(OPMV) is a recently discovered umbravirus in the family
OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the ...RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA
). sgRNA1 codes for a 30-kDa protein
that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a
-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences
, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.
(OPMV) is an umbravirus in the family
We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNA
site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA
sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation
We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.
In the absence of a 5' cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3' untranslated region (UTR) to attract initiation factors and/or ribosomes to their ...templates. These 3' cap-independent translational enhancers (3' CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3' CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5' end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3' CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus's host range.
Translation of plant plus-strand RNA viral genomes that lack a 5' cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 3' untranslated region (UTR). ...3'CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 3' end that is nearly always transferred to the 5' end via a long-distance kissing-loop interaction between sequences in the 3'CITE and 5' hairpins. We report here the identification of a novel 3'CITE in coat protein-deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 3' UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 3'CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 5' end. We also report that the ∼30-nucleotide (nt) 5' terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 3'CITE or the 3'CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs.
Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 3' cap-independent translation enhancer (3'CITE), which does not connect with the 5' end by a long-distance RNA:RNA interaction that is typical of 3'CITEs. ulaRNA 5' terminal hairpins can also enhance translation in association with cognate 3'CITEs or those of related ulaRNAs and, to a lesser extent, with 3'CITEs of umbraviruses, or even independent of a 3'CITE. These findings introduce a new type of 3'CITE and provide the first information on translation of ulaRNAs.
Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through ...mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.
Abstract
Plus-strand RNA viruses frequently employ -1 programmed ribosomal frameshifting (-1 PRF) to maximize their coding capacity. Ribosomes can frameshift at a slippery sequence if progression is ...impeded by a frameshift stimulating element (FSE), which is generally a stable, complex, dynamic structure with multiple conformations that contribute to the efficiency of -1 PRF. As FSE are usually analyzed separate from the viral genome, little is known about cis-acting long-distance interactions. Using full-length genomic RNA of umbravirus-like (ula)RNA citrus yellow vein associated virus (CY1) and translation in wheat germ extracts, six tertiary interactions were found associated with the CY1 FSE that span nearly three-quarters of the 2.7 kb genomic RNA. All six tertiary interactions are conserved in other Class 2 ulaRNAs and two are conserved in all ulaRNAs. Two sets of interactions comprise local and distal pseudoknots that involve overlapping FSE nucleotides and thus are structurally incompatible, suggesting that Class 2 FSEs assume multiple conformations. Importantly, two long-distance interactions connect with sequences on opposite sides of the critical FSE central stem, which would unzip the stem and destabilize the FSE. These latter interactions could allow a frameshifting ribosome to translate through a structurally disrupted upstream FSE that no longer blocks ribosome progression.
Graphical Abstract
Graphical Abstract
Abstract
Two-dimensional drawing of nucleic acid structures, particularly RNA structures, is fundamental to the communication of nucleic acids research. However, manually drawing structures is ...laborious and infeasible for structures thousands of nucleotides long. RNAcanvas automatically arranges residues into strictly shaped stems and loops while providing robust interactive editing features, including click-and-drag layout adjustment. Drawn elements are highly customizable in a point-and-click manner, including colours, fonts, size and shading, flexible numbering, and outlining of bases. Tertiary interactions can be drawn as draggable, curved lines. Leontis-Westhof notation for depicting non-canonical base-pairs is fully supported, as well as text labels for structural features (e.g. hairpins). RNAcanvas also has many unique features and performance optimizations for large structures that cannot be correctly predicted and require manual refinement based on the researcher's own analyses and expertise. To this end, RNAcanvas has point-and-click structure editing with real-time highlighting of complementary sequences and motif search functionality, novel features that greatly aid in the identification of putative long-range tertiary interactions, de novo analysis of local structures, and phylogenetic comparisons. For ease in producing publication quality figures, drawings can be exported in both SVG and PowerPoint formats. URL: https://rnacanvas.app.
Graphical Abstract
Graphical Abstract
Exploring the 2,692 nt citrus yellow vein-associated virus genome structure in RNAcanvas. Currently selected sequence highlighted yellow. RNAcanvas has highlighted two putative tertiary pairings purple (hovered by cursor) and pink.
Background
Few publications in the literature examine enhanced recovery after scoliosis surgery (ERAS) in children, despite significant scientific interest in adults. The objective of the current ...study was to describe an ERAS protocol for surgical correction of adolescent idiopathic scoliosis (AIS) and its results.
Methods
ERAS outcomes were measured in two patient cohorts. Historical controls and ERAS groups were selected from patients managed for scoliosis surgery in 2015 and 2018, respectively. The ERAS protocol included fasting minimization, carbohydrate loading, the avoidance of background morphine infusions, perioperative opioid‐sparing protocols, the use of a cooling brace, early physiotherapy, feeding and oral medications, and the early removal of urinary catheters and surgical drains. The main outcome of the study was hospital length of stay.
