Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to genus Alphahypovirus of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining are heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite RNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of RNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of fungal satellite.Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus  Alphahypovirus of the family Hypoviridae was identified in a phytopathogenetic P. fici isolated from a tea leaf and tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1), which represents the first hypovirus from Pestalotiopsis fungi. Three RNA components  associated with PfHV1 including the genomic RNA, defective RNA and satellite-like RNA (SatL-RNA) were determined, and their molecular and biological traits were characterized. The SatL-RNA negatively regulates the accumulation of PfHV1 RNA1, and represents a novel class of satellite nucleic acids since it has substantial sequence homology with the host viral genome and is not encapsidated in the coat protein of its helper virus. This study contributes to our better understanding of fungal satellites, as well as the molecular and biological traits of hypoviruses.A novel hypovirus belonging to the genus  Hypoviridae is a family of capsidless viruses with positive-sense (+), single-stranded (ss) RNA genomes of 9.1–12.7 kb that possess either a single large ORF or another small ORF [Hypovirus, is included in this family, which contains Cryphonectria hypovirus 1–4 (CHV1-4) and among them, CHV1 is associated with biologic control of the filamentous fungus that causes chestnut blight [Hypovirus has been rapidly expanded into eight genera, namely Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Zeta-, Eta-, and Thetahypovirus [ypovirus –5. Hypovypovirus , Botrytiypovirus . Defectiypovirus , Sclerotypovirus , Fusariuypovirus , which wTotiviridae and Partitiviridae. Satellites do not constitute a homogeneous taxonomic group, and have no taxonomic correlation with their helper viruses, and have arisen independently a number of times during virus evolution [Satellites are subviral agents which lack genes that could encode functions needed for replication, and depend on the co-infection of a host cell with a helper virus, which consist of satellite viruses and satellite nucleic acids, discriminating by the presence of a structural protein . Satellivolution . Since sPestalotiopsis spp. belong to the family Amphisphaeriaceae that are distributed widely throughout tropical and temperate regions. They are common phytopathogens reducing production and causing enormous losses in either horticultural plants or forest trees [P. theae and P. longiseta are responsible for tea gray blight disease, which leads to large brown spots with the formation of apparent concentric rings in the late period of disease on mature and old leaves of tea plants, and even leads to death of tender branches of infected tea seedlings; P. theae, P. camelliae, and P. clavispora were also identified in association with small brown-black spots on tea tender leaves [Pestalotiopsis species can confer fitness to the host plants, and are also thought to be a rich source for bioprospecting compared to other fungal genera since they produce novel compounds with medicinal, agricultural and industrial applications [Pestalotiopsis fungi live in host plants with a complex lifestyle, being parasitic, commensal, or mutualistic. Mycoviruses might have participated in the transition of these lifestyles, since a chrysovirus, the first mycovirus isolated from Pestalotiopsis fungi, can convert its host fungus from a phytopathogenetic one to a nonpathogenetic endophyte on tea leaves, conferring high resistance to the host plants against the virulent P. theae strains [st trees . For exar leaves ,17. Howe strains . It is f strains .P. fici with three components (RNAs 1 to 3) was identified and characterized, which represent a novel class of satellite nucleic acids, illustrating some novel molecular and biological traits of a fungal satellite.In this study, a novel special satellite RNA associated with a novel hypovirus from P. fici strain AH1-1 was isolated from a tea leaf showing the typical symptoms of tea grey blight disease collected in Liu’an city, Anhui province, China, and was identified based on morphologies and multi-loci sequences as previously described [-, AH1-1-14 and AH1-1-24 are subisolates generated from single conidia of strain AH1-1. Strain AH1-1V- is a cured subisolate of AH1-1 by elimination of dsRNA components, and conferred hygromycin (hyg)-resistant ability by transformation of a hyg gene to generate strain AH1-1hygV-. P. theae TP-2-2W, JWX-3-1, and CJB-4-1 were collected from tea leaves in Yichang city, Hubei province, China, and were identified on their morphologies and internally transcribed spacer (ITS) sequence. P. theae strain LI41 was isolated from a healthy tea leaf collected in Xuan’en county, En’shi prefecture, Hubei province, China, and LI41-1P1 is a subisolate of P. theae LI41, both of which were infected by Pestalotiopsis theae chrysovirus-1 (PtCV1) [escribed , and str (PtCV1) . All strFor dsRNA extraction, fungal mycelial plugs were inoculated onto sterilized cellophane disks on PDA plates at 25°C in the dark for 4 to 5 days, and the mycelia were collected and subjected to dsRNA extraction as previously described . The resin vitro transcripts of complementary DNAs (cDNAs) of peach latent mosaic viroid (PLMVd) [The dsRNA preparation, BdRV1 dsRNAs (extracted from mycelia infected by BdRV1) , in vitr (PLMVd)  with its (PLMVd) .MAFFT alignment and NJ / UPGMA phylogeny (cbrc.jp)). The phylogenetic tree for RNA-dependent RNA polymerase (RdRp) sequences was constructed using MEGA 11 with the Maximum Likelihood method [https://www.ncbi.nlm.nih.gov/orffinder/). Conserved domains were predicted using Phyre2 (https://www.sbg.bio.ic.ac.uk/phyre2). Internal ribosomal entry site (IRES) elements were predicted