However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses. Replication of viral RNA is initiated de novo in a primer-independent manner. During the first step of replication, cRNA is synthesized and nascent cRNA molecules assemble into vRNP-like complementary ribonucleoprotein (cRNP) complexes with newly synthesized polymerase and NP  segments are coated by nucleoprotein (NP) and bound at their conserved 5′ and 3′ ends by the viral RNA-dependent RNA polymerase, forming viral ribonucleoprotein (vRNP) complexes. The conserved 5′ and 3′ vRNA ends are also referred to as the vRNA promoter. The viral polymerase consists of the three subunits polymerase acidic (PA), polymerase basic 1 (PB1) and polymerase basic 2 (PB2) proteins. During infection, the viral polymerase transcribes vRNA into mRNA and replicates it through a cRNA replicative intermediate , and se11–Avian influenza virus polymerases are severely restricted in their activity in mammalian hosts, resulting in impaired viral genome replication and mRNA synthesis and a reduced potential for acquiring beneficial mutations . For ins17–In addition to E627K, several other adaptive mutations have been found to cluster on the surface of the 627 domain or in the close vicinity of the 627 domain and the nascent RNA exit channel , 21. On 22–Although adaptive mutations have been demonstrated to enhance the activity of avian influenza virus polymerases in mammalian cells, there is disparity in the literature regarding the mechanism through which these mutations enhance polymerase activity. For instance, it has been proposed that 627E could impair vRNP assembly in mammalian cells by destabilizing polymerase and NP interactions –28, but –in vitro polymerase activity assays and cell-based vRNP reconstitution assays, we characterized the role of the flexible C-terminal two-thirds of PB2 in influenza virus polymerase activity. We report that the influenza virus polymerase has different requirements for PB2 domains, depending on whether it uses vRNA or cRNA as the template. We also show that the 627 domain of PB2 is not required for basic polymerase activities such as binding of viral RNA and transcription of viral RNA in vitro but that the domain is essential for viral RNA replication and transcription in a cellular context. Furthermore, we show that a replicating RNA polymerase that uses a vRNA as the template requires a second polymerase with a 627 domain to stabilize the nascent cRNA in a cellular context.In this study, using a combination of In order to address the function of the flexible C-terminal two-thirds of PB2  and to iIt is believed that polymerase complex formation takes place in the nucleus after separate nuclear import of the PB1-PA dimer and the PB2 monomer , 37–39. 37–in vitro activity assays using purified recombinant polymerase. First, we evaluated the ability of the polymerases with PB2 mutations to carry out the cap snatching and capped RNA primer extension steps that are required of viral transcription. Purified polymerase was bound to vRNA promoter, comprising a 15-nt-long RNA corresponding to the 5′ end of vRNA and a 14-nt-long RNA corresponding to the 3′ end of vRNA, and incubated with a 20-nt-long radiolabeled capped RNA in the absence (to assess cap snatching) or presence (to assess capped RNA priming) of nucleotides. In the absence of nucleotides, the wild-type and K627E mutant polymerases produced two major cleavage products  is active on the vRNA but not the cRNA template, revealing differential requirements of the PB2 structure for viral transcription and replication initiation on the vRNA and cRNA promoters.Taken together, these results show that a polymerase lacking the 627 domain is active in cap snatching, cap-dependent transcription initiation, and ApG extension in vitro, we next evaluated the role of the 627 domain in polymerase activity in a cellular context using a mini-replicon assay. RNPs were reconstituted by coexpression of the three polymerase subunits, NP, and segment 6 vRNA in human HEK-293T cells, and the accumulation of positive- and negative-sense viral RNAs was analyzed by primer extension. In contrast to results obtained in vitro , which allows RNA binding but no transcription or replication , was coeOverall, our data show that the 627 domain is essential for the transcription and replication of vRNA in a cellular context, independent of the host species and the requirement for NP. However, the mutant lacking the 627 domain is able to bind vRNA and cRNA. Furthermore, these data confirm previous findings that the amino acid at position 627 of PB2 plays a central role in determining the ability of the polymerase to transcribe and replicate vRNA in a mammalian host.in vitro but that it is essential for the accumulation of viral RNAs in the cell. In a cellular context, free polymerase needs to be recruited to replicating vRNPs to stabilize newly synthesized nascent cRNA and protect it from host nucleases  virus was generated using the pHW2000 eight-plasmid system (Human embryonic kidney 293T cells (HEK-293T) and chicken embryonic fibroblasts (DF-1) were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). Madin-Darby bovine kidney (MDBK) epithelial cells were cultured in minimal essential medium (MEM) supplemented with 10% FCS and 2 mM d system . Plasmidd system , pcDNA-Pd system , pcDNA-Pd system , and pcDd system , expressd system  and pPOLd system  have beed system . pcDNA-Pd system  as descrHEK-293T cells grown on sterilized glass coverslips in 24-well plates were transiently transfected with 0.25 μg each of pcDNA-PA, pcDNA-PB1, and pcDNA-PB2/pcDNA-PB2Δ535-667/pcDNA3A using Lipofectamine 2000 reagent (Invitrogen) and OPTIMEM (Invitrogen) according to the manufacturer's instructions. Forty-eight hours posttransfection, cells were fixed for 15 min in 4% formaldehyde in 250 mM HEPES (pH 7.9) and permeabilized for 15 min in 0.25% Triton X-100 in phosphate-buffered saline (PBS). Cells were blocked in PBS containing 10% normal goat serum, 0.5% Triton X-100, and 3% bovine serum albumin overnight at 4°C. Cells were stained with a polyclonal rabbit antibody raised against the N-terminal 180 amino acids of PB2  and an Ag for 5 min. The cleared cell lysate was diluted 1:5 in binding buffer  and incubated with washed IgG Sepharose  (50 μl per sample) at 4°C for 3 h. After binding, the IgG Sepharose beads were washed three times in wash buffer . Recombinant polymerase was released using tobacco etch virus (AcTEV) protease in elution buffer  at 4°C overnight and cleared from the beads at 17,000 × g for 5 min. Purified polymerase was analyzed by SDS-PAGE and silver staining  as well as Western blotting using a polyclonal rabbit antibody raised against the N-terminal 180 amino acids of PB2  using Lipofectamine 2000 reagent (Invitrogen) and OPTIMEM (Invitrogen) according to the manufacturer's instructions. Cells were harvested 48 h posttransfection, lysed in 500 μl of Tris lysis buffer  at 4°C for 1 h and centrifuged at 17,000 × s of PB2  as the p32P]GTP , 0.8 mM S-adenosylmethionine, 0.5 U/μl of vaccinia virus capping enzyme (New England BioLabs [NEB]), and 2.5 U/μl of 2′-O-methyltransferase (NEB) at 37°C for 1 h. The product was analyzed by 16% denaturing PAGE, excised, eluted overnight in deionized water, and desalted using NAP-10 columns . Capped-RNA cleavage assays were performed in a reaction mixture containing 5 mM MgCl2, 1 mM DTT, 2 U/μl of RNasin, 0.5 μM 5′ vRNA promoter, 0.5 μM 3′ vRNA promoter, ∼1,500 cpm of capped RNA, and 5 ng/μl of polymerase in 0.5× polymerase elution buffer. Extension assays were performed in a reaction mixture containing 5 mM MgCl2, 1 mM DTT, 2 U/μl of RNasin, 0.5 μM 5′ vRNA promoter, 0.5 μM 3′ vRNA promoter, 1 mM ATP, 0.5 mM UTP, 0.5 mM CTP, 0.5 mM GTP, ∼1,500 cpm of capped RNA primer, and 5 ng/μl of polymerase in 0.5× polymerase elution buffer. Reaction mixtures were incubated at 30°C for 4 h and stopped by addition of an equal volume of 80% formamide, 1 mM EDTA, and bromophenol blue and xylene cyan dyes and incubation at 95°C for 3 min. Reaction products were resolved by 20% denaturing PAGE containing 7 M urea in Tris-borate-EDTA (TBE) buffer and visualized by autoradiography. ImageJ was used to analyze the 32P-derived signal  (Chemgenes) was capped with a radiolabeled cap 1 structure in 20-μl reaction mixtures containing 1 μM RNA, 0.25 μM GTP , 0.5 μM 5′ vRNA (or cRNA), 0.5 μM 3′ vRNA (or cRNA), and 5 ng/μl of polymerase in 0.5× polymerase elution buffer were incubated at 30°C for 1 to 4 h. Reactions were stopped by adding an equal volume of 80% formamide, 1 mM EDTA, and bromophenol blue and xylene cyan dyes and incubation at 95°C for 3 min. Reaction products were resolved by 20% denaturing PAGE containing 7 M urea in TBE buffer and visualized by autoradiography. ImageJ was used to analyze the 32P-derived signal . The accumulation of viral mRNA, vRNA, and cRNA was analyzed by primer extension using 32P-labeled primers specific for negative- or positive-sense NA RNA  and OPTIMEM (Invitrogen) according to the manufacturer's instructions. One microgram each of pcDNA-PA, pcDNA-PB1, pcDNA-PB2, and pcDNA-NP as well as a vRNA-expressing plasmid (pPOLI-NA-RT or pPRC425-NA for full-length neuraminidase [NA] segment vRNA template or pPOLI-NA47 for a short [47-nt] vRNA template) was transfected. Cells were harvested 48 h posttransfection. Total RNA was extracted using TRI Reagent (Sigma-Aldrich) and dissolved in 10 μl of double-distilled water  and OPTIMEM (Invitrogen) according to the manufacturer's instructions. Recombinant polymerase was purified after 48 h as described above. RNA was extracted from purified polymerase using TRI Reagent (Sigma-Aldrich) and dissolved in 10 μl of ddHNA47 RNA . Primer d signal .293T cells were transiently transfected in 35-mm dishes as described above with 1 μg each of pcDNA-PA, pcDNA-PB1a, and pcDNA-PB2 with or without 2 μg of pcDNA-NP. Twenty-four hours posttransfection, cells were infected with influenza A/WSN/33 virus at a multiplicity of infection (MOI) of 10 in the presence of 10 μg/ml of actinomycin D. Total RNA was extracted 6 h postinfection and analyzed by primer extension as described above.