Results
Overall, 82 controls and 81 ERAS patients were recruited. ERAS protocols were observed in over 80% of patients for almost items. Median length of hospital stay was significantly lower in the ERAS group (‐ 3 95% confidence interval: −2; −4 days). Median morphine consumption was reduced by 25% and 35% on days 2 and 3, respectively. The incidence of PONV did not differ between the two groups, and the incidence of constipation decreased slightly but significantly in the ERAS group on day 2. Pain intensity at rest and movement were lower in the ERAS group at day 2 and 3.
Conclusions
The current study suggests an ERAS protocol after adolescent idiopathic scoliosis surgery is associated with reduced hospital length of stay and improved postoperative care.
Significance Many functions in the cell are performed by Brownian machines, macromolecular assemblies that use energy from the thermal environment for many of the conformational changes involved in ...their work cycles. Here we present a new approach capable of mapping the continuous motions of such nanomachines along their trajectories in the free-energy landscape and demonstrate this capability in the context of experimental cryogenic electron microscope snapshots of the ribosome, the nanomachine responsible for protein synthesis in all living organisms. We believe our approach constitutes a universal platform for the analysis of free-energy landscapes and conformational motions of molecular nanomachines and their dependencies on temperature, buffer conditions, and regulatory factors.
A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The free-energy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.
Background
EOSedge™* (EOS Imaging, Paris, France) is an X‐ray imaging system using automatic exposure control (AEC) with tube current modulation, in order to optimize dose deposition in patients.
...Purpose
This study aims at characterizing EOSedge organ dose deposition in comparison to a digital radiography (DR) system and the previous EOS system (EOS‐1st generation), in relation to their respective image quality levels.
Method
Organ doses were measured in an anthropomorphic female adult phantom and a 5‐year‐old pediatric phantom using optically stimulated luminescence (OSL) dosimeters, which were carefully calibrated within the studied energy range. Organ doses were recorded on the EOSedge and the Fuji Visionary DRF (Fujifilm Medical Systems U.S.A., Inc, Lexington, MA). The resulting effective doses were compared to the EOS‐1st‐generation values present in the literature. Image quality assessment was carried out on end‐user images. Quantitative image quality metrics were computed for all tested modalities on a quality assurance phantom. Qualitative assessment of EOSedge image quality was based on anthropomorphic phantom acquisitions against the EOS‐1st‐generation system, and on clinical images against the tested DR system.
Results
For a full‐spine exam, and on the female adult phantom (respectively, the pediatric phantom), an effective dose of 92 μSv (respectively, 32 μSv) was obtained on EOSedge, and 572 μSv (respectively, 179 μSv) on the DR system; these values were compared to effective dose values of 290 μSv (respectively, 200 μSv) from the literature on EOS‐1st generation, leading to an effective dose reduction factor of 6 with respect to the DR system, and of 3–6 with respect to EOS‐1st generation. EOSedge provides the best compromise between contrast‐to‐noise ratio (CNR) and dose, with more consistent CNR values than the other tested modalities, in a range of attenuation from 10 to 40 cm of poly(methyl methacrylate) (PMMA). Within this range, EOSedge is also comparable to DR for 10 and 20 cm of PMMA, and better than DR for 30 and 40 cm of PMMA, both in terms of spatial resolution and low‐contrast detection. The anatomical landmarks of interest in the follow‐up of spinal deformities can be detected in all tested modalities.
Conclusion
Results showed that EOSedge provides significant dose reduction factors for full spine imaging in both adults and children compared to the other tested modalities, without compromising image quality. We believe that this work could help raise awareness on the capabilities of modern X‐ray systems, when equipped with appropriate AEC strategies, to perform ultra‐low‐dose, long‐axis images.
3'UTRs of carmoviruses Simon, Anne E
Virus research,
08/2015, Letnik:
206
Journal Article
Recenzirano
Carmovirus is a genus of small, single-stranded, positive-strand RNA viruses in the Tombusviridae. One member of the carmoviruses, Turnip crinkle virus (TCV), has been used extensively as a model for ...examining the structure and function of RNA elements in 3'UTR as well as in other regions of the virus. Using a variety of genetic, biochemical and computational methods, a structure for the TCV 3'UTR has emerged where secondary structures and tertiary interactions combine to adopt higher order 3-D structures including an internal, ribosome-binding tRNA-shaped configuration that functions as a 3' cap-independent translation enhancer (3'CITE). The TCV 3'CITE also serves as a scaffold for non-canonical interactions throughout the 3'UTR and extending into the upstream open reading frame, interactions that are significantly disrupted upon binding by the RNA-dependent RNA polymerase. Long-distance interactions that connect elements in the 3'UTR with both the 5' end and the internal ribosome recoding site suggest that 3'UTR of carmoviruses are intimately involved in multiple functions in the virus life cycle. Although carmoviruses share very similar genome organizations, lengths of 5' and 3'UTRs, and structural features at the 3' end, the similarity rapidly breaks down the further removed from the 3' terminus revealing different 3'CITEs and unique virus-specific structural features. This review summarizes 20 years of work dissecting the structure and function of the 3'UTR of TCV and other carmoviruses. The astonishing structural complexity of the 3'UTRs of these simple carmoviruses provides lessons that are likely applicable to many other plant and animal RNA viruses.
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