the liver-specific microRNA, miR-122. miR-122 binds to two sites in the 5' untranslated region (UTR) and this interaction ...promotes HCV RNA accumulation, although the precise role of miR-122 in the HCV life cycle remains unclear. Using biophysical analyses and Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) we investigated miR-122 interactions with the 5' UTR. Our data suggests that miR-122 binding results in alteration of nucleotides 1-117 to suppress an alternative secondary structure and promote functional internal ribosomal entry site (IRES) formation. Furthermore, we demonstrate that two hAgo2:miR-122 complexes are able to bind to the HCV 5' terminus simultaneously and SHAPE analyses revealed further alterations to the structure of the 5' UTR to accommodate these complexes. Finally, we present a computational model of the hAgo2:miR-122:HCV RNA complex at the 5' terminus of the viral genome as well as hAgo2:miR-122 interactions with the IRES-40S complex that suggest hAgo2 is likely to form additional interactions with SLII which may further stabilize the HCV IRES. Taken together, our results support a model whereby hAgo2:miR-122 complexes alter the structure of the viral 5' terminus and promote formation of the HCV IRES.
The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of ...matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.
The objective of this study was to evaluate the chemical composition and fatty acid profile of broilers fed diets containing glycerol monolaurate (GML) in place of antimicrobials. Groups: T0 group ...used as control; T100, T200, and T300 groups received diets supplemented with 100, 200, and 300 mg/kg of GML, respectively. The feed mixture used in the poultry feed during the four phases of the production cycle (days 1 to 7; 8 to 21; 22 to 35; and 36 to 42 of birds age) showed similar levels of protein, lipid and ash, as well as fatty acid profiles. Samples of frozen breasts from chickens slaughtered at 42 days of age were used for chemical gross composition and fatty acid analysis. We observed lower lipid levels in the meat of broilers in the T200 and T300 groups than in the T0 group. Lower lipid peroxidation occurred in the meat of animals that consumed GML in respect to control. Total saturated fatty acid percentage was lower, while total polyunsaturated fatty acid percentage was higher in the meat of broilers fed GML than in the control group. We conclude that the increase in GML concentrations alters the lipid profile of broiler meat.
Abstract Co-transcriptional assembly is an integral feature of the formation of RNA–protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict ...order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the structural insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of 23 resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.
Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc ...knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.
Bacteria within the genus
can be abundant in showerheads, and the inhalation of aerosolized mycobacteria while showering has been implicated as a mode of transmission in nontuberculous mycobacterial ...(NTM) lung infections. Despite their importance, the diversity, distributions, and environmental predictors of showerhead-associated mycobacteria remain largely unresolved. To address these knowledge gaps, we worked with citizen scientists to collect showerhead biofilm samples and associated water chemistry data from 656 households located across the United States and Europe. Our cultivation-independent analyses revealed that the genus
was consistently the most abundant genus of bacteria detected in residential showerheads, and yet mycobacterial diversity and abundances were highly variable. Mycobacteria were far more abundant, on average, in showerheads receiving municipal water than in those receiving well water and in U.S. households than in European households, patterns that are likely driven by differences in the use of chlorine disinfectants. Moreover, we found that water source, water chemistry, and household location also influenced the prevalence of specific mycobacterial lineages detected in showerheads. We identified geographic regions within the United States where showerheads have particularly high abundances of potentially pathogenic lineages of mycobacteria, and these "hot spots" generally overlapped those regions where NTM lung disease is most prevalent. Together, these results emphasize the public health relevance of mycobacteria in showerhead biofilms. They further demonstrate that mycobacterial distributions in showerhead biofilms are often predictable from household location and water chemistry, knowledge that advances our understanding of NTM transmission dynamics and the development of strategies to reduce exposures to these emerging pathogens.
Bacteria thrive in showerheads and throughout household water distribution systems. While most of these bacteria are innocuous, some are potential pathogens, including members of the genus
that can cause nontuberculous mycobacterial (NTM) lung infection, an increasing threat to public health. We found that showerheads in households across the United States and Europe often harbor abundant mycobacterial communities that vary in composition depending on geographic location, water chemistry, and water source, with households receiving water treated with chlorine disinfectants having particularly high abundances of certain mycobacteria. The regions in the United States where NTM lung infections are most common were the same regions where pathogenic mycobacteria were most prevalent in showerheads, highlighting the important role of showerheads in the transmission of NTM infections.
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