Habitat degradation can affect trophic ecology by differentially affecting specialist and generalist species, and the number and type of interspecific relationships. However, the effects of habitat degradation on the trophic ecology of coral reefs have received limited attention. We compared the trophic structure and food chain length between two shallow Caribbean coral reefs similar in size and close to each other: one dominated by live coral and the other by macroalgae . We subjected samples of basal carbon sources  and the same 48 species of consumers (invertebrates and fishes) from both reefs to stable isotope analyses, and determined the trophic position of consumers and relative importance of various carbon sources for herbivores, omnivores, and carnivores. We found that both reefs had similar food chain length and trophic structure, but different trophic pathways. On the coral-dominated reef, turf algae and epiphytes were the most important carbon source for all consumer categories, whereas on the degraded reef, particulate organic matter was a major carbon source for carnivores. Our results suggest that the trophic structure of the communities associated with these reefs is robust enough to adjust to conditions of degradation. However, habitat degradation can modify the number of species interactions, potentially altering the trophic ecology4. For example, in a forest subjected to selective logging in Borneo, species of ground-feeding and understorey-feeding birds had significantly higher trophic positions than they had in a non-logged forest5. In Moorea, the stable isotopic signatures of marine carbon sources and consumers differed significantly between two bays as a result of different levels of anthropogenic activities causing differences in mean annual river flow to each bay6.One of most evident effects of habitat loss and degradation in terrestrial and aquatic ecosystems is a decline in the diversity of ecological communities via changes on species abundance and richness8. For example, butterfly species with a narrow feeding niche and low levels of mobility and reproduction were most strongly affected by habitat loss across a wide range of habitats in America and Europe9. Also, among bird species, long-lived, large, non-migratory, forest specialists were less likely to occur and less abundant in more intensively man-used habitats than were short-lived, small, migratory, habitat generalists10.Life history traits may also determine the response of species to habitat degradation, with specialist species generally being more affected than generalist species11. However, coral reefs are being widely affected by a combination of global and local stressors, including climate change-induced coral bleaching, diseases, overfishing, and eutrophication12. Habitat degradation on coral reefs is mainly manifested as declines in the abundance of reef-building corals and their replacement by macroalgae or other organisms15. Coral reef degradation is already affecting community structure by changing diversity and abundance of species17 as well as ecosystem functioning and services19. The removal of particular species  and the addition or increase in abundance of others may fundamentally change ecological feedbacks, resulting in a transition of the ecosystem to an alternate state20. As occurs with terrestrial species, coral reef specialists are expected to be more affected than generalists by reef degradation23, further altering the food webs16.Coral reefs are the most biologically diverse ecosystems in tropical waters and provide important ecosystem services to millions of people around the world25. Ecosystem size and disturbance have been examined as factors potentially determining food chain length in some aquatic and terrestrial ecosystems, with results pointing to ecosystem size as having a greater influence than disturbance27. Yet, at the global scale, food chain length showed weak or no relationships with ecosystem size28, probably because environmental variables interact in complex ways to structure a community and may affect metrics of food web structure other than food chain length29.Food-chain length is an important descriptor of community structure and ecosystem functioning30 and a measure of food-chain length that integrates the assimilation of energy or mass flow through all the trophic pathways leading to top predators 26. Carbon isotope ratios are used as a tracer of food carbon source, whereas nitrogen isotope ratios are indicative of consumer trophic position31. The use of stable isotope analyses and descriptors  to examine the trophic structure and functioning of coral reef organisms and communities has been increasing in the last few years34. For example, some studies have examined the stable isotope composition of reef organic matter sources and consumers along environmental gradients or in different seasons38. Others have examined the potential effect of habitat degradation on coral reef food webs via stable isotope analyses by focusing on species of higher order consumers34.Stable isotopes provide information on the flow of energy or nutrients through food websAcropora palmata39. Contrarily, Bonanza, which previously held abundant colonies of A. palmata, has sustained a substantial decline in live coral cover and increase in macroalgal cover from values estimated in 1985 40. We first compared the architectural complexity (rugosity index) and percent cover of live coral and different types of functional groups of algae between reefs. We then explored whether the trophic structure and food chain length of associated reef communities differed between Limones and Bonanza by comparing the stable isotopes (δ15N and δ13C) of several basal carbon sources and the same 48 reef-associated consumer species on both reefs as well as the trophic position of the latter. Using bi-plots of δ13C and δ15N values we compared the isotopic niche widths of the different trophic categories of consumers  between reefs by means of several metrics of trophic structure42. We hypothesized that, as the coral-dominated Limones provides more habitats and trophic niches than Bonanza, then it will also exhibit a more complex trophic web with a higher trophic position of predators than the degraded reef.Here, we analyse the effect of habitat degradation on food webs by comparing the trophic structure and food chain length between two shallow Caribbean coral reefs known as “Limones” and “Bonanza” that are similar in size and subjected to the same environmental conditions, but have contrasting levels of degradation Fig. . Limones14 = 5.46, p = 0.0003), as was reef rugosity ; whereas cover of brown and red fleshy, and green and red calcareous macroalgae was significantly greater on Bonanza than on Limones  was greater on Bonanza (69.9%) than on Limones (26.6%).As anticipated, the ecological condition of both reefs differed broadly Fig. . Live co15N values was very similar on both reefs, the range in δ13C values was wider on Bonanza than on Limones  , whereas three had a higher TP on Limones . Regardless, there was a high correlation between the TP values of individual consumers from Limones and Bonanza (r = 0.97)  Fig. . Moreove97) Fig. , indicat13C values on the degraded reef (Bonanza) relative to Limones reflects the broader range in δ13C values of basal carbon sources, which ranged from −18.76 ± 3.01 (mean ± SD) for turf algae to −8.78 ± 0.58 for the red coralline algae Amphiroa tribulus  on Limones and for eight species (seven invertebrates and one fish) on Bonanza  than the correlation between the corresponding TP values , whereas POM contributed much smaller percentages to the isotopic signature of all consumer trophic categories on Limones Fig. . In cont13C range (from −16.31‰ for S. aurofrenatum to −13.51‰ for the long-spined sea urchin Diadema antillarum) and a smaller standard ellipse area (SEAc) (Table 13C range (from −18.28‰ for A. coeruleus to −11.90‰ for M. coryphe), resulting in a greater niche size, as indicated by the convex hull area (TA) and SEAc   and a degraded, macroalgal-dominated reef (Bonanza). We chose two reefs of a similar size to minimize the potential effect of ecosystem size on food chain length27. Our results also do not imply similarities in community structure because we intentionally sampled the same species on both reefs. Yet, the structure of the coral reef food web and the trophic position of high-order consumers (mesopredators) were similar between both reefs despite the difference in degradation, although consumers showed greater enrichment of δ13C on the degraded reef.Habitat degradation is thought to be a major driver of change in the species composition and trophic structure of ecosystems44. We focused our samples of baseline carbon sources on erect macroalgae and turf, which cover a large percentage of reef area on both reefs  of two separate reefs distant from each other by ~2 km.On coral reefs, baseline carbon sources may include benthic macroalgae, reef-associated detritus, particulate organic matter (POM), coral tissue, and phytoplanktonsee Fig. , and on 47, our results strongly suggest that on the coral-dominated reef, turf algae and epiphytes constitute a key source of carbon for consumers of the different trophic categories, whereas macroalgae and POM are considerably less important. Turf is an important component of coral reef food webs because many more herbivorous fishes feed on turf algae than on erect macroalgae49, which they often find unpalatable. In addition, turf traps vast amounts of detritus, and some fishes that are considered as herbivorous actually feed on the detritus trapped in the turf50. Also importantly, algal turf constitutes one of the most productive habitats for small reef mesograzer invertebrates, in particular small crustaceans such as amphipods, isopods, ostracods, and tanaids52, which can be consumed by fishes54. Therefore, turf-feeding herbivores constitute an important trophic link between benthic primary production and secondary consumers56, especially since the use of stable isotopes has revealed that predators on coral reefs consume more herbivorous prey (both fishes and invertebrates) than previously reported57. This appears to be the case in Limones, where the isotopic signature of consumers in all trophic categories was consistent with a more benthic-derived carbon pathway. Similarly, benthic primary production was found to be an important source for consumer production, including apex predators, in some coral-dominated coral reefs of Australia and Hawaii38.Although results from mixing models should be taken with caution because they are based on fixed trophic enrichment factors13C) at the base of the food web41. Among consumers, herbivores in particular exhibited a broader isotopic niche along the carbon axis on Bonanza compared with Limones. On Bonanza, macroalgae, algal turf, and POM were identified as being more or less equally important as carbon sources for herbivores and omnivores, but not for carnivores. Degraded reefs are characterized by increases in algal cover that can benefit some herbivorous fishes but only in the short term, as some large erect macroalgae  are often unpalatable to fishes59. These macroalgae are readily consumed by small mesograzers and omnivores, such as sea hares, some snails, amphipods, and