The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5' end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral ...RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5' terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.
RNA is a versatile macromolecule that accommodates functional information in primary sequence and secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, ...comparative sequence analysis, and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus’ life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a “code within the code” for controlling biological processes.
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•The genome of HCV is folded into specific RNA structures•HCV genomic structures are conserved across multiple genotypes•Elaborate RNA structures are present within protein-coding sequences•Genetic manipulation of these structures affects replication and infectivity
Using biochemistry and viral genetics, the RNA genome of HCV was shown to contain numerous well-conserved RNA structures within protein-coding regions. Genetic manipulation of these structures alters the ability of HCV to replicate and infect, thereby demonstrating that RNA sequences can store multiple layers of information.
Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly ...immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/
) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment
These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers.
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RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, ...we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.
NS3, an essential helicase for replication of hepatitis C virus, is a model enzyme for investigating helicase function. Using single-molecule fluorescence analysis, we showed that NS3 unwinds DNA in ...discrete steps of about three base pairs (bp). Dwell time analysis indicated that about three hidden steps are required before a 3-bp step is taken. Taking into account the available structural data, we propose a spring-loaded mechanism in which several steps of one nucleotide per adenosine triphosphate molecule accumulate tension on the protein-DNA complex, which is relieved periodically via a burst of 3-bp unwinding. NS3 appears to shelter the displaced strand during unwinding, and, upon encountering a barrier or after unwinding >18 bp, it snaps or slips backward rapidly and repeats unwinding many times in succession. Such repetitive unwinding behavior over a short stretch of duplex may help to keep secondary structures resolved during viral genome replication.
Deep carbon emissions from historically inactive volcanoes, hydrothermal, and tectonic structures are among the greatest unknowns in the long‐term (∼Myr) carbon cycle. Recent estimates of diffuse CO2 ...flux from the Eastern Rift of the East African Rift System (EARS) suggest this could equal emissions from the entire mid‐ocean ridge system. We report new CO2 surveys from the Main Ethiopian Rift (MER, northernmost EARS), and reassess the rift‐related CO2 flux. Since degassing in the MER is concentrated in discrete areas of volcanic and off‐edifice activity, characterization of such areas is important for extrapolation to a rift‐scale budget. Locations of hot springs and fumaroles along the rift show numerous geothermal areas away from volcanic edifices. With these new data, we estimate total CO2 emissions from the central and northern MER as 0.52–4.36 Mt yr−1. Our extrapolated flux from the Eastern Rift is 3.9–32.7 Mt yr−1 CO2, overlapping with lower end of the range presented in recent estimates. By scaling, we suggest that 6–18 Mt yr−1 CO2 flux can be accounted for by magmatic extension, which implies an important role for volatile‐enriched lithosphere, crustal assimilation, and/or additional magmatic intrusion to account for the upper range of flux estimates. Our results also have implications for the nature of volcanism in the MER. Many geothermal areas are found >10 km from the nearest volcanic center, suggesting ongoing hazards associated with regional volcanism.
Plain Language Summary
The amount of carbon dioxide seeping out of the Earth's surface is poorly understood. Magma carries dissolved carbon dioxide from the deep earth toward the surface, where it is released and travels along fractures in the crust. This study attempts to quantify this phenomenon in central Ethiopia, where a continental rift is splitting one tectonic plate in two. In Kenya and Tanzania, a similar study suggested that the flow of carbon dioxide through the East African Rift was much larger than previously thought. We undertook new surveys and found that it varies greatly, which makes estimating a total flow through the rift very difficult. The distribution of hot springs and volcanic vents provides clues concerning where heat and carbon dioxide come to the surface—by compiling the locations of these features we were able to extrapolate from our surveys for a new estimate. Our results suggest that the East African Rift releases less carbon dioxide than was thought from Kenya and Tanzania, but still a substantial amount. If the rift does emit as much carbon dioxide as suggested, either more carbon is below the crust in East Africa than we thought or more magma is involved.