majoid crabs61, but the ecological effects of these small animals tend to be more localized than those of herbivorous fishes or urchins62. Therefore, macroalgal-feeding herbivores may not represent a major food source for carnivores on Bonanza, for which POM was identified as the main carbon source. On coral reefs, POM includes phytoplankton as well as other edible particles such as phytobenthos debris, mucus, and faeces45. Small pelagic and benthic fishes associated with coral reefs, such as silversides (Atherinidae) and basslets (Grammatidae), feed on POM and zooplankton, as do juveniles of surgeonfishes (Acanthuridae), grunts (Haemulidae), jacks (Carangidae), and yellowtail snappers (Ocyurus chrysurus)63. Other fishes that are mainly herbivorous can change feeding modes depending on the ecological context64. These fishes are prey for many reef mesopredators, which may further use resources differentially depending on the seascape configuration. For instance, the blackspot snapper Lutjanus ehrenbergii shifted from a benthic macroalgal food web on shelf reefs to a phytoplankton-based food web on oceanic reefs in the Red Sea44. Planktonic-derived production was also identified as important for sustaining key predatory species in reef fisheries in Australia65. On Bonanza, reef degradation appears to be causing a temporal shift from a benthic algal-based food web to a phytoplankton-based food web.In contrast to Limones, the distribution of the carbon signal on Bonanza was broader and originated from different sources, potentially increasing trophic diversity  δ13C values67. French angelfish can consume algae and invertebrates, but feed mostly on sponges63, which are filter feeders and hence display low values of δ13C. For example, an average δ13C value of −17.2‰ was recorded for coral reef sponges in Florida68. Nevertheless, even after accounting for outliers such as P. paru, omnivores from Bonanza had the largest overall isotopic niche as revealed by the size of the SEAc. Omnivores play an important role in dampening potential trophic cascades69 and would be expected to have a greater potential of short-term adaptive response to changes in habitat degradation.Omnivory is common in marine food webs71. The current paradigm is that habitat loss and degradation are the main drivers of biodiversity loss, but our results cannot be discussed in terms of diversity or abundance because that was not the aim of our study. We found that coral cover was much lower and macroalgal cover much higher in Bonanza than in Limones. Yet, despite the differences in habitat degradation, food chain length was similar on both reefs, suggesting that the trophic structure of the communities associated with these reefs is robust enough to adjust to conditions of degradation. However, we do not interpret our finding as indicative of a lack of effect of habitat degradation on the trophic ecology of these reefs, because the range in δ13C values for carbon sources and herbivorous species was wider in Bonanza than in Limones, and the carbon pathways appear to differ between both reefs despite their proximity. Future research should include sampling on more coral- and algal-dominated reefs to examine the generality of our findings, and exploring the presence of potential sublethal effects  of habitat degradation on high-order consumers.Phase shifts are becoming increasingly common in coral reefs with different levels of habitat degradation40. Habitat degradation varies among reef units and patches23. We selected two reef units exhibiting visibly contrasting levels of degradation: “Limones” and “Bonanza”. Limones  is considered an exceptional site for Acropora palmata within the Mesoamerican Reef System due to its large populations of this branching coral species that cover nearly 40% of the reef substrata39 , depth range (1–5 m), and distance from the shoreline. Samples for stable isotope analyses  were collected on the back reef to crest zones within an area of ~500 m2 near the centre of each reef.The study was conducted in the Puerto Morelos Reef National Park, located on the Mexican portion of the Mesoamerican Reef System see Fig. . The Pue18. A perfectly flat surface would have a rugosity index of one, with larger numbers indicating more complex surfaces.To compare the current status of Limones and Bonanza, benthic habitat composition and architectural complexity were described for each reef using eight randomly selected 10-m transects within the same area in which samples were collected on each reef. At each transect, coral cover was measured by means of the line-point counts method. A surveyor recorded the benthic component  intercepting the line every 10 cm , and cover of each component was estimated as a percentage of the number of points overlaying the transect. Also, at each transect, reef complexity was measured using the rugosity index, which is the ratio of a length of a chain moulded to the reef surface to the linear distance between its start and end pointAmphiroa tribulus, Caulerpa racemosa, Dictyota cervicornis, Halimeda tuna), epiphytes, and turf algae. For all consumer species and basal carbon sources, at least three to five replicates were sampled from each reef, yielding 514 samples in total. To minimize potential seasonal effects on the isotopic composition of organisms, all samples were obtained between late September 2015 and early February 2016, and samples from both reefs were interspersed throughout this period.The same species of consumers and types of basal carbon sources were sampled on both reefs by SCUBA diving and transported to the laboratory at UASA-UNAM  to be processed for stable isotope analyses. Consumers included 48 species, of which 32 were of invertebrates (including annelids (1 species), bivalves (4), gastropods (8), chitons (1), octopuses (1), decapod crustaceans (10), sea urchins (4), brittle stars (2), and sea cucumbers (1 species)) and 16 were of fishes  were identified in situ and caught with Hawaiian spears. A sample of muscle was taken from the dorsal region and immediately frozen until processing75. All samples were acidified with HCl (1 N) to remove inorganic carbon76 and then rinsed with Milli-Q water. Samples were dried at 60 °C for 48 h in aluminium foil trays, then ground to a fine powder and homogenized with agate mortar and pestle. Powdered sub-samples were weighed and sent for isotopic analysis in ultra-pure tin capsules.POM was obtained from seawater collected with a dark 5-L carboy. Then, 1 L of seawater was gently vacuumed using Whatman GF/F (0.7 µm) filters previously incinerated at 450 °C for 4 h. Samples of algal turf were taken from the top of dead coral using scalpel and forceps under a magnifying glass to avoid the presence of contaminants 15N and 0.02‰ for δ13C. Carbon and nitrogen ratios were expressed in delta (δ) notation, defined as parts per mil (‰) of difference relative to an international standard:13C and 15N) to the lighter, more common isotopes . Delta values are reported relative to the international standards of Vienna Pee-Dee Belemnite (VPDB) carbon and atmospheric nitrogen26.Elemental and stable isotope analyses were carried out in a Finnigan Delta V Plus mass spectrometer (Thermo Scientific) interfaced with an elemental analyser  at the Mass Spectrometry Laboratory of Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Mexico. The average precision across runs was 0.1‰ for δt tests were used to test the null hypotheses of no significant differences in the mean percent coral cover, rugosity index, and δ15N and δ13C composition of each basal resource and consumer species between Limones and Bonanza. Bi-plots of δ13C and δ15N values of basal carbon sources and consumers were used to visualize the food web structure at each reef and the differences in isotope values of basal carbon sources and consumers among reefs.Student’s et al.77:base was set at a trophic level 2 baseline using the mean δ15N of the epifaunal reef clam Barbatia domingensis (=B. cancellaria), which we chose as the baseline organism given that filter-feeding bivalves are good integrators of the isotopic variation at the base of pelagic and benthic food webs35; δ15Nlim is the saturating isotope limit as TP increases; δ15Nbase is the isotope value for a known baseline consumer in the food web , and k is the rate at which δ15NTP approaches δ15Nlim per TP step. Estimates of k and δ15Nlim are given by:0 = 5.924 and the slope β1 = −0.271. These values characterize the change in δ15N as dietary δ15N values increase77.The relative trophic position (TP) of all species of consumers from each reef was calculated using the following equation from the meta-analysis performed by Hussey 79. Mixing models require the use of trophic enrichment factors and for these purposes we used the mean (±SD) values proposed by Post26 for δ13C (0.40 ± 1.30‰) and δ15N (3.40 ± 1.00‰). The estimated values of the dietary proportion were obtained via a Markov-Chain Monte Carlo (MCMC) simulation78 on stable isotope data from consumers. Each consumer species was previously categorized as herbivore, omnivore, or carnivore based on literature reports on its type of diet (see Supplementary Table 15N range) and CR (δ13C range), measures that provide information on the food chain length and maximum trophic position within the community, and the diversity of basal carbon sources, respectively. We also inferred the total isotopic niche width using the total area (TA) index, which is a measure of the area of a polygon drawn through the most extreme data points of the population in the isotopic niche space 41, and the standard ellipse area (SEA), which measures the isotopic niche width of the mean core community and contains approximately 40% of the data42. To compare the total trophic niche area for each consumer trophic category  between reefs, we used the Bayesian standard ellipse area corrected for sample size (SEAc), estimated and plotted using the SIBER routine for the SIAR package in R42. For each trophic category, distinction between the two SEAc for each reef was inferred by posterior probabilities derived by Bayesian inference based on 100,000 MCMC draws, and the trophic niche overlap was calculated as the proportion of SEAc overlapping between the two reefs.To quantify the relative importance of basal carbon sources supporting the food webs on each reef, we applied a Bayesian mixing model using SIAR  version 3.0.2All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary information


This paper reports on the various nuances of the origins of life on Earth and highlights the latest findings in that arena as reported at the Network of Researchers on Horizontal Gene Transfer and the Last Universal Common Ancestor (NoR HGT and LUCA) which was held from the 3–4th November 2016 at the Open University, UK. Although the answers to the question of the origin of life on Earth will not be fathomable anytime soon, a wide variety of subject matter was able to be covered, ranging from examining what constitutes a LUCA, looking at viral connections and “from RNA to DNA”, i.e., could DNA have been formed simultaneously with RNA, rather than RNA first and then describing the emergence of DNA from RNA. Also discussed are proteins and the origins of genomes as well as various ideas that purport to explain the origin of life here on Earth and potentially further afield elsewhere on other planets. The latest research suggests that, after a few 100 million years of accretion of the Solar System at approximately 4.67 billion years , thereby positioning RNA as the more ancient molecule. As a result of RNA’s two “activities”, DNA is relegated to second place, meaning that it would have been made from the RNA which predated it. Krishnamurthy  and his team thought otherwise. Their counter-intuitive argument runs along the lines that a transition of homogeneous RNA → homogeneous DNA seems unrealistic in the absence of separating and editing mechanisms in a prebiotic context. Therefore, the simultaneous presence of RNA and DNA molecules with the pre-existing alternative building structures ; alternative sugars; alternative linker units and alternative recognition elements would have removed the need for “clean reactions” with high chiral purity demand during homogeneous → homogeneous transition. In the milieu of the prebiotic epoch, gradual chemical evolution of heterogeneous → homogeneous during the oligomeric/polymeric transitions led to the eventual emergence of homogeneous backbones of RNA:DNA → DNA/protein world as we recognise today. The net result being that it was a “chimeric oligonucleotide system” from which life emerged rather than a “pure” RNA world ,50.If there is life elsewhere in the universe, will it also use DNA? This was the question put forward by Devine  at the beginning of his oral presentation. In order to answer this point, he proceeded to examine the synthetic analogues of nucleic acids which differ from their natural counterparts in three key areas. These being (a) replacement of the phosphate moiety with an uncharged analogue; (b) replacement of the pentose sugars, ribose and deoxyribose, with an alternative pentose and hexose derivatives; and finally (c) replacement of the two heterocyclic base pairs adenine/thymine and guanine/cytosine with non-standard analogues that obey the Watson-Crick pairing rules. The conclusion was that a polyphosphate backbone was crucial to generating viable genetic biomolecules; sugar moiety can vary to some degree; and extra-terrestrials may not necessarily have DNA, but will have a sugar-phosphate backbone in their nucleic acid . In presenting “the origin and early evolution of information transfer in biological systems”, Pohorille  explored the question of whether linear, genomic information transfer might have been preceded by a simpler, non-linear information transfer system that operated at the origin of life. Based on both experimental and theoretical studies he advanced a thesis that the earliest information transfer was based on molecular self-assembly and recognition. Darwinian evolution could have progressed without a genome, leading to the formation of simple metabolic networks. However, it encountered barriers. The requirement for increasing efficiency, diversity and specificity created an imperative for the emergence of the modern, more precise linear information storage and transfer system. Thus, the genome itself was a product of evolution. Next, Pohorille discussed the evolution of simple genomes, focusing on questions such as: was the outcome of early evolution predictable or was it, instead, governed by chance? In addition, what was the role of “neutral mutations” in the evolution of increasingly complex systems? To address these questions, he and his collaborators explored fitness landscapes of RNA ligases that differed in length. The results support the existence of near-neutral networks and possibilities for evolutionary optimisation. Also, their findings, based on combined phylogenetic and structural data, suggest that complex modern RNA structures evolved from simple shared structures that were present in much shorter, although less efficient RNA molecules ,53.RNAs are widespread in all biological systems (except for DNA viruses) and are involved in multi-laterally adapted systems that control numerous cellular processes, the magnitudes of which are still being explored. Principally, there are two broad categories of RNAs, namely coding and non-coding (ncRNA)—this summary refers only to the latter. glmS ribozymes), whereby they regulate their own activity and perform genetic control by a metabolite binding mRNA [E. coli) via antisense RNA as in the hok/sok system [ncRNA molecules, such as housekeeping RNAs  and the thousands of regulatory RNAs that are the subject of ongoing intense studies, can form structures ranging from primary to quaternary levels. These are as follows: primary structures of approximately 22 nucleotides, as in guided single stranded microRNAs (miRNA); double stranded miRNA interference segments exist as secondary shapes; tertiary level structures which are broadly composed of RNAs and protein complexes called ribonucleoproteins as in RNase P and RNase MRP  complexes; at the quaternary level, again in conjunction with proteins, various co-factors and metal ions , RNAs form huge ncRNA-nanomachines as in spliceosomes, the Varkud satellite (VS), RNA-induced silencing complex (RISC) and ribosomes. These structures are multifunctional and are broadly regulatory, being involved in gene regulation as well as interfering with and processing both small and large RNAs . Such pring mRNA . They cak system . It is sk system . Furtherk system . ncRNAs,k system . A uniquThe conserved nature of ncRNAs allowed Jheeta  to postulate that it is highly feasible that these ncRNA molecules could still have overall control of cellular activity and perhaps this is the reason why DNA replication still requires this ncRNA primer. This is particularly relevant as there are large numbers of newly discovered ncRNAs whose functions are still to be explained and validated. Neisseriaceae family [Horizontal gene transfer (HGT) was a pre-requisite during the LUCA epoch and probably during the preLUCA era as well. It certainly continues to be widespread in cellular life forms, particularly in prokaryotes, on Earth today. HGT is important in that it is a necessary source of the genetic innovations occurring throughout the history of cellular life forms. Such innovations could be achieved via three main mechanisms, namely conjugation (plasmid-mediated), transduction (virus-mediated) and transformation (naked DNA uptake directly). Of these, the two former mechanisms resemble “infections”, whereas transformation is a highly-evolved feature of the recipient cell. The term HGT encompasses all three mechanisms despite their intrinsic differences and evolutionary consequences which were reported in the 2014 NoR HGT and LUCA meeting . During e family  he reveae family . These aIt is difficult to specify an exact point as to when eukaryote cells emerged in the time frame of the evolution of life on the Earth, but it can be surmised that there are eukaryotes that existed at least 1 billion years ago, as testified by the triploblastic animal fossil evidence . HoweverGene loss from organellar genomes to the nucleus has occurred throughout the evolutionary history of eukaryotes. The transfer of such genes to the nucleus is mainly governed by DNA-directed mechanisms; however, ncRNA-directed movements of genetic material to the nucleus have also been uncovered. This is particularly true of chloroplasts. Recent technical advances in organellar biotechnology, genome engineering and single-molecule tracking have given some novel insights into ncRNA chemistry at both cellular and organismal survival levels. From this research, Kotakis  posited that an ncRNA-directed route in Archaea, although a rare occurrence, could have contributed to endosymbiotic gene transfer under certain micro-environmental conditions-encouraging “photosynthetic” genomes to migrate to the nucleus during the process of endobiosynthesis. It was proposed that ncRNA molecules with a particular structural configuration and respective functional attributes can drive evolution by reacting to environmental pressures while drowning-out epigenetic aberrations. The structural and functional aspects of ncRNA also affect redox and genetic characters leading to co-evolution. He surmised that RNA could substitute every step in the dogma of molecular biology concerning the flow of genetic information ,64.Desulfotalea psychrophila, Desulfotomaculum arcticum, and Desulfuromusa ferrireducens) which all demonstrate highly efficient survival mechanisms. After preliminary tests, growth was observed in each of the experiments for all three organisms; further DNA purification and isolation has led to successful PCR analyses for future DNA amplification. She concluded that such research is important not only for improved understanding of biology in harsh environments, but also to aid NASA’s planetary protection policy by assisting in the recognition and mitigation of potential contamination on extra-terrestrial bodies [It is without a doubt that life exists on Earth, but what is not known is whether there is any life in our solar system  or even further afield in the Universe, as none has been discovered to date. Shepherd  investigated the potential of this issue by working with extremophiles; hardy microbes that exhibit a high potential to flourish in hostile environments such as −50 °C temperatures, pressures of 0.006 bar, and low water activity, among other stressors, which are all representative of the conditions on present-day Mars . She stul bodies . Such coThe recent discovery of a large number of exoplanets raises a question: where does a newly-discovered exoplanet stand in its capability to develop life as we know it on Earth? Maccone’s  answer to this question is to describe recent developments in a new statistical theory describing Evolution and SETI by mathematical equations. His theory deals with the mathematics of lognormal stochastic processes and he capitalises on the basic numbers involved with evolution: the time when life first appeared on Earth (3.5 billion years ago?); the number of species living on Earth nowadays (50 million or more?); and the Shannon Entropy to quantify molecular complexity between the first species (RNA organism?) and today’s humans, resulting in an overall molecular complexity increase of 25.575 bits/individual if the growth was exponential in the time, and 12.074 bits/individual if the growth was of Markov-Korotayev type (a cubic in the time) ,68. Are we any nearer to obtaining a complete answer to the pertinent question of the origin of life? The information disseminated at this latest meeting basically yields up a miniscule yes, but a humungous no. On a positive note, at every NoR HGT and LUCA meeting something new is brought to the table. For example, Krishnamurthy’s announcement that RNA and DNA may have developed simultaneously from XNA, as well as Jheeta’s hypothesis that RNA may still be in overall control of cellular activities and, also, the assertion by Kotakis that RNA can control the movement of genes from an organelle to a nucleus—a question here is what else is RNA capable of doing? These are still early days in the quest for a total understanding of the exact mechanisms of life’s emergence on Earth; however, it is expected that with the continuance of such meetings as NoR HGT and LUCA we can collectively begin to make serious headway.