Key Points
New gas surveys show that diffuse CO2 degassing in the Main Ethiopian Rift is highly variable, concentrated along faults in geothermal areas
Locations of hot springs and fumaroles indicate numerous (∼14–20) geothermal areas some distance from major volcanic edifices
Extrapolation from our data suggests a total diffuse CO2 flux from the Main Ethiopian Rift of 0.5–4.4 Mt yr−1 (whole Eastern Rift: 4–33 Mt yr−1)
Mercury is a highly volatile, bioaccumulating toxic trace metal with a long (∼1
yr) atmospheric residence time. Hg is strongly enriched in volcanic emanations, and volcanoes are the only natural ...sources of direct Hg emission to the free troposphere and stratosphere. However, there is considerable uncertainty over the annual emission rate of mercury from volcanoes. Previous estimates, based on limited measurements from volcanic plumes, span three orders of magnitude (∼10
0–10
3
Mg
Hg/yr), or from <1% to ∼50% of total natural Hg emissions.
Here we critically evaluate published data from volcanic plumes, and combine this with information from natural archives to show unequivocally the significance of volcanoes for the global biogeochemical mercury cycle. ‘Low’ global volcanic flux estimates (<∼50
Mg/yr) are based on the inappropriate extrapolation of data from low-temperature fumarolic degassing at non-erupting volcanoes to the high-temperature emissions from active volcanoes. Based on data from active volcanoes, we estimate that the time-averaged volcanic Hg emission is ∼700
Mg/yr, or 20–40% of total natural emissions. Continuous degassing accounts for only ∼10% of this flux, while 75% of volcanic Hg is released during ‘smaller’ sporadic eruptions (<10–10
2
Mg/event). Rare, large (>10
3
Mg) explosive eruptions overwhelm the total atmospheric burden several times per century, and account for ∼15% of total volcanic Hg emissions.
Several lines of evidence have previously been used to suggest that ice retreat after the last glacial maximum (LGM) resulted in regionally-increased levels of volcanic activity. It has been proposed ...that this increase in volcanism was globally significant, forming a substantial component of the post-glacial rise in atmospheric CO2, and thereby contributing to climatic warming. However, as yet there has been no detailed investigation of activity in glaciated volcanic arcs following the LGM. Arc volcanism accounts for 90% of present-day subaerial volcanic eruptions. It is therefore important to constrain the impact of deglaciation on arc volcanoes, to understand fully the nature and magnitude of global-scale relationships between volcanism and glaciation.
The first part of this paper examines the post-glacial explosive eruption history of the Andean southern volcanic zone (SVZ), a typical arc system, with additional data from the Kamchatka and Cascade arcs. In all cases, eruption rates in the early post-glacial period do not exceed those at later times at a statistically significant level. In part, the recognition and quantification of what may be small (i.e. less than a factor of two) increases in eruption rate is hindered by the size of our datasets. These datasets are limited to eruptions larger than 0.1km3, because deviations from power-law magnitude–frequency relationships indicate strong relative under-sampling at smaller eruption volumes. In the southern SVZ, where ice unloading was greatest, eruption frequency in the early post-glacial period is approximately twice that of the mid post-glacial period (although frequency increases again in the late post-glacial). A comparable pattern occurs in Kamchatka, but is not observed in the Cascade arc. The early post-glacial period also coincides with a small number of very large explosive eruptions from the most active volcanoes in the southern and central SVZ, consistent with enhanced ponding of magma during glaciation and release upon deglaciation.
In comparison to non-arc settings, evidence of post-glacial increases in rates of arc volcanism is weak, and there is no need to invoke significantly increased melt production upon ice unloading, as occurred in areas such as Iceland. Non-arc volcanoes may therefore account for a relatively higher proportion of global volcanic emissions in the early post-glacial period than is suggested by the relative contributions of arc and non-arc settings at the present day.
The second part of this paper critically examines global eruption records, in an effort to constrain global-scale changes in volcanic output since the LGM. Accurate interpretation of these records relies on correcting both temporal and spatial variability in eruption recording. In particular, very low recording rates, which also vary spatially by over two orders of magnitude, prevent precise, and possibly even accurate, quantitative analysis. For example, if we assume record completeness for the past century, the number of known eruptions (volcanic explosivity index≥2) from some low-latitude regions, such as Indonesia, is approximately 1 in 20,000 (0.005%) for the period 5–20ka. There is a need for more regional-scale studies of past volcanism in such regions, where current data are extremely sparse. We attempt to correct for recording biases, and suggest a maximum two-fold (but potentially much less) increase in global eruption rates, relative to the present day, between 13 and 7ka. Although volcanism may have been an important source of CO2 in the early Holocene, it is unlikely to have been a dominant control on changes in atmospheric CO2 after the LGM.
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