However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements.Internal ribosome entry site (IRES) elements are  In addition, eIF4F mediates the assembly of the 43S preinitiation complex, which consists of the 40S ribosomal subunit bound to the multi-subunit factor eIF3, and the ternary complex (composed of eIF2-GTP and the initiator Met-tRNAi). The 43S complex scans the 5′ untranslated region (UTR) of the mRNA in 5′–3′ direction until an AUG triplet is found in the appropriate context to start protein synthesis. Upon start codon recognition via base pairing of Met-tRNAi with mRNA in the P site of the ribosome, conformational rearrangements trigger the formation of the 48S preinitiation complex. In this step, eIF5B displaces eIF2-GDP from Met-tRNAi promoting 60S ribosomal subunit joining together with eIF1A, leading to the formation of the elongation competent 80S ribosome.Regulation of protein synthesis is a key step of gene expression in all organisms. The process of RNA translation occurs in four basic steps, initiation, elongation, termination, and ribosome recycling. In eukaryotes, the vast majority of mRNAs initiate translation by a cap-dependent mechanism . This geFigure 1B).Beyond this general manner to initiate translation, under certain physiological conditions specific mRNAs initiate protein synthesis using a cap-independent mechanism, which eventually could lead to the synthesis of a different polypeptide. Research conducted to understand translation control operating in specific mRNAs provided evidence for different cap-independent mechanisms . One of Figure 1B), first reported in the genomic RNA of picornaviruses  have been described , probably because two ORFs must be separately regulated in dicistrovirus infection. The structural features of the 5′ end IRES governing translation of ORF1 differ between members of the dicistrovirus genus  is governed by distinct IRES elements . High-resolution studies of the three-dimensional structure of IGR–ribosome complexes showed that the PKI of the cricket paralysis virus (CrPV) IGR resembles a tRNA/mRNA interaction in the decoding center of the A site, blocking tRNA binding, and mimicking a pre-translocation rather than initiation state of the ribosome (The dicistrovirus IGR (about 190 nt) adopts a compact three-dimensional structure, which is essential for IRES activity . It consribosome . PKII isribosome . Yet, suribosome . These dNear atomic-resolution studies of the structural conformation of the dicistrovirus IGR IRES assembled on ribosome complexes illustrate the active role of the RNA structure in manipulating the ribosome. Structural analyses of the ribosome-bound Taura syndrome virus (TSV) IRES revealed that PKI occupies the ribosomal decoding center at the A site resembling the tRNA anticodon–mRNA codon interaction. However, in contrast to conventional mRNA, the ORF of the IGR-driven mRNA is placed in the A site, whereas the 40S peptidyl (P) site remains unoccupied . More reFigure 2). Why the 5′ UTR IRES elements of dicistroviruses are more variable than the IGRs remains elusive. Yet, the strong conservation of the IGR structure shows that its function intimately relies on a defined three-dimensional structure. Instead, the function of the 5′ UTR IRES is determined by the concerted action of the RNA conformation and the interaction with eIF3 . Each of these domains performs a distinct function during internal initiation. Domain II, which adopts an L-shape structure consisting of subdomains IIa and IIb, is involved in eIF5-induced GTP hydrolysis of eIF2. In addition, the apical loop of subdomain IIb contacts the ribosomal proteins RPS5, RPS7, and RPS11 . Additionally, eIF4E–eIF4G binding stimulates the rate of IRES RNA unwinding by eIF4A  because of the similarity with the HCV IRES (Type I IRES elements occur in the RNA genome of enterovirus . Representative members of type II IRES elements occur in cardiovirus (EMCV) and aphthovirus (FMDV) RNAs. The IRES elements classified as types I and II require the C-terminal region of eIF4G, eIF4A, eIF2, and eIF3 to assemble 48S initiation complexes in vitro , but arein vitro . More reby eIF4A . Type IVHCV IRES . A membeHCV IRES .In the rest of this review, we will concentrate on structural motifs present in types I and II picornavirus IRES elements. Representative members of types I and II IRES elements have been studied in great detail , which adopt stem-loop structures generally including internal bulges . This interaction requires the concerted action of all subdomains, since subtle changes in the orientation abrogate the interaction with eIF4G  rearrangements upon incubation of the EMCV IRES with eIF4G and eIF4A in the presence of ATP . These dth eIF4G . RemarkaFigure 4). Noteworthy, the ability of RNA molecules to acquire distinct conformations in response to specific ligands, as well as environmental signals, determines their function. In addition, conformational transitions could be spatially and/or temporally tuned enabling the assembly of ribonucleoprotein complexes (RNPs) in a hierarchical and sequentially ordered manner. Thus, IRES elements could exploit RNA structure flexibility, and therefore plasticity, as a core functional element.RNA flexibility appears to be a key feature of picornavirus IRES elements, as mentioned above for the HCV IRES. In this regard, the implementation of novel di-metallic chemical compounds able to detect flexible regions on the RNA structure in solution have allowed the identification of four-way and three-way junctions within flexible regions of the FMDV and HCV IRES conformation , which aIn contrast to the IRES elements described above, the activity of several plant viral IRES elements depends on RNA sequences mostly consisting of single-stranded regions. For instance, the IRES placed upstream of the coat protein ORF of crucifer-infecting tobamovirus (CrTMV) was mapped to A-rich regions . In thisFigure 5A), a positive-strand RNA virus with a dicistronic genome  RNA . UltimatCollectively, the heterogeneity of IRES structural organization described above points to different strategies developed by viruses to exploit the host translation machinery. At the same, this diversity illustrates the challenges to accurately predict genome-wide the presence of functional IRES elements, or IRES-like elements, in eukaryotic organisms.Figure 5B), the RNA helicase DEAD box protein 3 (DDX3) is involved in translation of this atypical mRNA . This IRES element is surrounded by other RNA structural motifs, the trans-activating region (TAR) and the polyadenylation signal (PBS) at the 5′ end, and the packaging signal (Psi) at the other end , or by using functional bridges, generally eIFs and RBPs. It is well established that eIFs and auxiliary RBPs stimulate 48S complex formation in reconstitution assays using purified factors with picornavirus IRES elements . LikewisFigure 6) can be summarized in several ways: RNA chaperones stabilizing specific conformations of the IRES structure, stabilizing the interaction of eIFs with the IRES facilitating the recruitment of the ribosomal subunits, mediating a direct recruitment of the ribosome, helping to remove RNA secondary structure near or at the start codon, and/or titrating IRES ligands, either stimulator or repressor molecules. For reasons of space, we will concentrate on RBPs recognizing specific structural motifs of representative members of picornavirus IRES elements and their impact on IRES function.The role of RBPs on the activity of viral IRES elements has been studied extensively. Current knowledge on how RBPs modulate IRES function . Soon after the report of PTB as an EMCV IRES-interacting factor -binding protein 2 (PCBP2), a factor expressed in most cell types that harbor three ribonucleoprotein K homology (KH) domains. KH domains bind RNA and DNA and are found in proteins controlling transcription and translation, along with other cellular processes. The PCBP2 binding site in the PV IRES is the ACCCC loop, a conserved motif located in domain IV in close proximity to the GNRA tetraloop . PCBP2 pFigure 4) proposed for the FMDV IRES , Far upstream element-binding protein 1 (FBP1) and FBP2 , Ras GTPase SH3 domain binding protein 1 (G3BP1), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), cold-shock domain containing E1 , or glycil-tRNA synthetase (GARS), to name a few  methodologies to analyze the RNA local flexibility of the FMDV IRES and the 3′ UTR in living cells confirmed the existence of long-range interactions (in cis altered the 3′ UTR local flexibility leading to overall enhanced reactivity. Unlike the reactivity changes observed in the IRES element, the SHAPE differences of the 3′ UTR were not statistically significant, suggesting a dynamic RNA structure. Covariation analysis predicted IRES-3′ UTR conserved helices in agreement with the protections observed by SHAPE probing. In support of this notion, engineering alternative base pair interactions compensated experimental disruption of the predicted base pairs. These results provided direct evidences for dynamic long-range interactions between these distant elements of the viral genome.RNA virus genomes fold into complex structures that include long-range RNA–RNA interactions relevant to control critical steps of the viral cycle (for recent reviews on this topic, see nscripts . More reractions . In compLong-range interactions in viral RNAs controlling fundamental processes of the viral replication cycle, such as translation and replication, have been extensively documented. Readers are directed to recent reviews discussing examples on cap-independent translation enhancers and other RNA-looping examples .RNA viruses have small, compact genomes. Thus, expression of their genome strongly depends on the host cell. This feature ensures that viral genomes evolved to increase their replication potential in the infected host, generally using unique mechanisms usurping the cellular machinery in their own benefit. In turn, this characteristic provides useful therapeutic targets. Because IRES elements are essential for viral infection and harbor conserved RNA motifs, they constitute specific targets for antiviral compounds, as exemplified by a large variety of modified antisense oligonucleotides, siRNAs, morpholino oligomers, aptamers, and small molecules based on benzimidazole .in vitro assembly of 48S complexes with picornavirus type II IRES elements (The evolutionary plasticity of RNA viruses makes them an ideal system for identifying mechanisms used not only by viruses, but also by the host cell. The observation that IRES-like sequences were described in cellular RNAs  provideselements , direct elements .Given the diversity of IRES-driven mechanisms to govern protein synthesis among viral RNAs, it is also conceivable that some type of IRES elements could arise by convergent evolution. Possible events resulting in the generation of novel IRES elements could be the assembly of RNA modules derived from different molecules by RNA recombination, integrative events, or RNA ligation. These events, although infrequent, could render structural elements with functions unrelated to the RNA molecules harboring the original modules. Indeed, RNA molecules have unique structural attributes, which include the ability to self-assemble through the arrangement of building blocks . An examThe relationship between RNA structure and biological function is generally inferred from conservation of structural motifs. Because RNA structure plays a fundamental role in most viral IRES elements , it is pIn this review, we have discussed examples of conserved RNA motifs impacting on a different extent on the activity of viral IRES elements. The influence of conserved motifs could be due to their impact on the IRES structure organization, but also on the influence on RBPs recognition and ribosome recruitment. Furthermore, although IRES elements from genetically distant viral RNAs lack overall conserved features, most of them have RNA structure flexibility. This characteristic increases in correlation with the requirements of factors to assemble competent translation initiation complexes. In this regard, it is important to note that the functional features of RNA molecules are established in their three-dimensional structure, but also in their ability to acquire distinct conformations in response to specific signals. In addition, conformational transitions could be spatially and temporally tuned to achieve distinct functions enabling assembly of RNPs in a hierarchical and sequential ordered manner. Thus, IRES elements could exploit RNA structure flexibility, and thus plasticity, as a core functional element. Future studies aimed to understand the structural organization and function of diverse viral IRES elements will help to improve the accuracy to predict IRES-like motifs in other genomes, regardless of the lack of a universal structural motif unique to all viral IRES elements.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PD patients were evaluated in the morning after a 12-hour therapy wash-out in two experimental conditions: i) after levodopa assumption (“on”); (ii) without drug administration (“off”). The experimental task required to execute three uncorrelated actions after 10' for three consecutive trials. Distinct scores for the spontaneous recall of the intention to perform the actions (prospective component) and for the correct execution of the actions (retrospective component) have been computed. Results showed that in the “off”condition PD patients were selectively impaired on the prospective component of the task. However, L-dopa administration significantly improved PD patients’ performance actually restoring the prospective memory deficit. These results support a critical role of dopaminergic modulation in prospective memory processes in PD patients, possibly through the replacement of dopamine levels in fronto-striatal pathways.Growing interest is present in literature on the study of prospective memory functioning in Parkinson’s disease (PD). Current data indicate that prospective memory may be impaired in PD and a relationship with general executive dysfunctioning has been suggested. However, although the dopamine dependency of cognitive dysfunction in PD has been widely investigated, poor is known on the dopaminergic modulation of PM. In the present study we explored the effect of acute administration of levodopa on the performance of a PD sample (


To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by  The rapid development and implementation of fluorescence microscopy techniques has led to the realization that, even in tiny bacterial cells, key molecular processes can be highly orchestrated and organized to temporarily or statically occur at specific subcellular sites . Most maInitially, gene expression, like many other cellular processes, could only be studied in cultures of cells and, thus, in an ensemble fashion. In the latter approach, total RNA is usually extracted from tissue samples or cell cultures, after which it is further analyzed by Northern blotting, quantitative reverse transcriptase (qRT)-PCR, DNA microarrays or RNA-sequencing. This type of data yields important information but typically represents averaged and normalized values that cannot be regarded as quantitative or absolute. In addition, ensemble RNA measurements do not provide information on cell-to-cell variations in RNA content and intracellular spatiotemporal distributions of transcripts. Biological processes such as transcription are inherently stochastic and can show great differences within clonal populations, such as bacterial cultures . TissuesEscherichia coli) and therefore require a potentially bias-introducing pre-amplification step to prepare cDNA libraries  recognizes distinct RNA sequences with high affinity. One of the first high-specificity RNA-RBP pairs that was characterized originates from the S2 phage . The MS2S2 phage . RepressS2 phage . RealiziS2 phage . By intrst cells . These sst cells . More re embryos . The MS2 embryos . In 2016peptides . A summaAlthough protein-based RNA labeling is now widely employed in eukaryotic organisms, several caveats have thwarted the extensive use of MS2-based RNA labeling and similar systems in bacteria. The main problem is that most employ intact autofluorescent proteins, causing substantial background noise that obscures the proper visualization of single transcripts. The background noise is effectively reduced in eukaryotic cells by tagging unbound MS2-GFP proteins with a nuclear localization signal (NLS) sequence, which directs the surplus of NLS-MS2-GFP proteins to the nucleus . Such coE. coli cells to study transcription kinetics (ms2) transcriptionally fused to the transcript under study resulted in sufficient fluorescent signal to visualize and follow single mRNA molecules over time in individual E. coli cells. In comparison, the incorporation of 24 binding sites typically suffices to monitor single transcripts in eukaryotic cells. It was recognized that extensive tethering of MS2-GFP reduces the diffusion rate of the transcript under study and renders the molecule perpetual . This can be a laborious task and, as discussed above, may influence the endogenous properties of the transcript under study. The RNA-labeling system employing PumHD and homologous proteins does not suffer from the latter drawback . It remains to be elucidated whether this system is functional in bacteria to track individual transcripts due to a potentially insufficient signal amplification.The RNA-guided Cas9 protein of the bacterial CRISPR-Cas9 immune system is a DNA endonuclease that was long thought to solely recognize DNA targets. The site where Cas9 interaction with DNA occurs is dictated by the combination of the protospacer adjacent motif (PAM) domain in the target DNA and the Cas9-bound single-guide (sg) RNA. Recently, Cas9 was found to also recognize single-stranded RNA after slight modifications of the CRISPR/Cas9 system : The sgRrinciple . It was lization . As a prlization . SubsequTo reduce signal-to-noise ratios, care needs to be taken in expressing the proper number of RBP-GFP molecules in relation to the estimated number of available RNA target binding sites. Even low expression of MS2-GFP may lead to a surplus of unbound MS2-GFP proteins in the cytoplasm, which would thwart capturing single mRNA molecules using fluorescence microscopy. This is true for all RBP-FPs expressed in bacterial cells in which RBP-FPs cannot be contained within a specific compartment. Even though Pumby and Cas9 have the advantage of targeting endogenous mRNAs, the background noise issue remains. It is essential to carefully consider which RBP is the right choice for RNA visualization in bacteria. For instance, a detailed analysis of the binding kinetics of MS2 and PP7 to their respective aptamers revealed that PP7 binds with a higher affinity than MS2 because PP7 occupies more aptamer repeats. Importantly, the construction of a tandem dimer of both MS2 and PP7 increased protein-RNA interactions in various eukaryotic cell lines, effectively improving signal-to-noise ratios . BacteriFigure 1C; Figure 1C; Figure 1C; Figure 1C; Protein complementation employs split fluorescent proteins that reconstitute into a functional fluorescing protein upon reunion via the interaction of their respective fusion partners . The impE. coli are degraded within 3–10 min post-synthesis , followed by cyclization and a final oxidation step in order for the active site to fluoresce. In addition to folding, the last step can take a relatively long time and greatly depends on the presence of sufficient molecular oxygen. Maturation of full-length super-folder GFP is estimated to take around 11 min under optimal conditions . A commoynthesis . Thus, tynthesis . Both shFigure 1D). Rather, RNA binding mediated FRET (RB-FRET) was developed to detect the interaction between eukaryotic RBPs and their cognate RNA (Förster Resonance Energy Transfer fluorescence microscopy makes use of a donor fluorophore that dissipates energy to and thereby excites a nearby (<10 nm) acceptor fluorophore upon excitation. Donor-acceptor pairs can exist of fluorophores and/or fluorescent proteins. Live single-cell and single-molecule FRET studies have been executed and well-performing FRET pairs consisting of fluorescent proteins have been carefully selected for various bacterial species . A combinate RNA . FRET isFigure 1E). The MS2 protein can be fused to, for instance, a trimethoprim (TMP)-tag  or a SNAP-tag   . The TMPsferase) ]. The STembranes . Many meFigure 1F). Hence, self-labeling protein tags hold great promise for both the visualization of RNA molecules and proteins, also because the diversity of commercially available tags and fluorophores is rapidly growing.Although the genetically encoded self-labeling protein tags can be relatively bulky and may require longer incubation times depending on the tag and cell type, they allow detecting mRNAs in live cells with photostable fluorophores. The latter is key to achieving single-molecule tracking and super-resolution microscopy . FluoropFigure 2A; in vitro  is a combinatorial method for the generation of nucleic acids binding to virtually any molecule, including proteins and small molecules such as organic dyes and metabolites  have been designed to visualize RNA through fluorophore-based FRET , of which the Spinach RNA-fluorophore pair was reported first . Spinach2 shows improved folding kinetics through the presence of stabilizing RNA hairpins but still requires a tRNA scaffold for optimal functionality (E. coli using fluorescence-activated cell sorting (FACS). In this way, the 49-nt RNA molecule Broccoli was developed , it would be highly desirable to be able to detect these molecules through RNA labeling. A recent study shows that arranging multiple Spinach aptamers in a tandem array increases fluorescence  is a triphenylmethane dye with low quantum yield in solution. The fact that the autofluorescence of MG increases in cold or viscous environments led Babendure and colleagues to develop a 38-nt-long MG-binding aptamer (MGA) that resulted in an over 2000-fold increase and red-shift in MG fluorescence  binds derivatives of thiazole orange (TO) at high binding affinities in the presence of potassium , resulting in a 100-fold increase in SB fluorescence  quenching is most effective. Static quenching is completely abolished when the quencher-fluorophore complex becomes dismantled . Quencherescence . SRB-2-tFigure 2B). Importantly, non-conditional fluorophores with different spectral properties, such as rhodamine green, tetramethylrhodamine or sulforhodamine can be used in combination with the same DNB aptamer  aptamer was introduced . Binding aptamer . To thisin vivo imaging of RNAs in bacterial cells or whether further advances of current or new aptamers are needed to obtain systems that accurately function in an intracellular environment.Future studies will have to show if recent improvements of existing aptamers are applicable and truly robust enough for in situ hybridizations of oligonucleotide probes to complementary nucleic acid sequences (in situ hybridization (FISH) has remained the predominant tool for the examination of cell-to-cell variations in transcript numbers. The technique itself was initially developed to visualize DNA but adapted to allow quantification and localization of mRNA in single cells using fluorescence microscopy. Contemporary RNA FISH protocols use oligonucleotides of 17–22 nucleotides that are complementary to the RNA under study (in vivo hybridization (FIVH) for live-cell mRNA imaging in mammalian cells (discussed below) and Turbo RNA FISH in which the hybridization time is increased by changing to alcohol-based fixation and increasing probe concentration  in individual cells . Both strategies have been successfully applied to quantify endogenous transcripts in E. coli  hybridization (BDH) , multiplFigure 3C). Locked nucleic acid (LNA) probes are more rigid because the 2′ oxygen and the 4′ carbon in the RNA nucleotide are linked, leading to highly specific Watson-Crick base pairing . Importantly, this technique has been successfully applied in single E. coli cells, in which it has led to the acknowledgment that mRNAs that include genes encoding membrane proteins are generally localized in proximity of the cytoplasmic membrane, whereas mRNA species that only contain open reading frames for soluble, secretory or outer membrane proteins are scattered throughout the cytoplasm (Multiplexed error-robust FISH was introduced by the group of Zhuang in 2015 and is a strategy based on multiplexed fluorescence formance . Elaboraytoplasm .in vivo hybridization (FIVH) can be achieved but requires alternative probe delivery and circumvention of at least the degradation of probes by cellular nucleases. Synthetic probes, such as LNA and PNA probes, but also 2′-O-methylated oligonucleotides  display improved nuclease stability, which make them especially suited for in vivo imaging of RNA . One end of the probe contains an organic fluorophore, while the other is labeled with a quencher that keeps the fluorophore in a dark state as long as the termini of the probe are in close proximity. Quenching is alleviated when the molecular beacon adopts a linear structure upon interaction with target RNA, yielding fluorescence. mRNAs have been visualized in vivo via microinjection of 2OMe-based molecular beacons in a variety of mammalian cells  and Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes consist of PNA or 2OMe-based backbones that carry a fluorogenic base . The incic cells . Interes oocytes .Figure 3D; The so-called black hole quencher (BHQ) aptamer initially showed insufficient functionality as mRNA appendix and was modified into a probe that anneals to target RNA  were used to detect 16S rRNA sequences in living E. coli cells with high specificity  and sodium dodecyl sulfate (SDS) surfactant  probes  can therefore be used in mammalian 293T cells to specifically incorporate an azide-carrying agmatine in 5S RNA carrying a genetically encoded archaeal tRNAIle2-tail, after which the tRNAIle2-5S fusion RNA can be fluorescently labeled with Sulfo-Cy5-azide via AAC , which requires the incorporation of toxic Cu(II) molecules. Therefore, the studies presented above utilized cell fixation prior to CuAAC. However, copper-free AAc reactions, like strain-promoted AAc (SPAAC), could be employed to realize site-specific and covalent RNA labeling in living cells.Last but not least, efforts are undertaken to covalently label RNAs in cells with reporter molecules via so-called bio-orthogonal “click” chemistry, which often makes use of one-pot azide-alkyne cycloaddition (AAC) to achieve bioconjugation. The RNA of interest can be post-synthetically and chemo-enzymatically modified by transferases to contain click-compatible moieties that are then joined to other substrates through biocompatible AAC  aptamer is a bright light in bacterial mRNA detection as the system combines the advantages of targeting native transcripts with high specificity and bright and stable fluorophores.RNA-binding protein-based techniques, such as the MS2, PP7 and eIF4A systems have been adapted for use in bacterial cells more than a decade ago but have, unfortunately, met with limited success. Although MS2 can be used to follow transcript production in time, one has to keep in mind that the diffusion and localization of MS2-labeled transcripts are severely estranged from the natural situation due to the addition of bulky complexes. The number of MS2-binding appendices can be reduced when studying high-abundant RNAs and/or by careful fine-tuning of the ratio between target RNAs and RBP levels. However, this would still not allow quantifying low-abundant or single RNA molecules to reflect native cell genetics. New RBP-based methods have recently emerged that make use of Cas9 or Pumby modules . Their uin vivo imaging of bacteria. Rather, it conveys static data of - in fact – highly dynamic processes. On top of that, cell fixation required for FISH could also influence the localization of the studied RNA by itself.To date, smFISH is the only available technique to reliably count the number of mRNA molecules in bacterial cells while at the same time acquiring data with high spatial resolution . Recent E. coli while at the same time keeping cells in a viable state  compatible with the small-sized and cell wall-enclosed bacteria. Electroporation can be used to internalize short DNA fragments or proteins up to 100 kDa in le state . It woulle state . More rele state . Alternain vivo RNA studies. Moreover, the recently developed aptamers and their organic dye ligands are, in theory, compatible with other aptamer-dye pairs by taking advantage of the variation in spectral characteristics of the fluorophores. Especially those techniques that would allow detecting single RNA molecules for prolonged periods of time would boost the knowledge on bacterial cell biology. So far, all fluorogen-binding aptamer methods that have been used to detect transcripts in bacteria have employed multicopy plasmids to express the mRNA. Also, all of the studies have so far been undertaken in E. coli only. These are clear indications of the fact that most techniques are still under development and none have yet yielded physiological information on endogenous bacterial transcripts, many of which are typically low-abundant and short-lived.The shortfall of FISH in imaging RNAs in living bacterial cells has led to the development of methodologically different RNA-labeling techniques. Approaches that have received the most attention in recent years make use of aptamer-binding fluorogenic dyes. Many small organic dyes are able to diffuse into cells and are good candidates for less intrusive RNA imaging strategies. A number of fluorogen-aptamer pairs have been reported for live-cell RNA imaging, of which most required considerable optimization after their first introduction. Next-generation aptamers are now coming of age. Additional technical improvements and development of new fluorogen-binding aptamers circumvent many of the problems that are encountered with protein-based RNA-labeling techniques and, thus, hold great promise for future E. coli at the single-cell level (Although monitoring of single mRNA molecules in bacteria has proven difficult, important information has been gained regarding the spatial behavior of RNA species inside bacterial cells. The techniques have allowed uncovering the transcription kinetics of promoter systems in ll level  and led ll level . The sysLvG has drafted the manuscript. JK and LvG have reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Methanothermobacter thermoautotrophicus RNA ligase (MthRnl) catalyzes formation of phosphodiester bonds between the 5′-phosphate and 3′-hydroxyl termini of single-stranded RNAs. It can also react with RNA with a 3′-phosphate end to generate a 2′,3′-cyclic phosphate. Here, we show that MthRnl can additionally remove adenosine from the 3′-terminus of the RNA to produce 3′-deadenylated RNA, RNA(3′-rA). This 3′-deadenylation activity is metal-dependent and requires a 2′-hydroxyl at both the terminal adenosine and the penultimate nucleoside. Residues that contact the ATP/AMP in the MthRnl crystal structures are essential for the 3′-deadenylation activity, suggesting that 3′-adenosine may occupy the ATP-binding pocket. The 3′-end of cleaved RNA(3′-rA) consists of 2′,3′-cyclic phosphate which protects RNA(3′-rA) from ligation and further deadenylation. These findings suggest that ATP-dependent RNA ligase may act on a specific set of 3′-adenylated RNAs to regulate their processing and downstream biological events. A variety of ribonucleases can catalyze this step, including: the tRNA splicing endonuclease, which removes introns in tRNAs2; Ire1 endonuclease, which cleaves the HAC1 mRNA during the unfolded protein response4; Usb1/Msp1 3′-5′ exoribonuclease, which trims the oligouridine tail for maturation of the U6 snRNA8; and type 1 DNA topoisomerase, which can cleave RNA through formation of covalent topoisomerase–RNA intermediate9. The 2′,3′-cyclic phosphate end can also be generated by conversion of an RNA terminating in a 3′-PO4, by the RNA cyclase RtcA12 and a number of thermophilic polynucleotide ligases13. This reaction involves transfer of AMP from ATP to the RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate, which is subsequently attacked by the 2′-OH to yield cyclic ends, a reaction that liberates AMP.2′,3′-cyclic phosphates at the 3′-termini of RNAs play important roles in RNA metabolism. This cyclic phosphate and a 5′-OH end are formed by a transesterification reaction, in which the 2′-OH on the ribose attacks the adjacent 3′-phosphate, breaking the phosphodiester backbone of the RNA, during either chemical or enzymatic cleavage22. RtcB reacts with GTP to form RtcB-GMP, converts the 2′,3′-cyclic phosphate into 3′-PO4, transfers the GMP to the 3′-PO4 end to form an RNAp(pG) intermediate25. It then forms a phosphodiester linkage by nucleophilic attack, with 5′-OH liberating GMP. ATP-dependent RNA ligation involves a series of nucleotidyl transfer steps similar to those that occur during ligation via the RtcB pathway, with the ligase reacting with ATP to form a covalent ligase-AMP, followed by transfer of the AMP to the 5′-PO4 end of the RNA to form an AppRNA intermediate, and subsequent formation of a phosphodiester bond by nucleophilic attack, with 3′-OH releasing AMP28.Interest in the 2′,3′-cyclic phosphate resurged following identification of a GTP-dependent RNA ligase, RtcB, that acts specifically on cyclic ends. The 2′,3′-cyclic phosphate and 5′-OH terminus generated during bacterial RNA repair, mammalian and archaea tRNA splicing, and metazoan unfolded protein response are joined to form a phosphodiester bond by RtcBMethanothermobacter thermoautotrophicus RNA ligase (MthRnl) is an ATP-dependent RNA ligase that belongs to the Rnl3 family and catalyzes the circularization of single-stranded RNA, as well as DNA29. Under non-optimal conditions, MthRnl can remove 5′-AMP from the AppRNA intermediate to generate a 5′-PO4 RNA30. MthRnl can also transfer AMP to RNA containing 3′-PO4 termini to form 2′,3′-cyclic phosphate13. While there is growing interest for the use of thermostable RNA ligases in constructing sequencing libraries of small RNAs, including microRNAs, its physiological/optimal polynucleotide substrate is not known. In this report, we show that MthRnl cleaves the adenosine residue from the 3′-OH end and leaves RNAs with 2′,3′-cyclic phosphates. Notably, cleavage is selective, with only a single adenosine residue removed. These findings raise the possibility that the RNA ligase acts as a surveillance/editing enzyme that selectively converts the reactive 3′-OH into a 2′,3′-cyclic phosphate end, thereby regulating their processing and downstream biological events.Several archaea species encode both GTP-dependent and ATP-dependent RNA ligases. 29 32 was capable of forming RNA* from any of the RNA substrates tested , and demonstrated that the recombinant protein is likewise capable of generating RNA*, specifically from RNA with a 3′-adenine  overhang and dual overhang pRNA substrates were sensitive to alkaline phosphatase treatment, indicating that the 5′-radiolabeled phosphate on RNA* is exposed Fig. . These rAs described above, RNA* was observed on pRNA substrates that contain adenine, but not a uracil at the 3′-terminus Fig. . This sp30. The 3′-deadenylation activity with 3′-(5nt) overhang pRNA substrate was ~ 2-fold higher than that with the dual overhang pRNA substrate  overhang pRNA is converted to pRNA(-3′A) in 60 min Fig. . The iniate Fig. . In the 30; only trace amounts of pRNA(-3′A) were detected in the presence of the K97A and N99A mutant forms of the enzyme. On the other hand, MthRnl variants that catalyze EpA formation but are defective for the step 2 or step 3 ligation reaction (T117A and R118A) were capable of forming pRNA(-3′A). We speculate that adenosine at the 3′-end of the RNA is recognized by the active site that recognizes the ATP in step 1 of the ligation reaction.Like the ligation reaction, the 3′-deadenylation activity requires a divalent cation. This requirement was satisfied by magnesium, manganese, cobalt or, to a lesser extent, calcium Fig. . MthRnl 29. To determine whether MthRnl is additionally capable of removing deoxyadenosine, we prepared RNA substrates in which the 3′-terminus was replaced with 2′-deoxyadenosine . However, this was not the case. The 3′-(5nt) overhang RNA substrate with two consecutive 3′-adenosines did not generate a shorter 3′-deadenylated species Fig. . To conf4 into 3′-OH  lacks a reactive 3′-OH that is necessary for the strand-joining reaction. We hypothesize that the adenosine is released and the phosphate group is retained on the 3′-end of pRNA(-3′A) by MthRnl. To test this hypothesis, we purified pRNA(-3′A) generated by MthRnl from the 3′-5nt) overhang substrate and incubated it with T4 polynucleotide kinase (Pnk), which can convert 2′,3′-cyclic phosphate or 3′-POnt overha4 is required for 3′-deadenylation by MthRnl, we incorporated radiolabeled phosphate into the RNA between the 3′-terminal adenosine and its penultimate nucleoside by ligating [α-32P]pAp with T4 Rnl1 ; one with both 5′- and 3′-PO4 (pRNAp); and one without a phosphate group (OHRNAOH)  Fig. . MthRnl nce Fig. ; lane 8.4 or a 3′-PO4. To distinguish between these possibilities, we isolated the OHRNA(-3′A) product generated by MthRnl . Treatment of OHRNA(-3′A) with acid, which converts the 2′,3′-cyclic phosphate into 2′-PO4 and 3′-PO4, sensitizes OHRNA(-3′A) to alkaline phosphatase  likely possesses a 2′,3′-cyclic phosphate group at its 3′-end. Furthermore, we show that OHRNA(-3′A) can be ligated by RtcB, which can join 5′-OH terminated RNA with either 2′,3′-cyclic phosphate or a 3′-PO4 end, to form a 20-mer circular RNA  with HCl inhibits circularization by ~50%; this likely reflects a conversion of 2′,3′-cyclic phosphate into ligatable 3′-PO4 and unligatable 2′-PO4 ends  could be occupied by either a 2′,3′cyclic phosphate, a 2′-PORnl Fig. , lane 8 Rnl Fig. , lane 6.ase Fig. , lane 8.RNA Fig. , lane 6.nds Fig. , lane 8.4 end. Once the RNA is 3′-deadenylated, subsequent deadenylation is inhibited due to the presence of a 2′,3′-cyclic phosphate end. Similar to the ligation activity, the 3′-deadenylase activity requires a divalent cation, and excess ATP inhibits the reaction. MthRnl mutant proteins that fail to form the ligase-AMP complex are defective for 3′-deadenylation. In contrast, Thr-117 and Arg-118, which do not affect formation of the ligase-AMP complex (step 1) but are required for formation of the phosphodiester bond (step 3), did not affect the 3′-deadenylation activity30. The fact that ATP can inhibit the 3′-deadenylation reaction suggests that the 3′-adenosine on the RNA may compete for the same catalytic site for binding of the ATP. We propose that 3′-adenosine is recognized by the ATP binding pocket in the MthRnl and catalyzes the deadenylation through a transesterification reaction, by stimulating the penultimate ribose 2′-OH to attack the adjacent 3′-5′ phosphodiester to form a 2′,3′-cyclic phosphate and releasing adenosine -rich tails are synthesized and removed by the exosome35. As in the case of the mammalian U6 snRNA, the formation of 2′,3′-cyclic phosphates could preclude the elongation of poly(A)-rich tails and protect them from degradation machinery from regulating stability of the mRNA. It is also plausible that MthRnl acts as a surveillance enzyme that prevents undesirable intramolecular RNA circularization and intermolecular ligation with the 5′-PO4 RNA, by converting the reactive 3′-OH end into a nonreactive 2′,3′-cyclic phosphate. MthRnl may also function in forming a 2′,3′-cyclic phosphate ends that serve as substrates for subsequent ligation with 5′-OH RNA by RtcB for a production of a novel RNA species, or generating an adenylated capped RNA (5′-AppRNA) with a 2′,3′-cyclic phosphate end. In summary, our findings raise possibilities for unexpected mechanisms whereby the MthRn1 could act as a surveillance or editing enzyme, to selectively remove the 3′-adenosine of an RNA to convert the reactive 3′-hydroxyl group into 2′,3′-cyclic phosphate and thereby regulate its metabolism.The present study raises the interesting prospect that RNA ligation might not be the only biochemical pathway for MthRnl. We speculate that MthRnl acts on a specific set of 3′-adenylated RNAs to regulate their processing and downstream biological events. RNAs that are exclusively terminated by 3′-adenosine include tRNAs and mRNAs. In the final step of tRNA maturation, the 3′-end is modified by the addition of a -CCA32P] ATP at 37 °C for 1 hr. Radiolabeled pRNA and pDNA were purified on 13% native polyacrylamide gels.Synthetic RNAs were purchased from Integrated DNA Technologies, Inc. . Oligoribonucleotides (1 nmole) were labeled at the 5′-end using 20 units of T4 Pnk in a 20 µl reaction containing 3 nmoles of  was prepared from 100 pmol ofE. coli BL21(DE3). MthRnl production was induced with IPTG, and His tagged-MthRnl proteins were purified from soluble bacterial extracts by Ni-agarose chromatography as described previously30. His tagged-T4 RNA Ligase 132 and T4 RNA Ligase 233 were produced in E. coli and purified as described. Protein concentrations were determined with the BioRad dye reagent, using bovine serum albumin (BSA) as the standard.Plasmids that encode wild-type and mutant MthRnl were transformed into 2, 1 pmol of 32P-labeled pRNA and the indicated amount of MthRnl, with or without ATP, were incubated at 55 °C. The reactions were terminated by adding an equal volume of formamide gel loading buffer . Products were resolved on denaturing 18% (w/v) polyacrylamide (19:1) gels containing 7 M urea in 0.5 × TBE . The extent of ligation and 3′-deadenylation were determined by scanning the gel with a Storm Molecular Imager and analyzed by ImageQuant software.Standard reaction mixtures (20 µl) containing 50 mM Tris-HCl (pH 6.5), 0.5 mM MgClSupplementary Figures