by aligning with IRESite database online  [The sequences of the dsRNAs were determined by cloning and sequencing amplicons generated by reverse transcription and polymerase chain reaction (RT-PCR) using the random primers 05RACE-3RT and 05RACE-3  as previd method . Open reuctures) .RT-PCR amplification was performed using a specific primer pair derived from the dsRNA 1 sequence  , generatin vitro transcription based on the corresponding cDNAs inserted into the pGEM-T easy vector with T7 RNA polymerase  as previously described [Digoxigenin (DIG)-labeled riboprobes 1 (binding to positions from 1588 to 1934 nt of dsRNA1), 2 and 3  were synthesized escribed . Nucleicescribed .Co-precipitate analysis of dsRNAs was conducted by sucrose gradient centrifugation according to the methods used for the purification of viral particles previously described . The resRNase A treatment was conducted as previously described . BrieflyPst I [To generate RNA3-over-expressed strains, RNA3 cDNAs were inserted into the multi-cloning sites of vector pCAMBgfp after linearization by Pst I . The conPst I .Actin gene was used to normalize the RNA samples for each qRT-PCR, and each treatment was conducted with three technical replicates.The total RNAs from mycelia were extracted by Trizol agent and subjected to digestion by 5×gDNA digesture  to remove the genomic DNAs, followed by cDNA synthesis using 2× reverse transcriptase  and amplification using 2×TSINGKE master qPCR mix  using specific primers designed for quantitative analysis in CFX96 Real-Time PCR Detection System . Amplification was conducted for 40 cycles of 95°C for 10 s, 56°C for 10 s and 72°C for 15 s after initial heating at 95°C for 1 min. A partial fragment of the Horizontal transmission of the virus was conducted as previously described . BrieflyCamellia sinensis, C. sinensis vars. Echa no.1 or Fuyun no.6) as previously described [Fungal growth rates and morphologies were assessed as previously described . Three bescribed . At 4 dahttp://www.winwrap.com). The mean values for the biological replicates are presented as column charts with error bars representing standard error of mean (SEM). The graphs were produced in Excel (Microsoft) and GraphPad Prism 7 (GraphPad software). P-values < 0.05 were considered to indicate statistical significance, while P-values < 0.01, extremely significant.The biological data were statistical analysis using SPSS Statistics 17.0 with one-way analysis of variance (ANOVA) and Tukey post-hoc tests  were detected in preparations of strain AH1-1 but not in a control strain TP-2-2W (Nucleic acid preparations enriched in dsRNA were obtained from  TP-2-2W . Of thes TP-2-2W . The seqin vitro dimeric transcripts of peach latent mosaic viroid (PLMVd) and dsRNAs from a dsRNA mycovirus . The RNAs extracted from strain AH1-1 together with BdRV 1 dsRNAs were digested by RNase A in 0.1×SSC, but they resisted digestion by DNase I, S1 nuclease, and RNase A in 2×SSC. In sharp contrast, PLMVd transcripts were completely degraded by S1 nuclease and by RNase A under both ionic conditions, but resisted digestion by DNase I; the genomic DNAs (extracted together with BdRV 1) and the cDNA plasmid of PLMVd (used for in vitro transcription) were completely degraded by DNase I while resistant to RNase A and S1 nuclease , together with an ssRNA control (nuclease . These dThe complete nucleotide sequence of dsRNA1 is 10316 base pair (bp) in length, consisting of a single putative ORF beginning at AUG (nt positions 513–515) and terminating at UAG (9600–9602), coding for a polyprotein of 3029 aa with an approximate molecular mass of 345.9 kDa.The deduced polyprotein aa sequence contains conserved domains of RNA dependent RNA polymerase (RdRp), viral RNA Helicase (Hel), and a most likely papain-like protease (Pro) . Within E value = 0) encoded by Wuhan insect virus 14 (WIV14), as well as high identities  to those of Fusarium sacchari hypovirus 1  [Hypoviridae illustrated that the sequence clustered with FsHV1 and AaHV1 in the genus Alphahypovirus  with the polyprotein  , Apis hy% with thThe complete nucleotide sequence of dsRNA2 is 5511 bp in length excluding the poly (A) tail, consisting of a single putative ORF (nt positions 510–4043), coding for a polyprotein of 1177 aa with an approximate molecular mass of 133.5 kDa. Alignment with dsRNA1 indicated that the total sequence of dsRNA2 is separately identical with dsRNA1 in five regions, i.e., nt position 4 to 3939, 5988 to 6225, 6220 to 6508, 6512 to 6770, and 6774 to 7562. Namely, dsRNA2 is a D-RNA generated from dsRNA1 by deletion three regions from nts 1 to 3, 3940 to 5987 and 7563 to 10316 . Moreovein vitro transcription and hybridization with the nucleic acid preparations of P. fici strain AH1-1. As expected, two hybridized signals were observed corresponding to the electrophoresis position of dsRNAs 1 and 2, further supporting the D-RNA nature of dsRNA2.To further confirm that dsRNA2 as a D-RNA generated from dsRNA1, a digoxigenin (DIG)-labeled riboprobe (termed riboprobe 1) binding to the nt 1588 to 1934 of dsRNA1 was synthesized P. fici strain AH1-1 without protein-denaturing treatment process were subjected to RNase A treatment at 37°C or on ice, and P. theae strain LI41-1P1 infected by PtCV1 (an encapsidated dsRNA mycovirus) was involved in parallel as a control. The results showed that PfHV1 dsRNAs, similar to PtCV1, are resistant to RNase A treatment, suggesting that they are encapsulated  tail, and it has a small putative ORF (nt positions 248–400) on its positive strand coding for a protein of 50 aa with an approximate molecular mass of 5.75 kDa. dsRNA3 contains nine putative IRES elements in nt positions 1–247  as aligned with those deposited in IRESite database . Alignmef dsRNA3 . Thus, tTo further confirm that dsNRA3 is a SatL-RNA hosted by PfHV1, the presumed viral vesicles were purified by centrifugation in stepwise sucrose gradients (10–35% with 5% sucrose increments), and subjected to nucleic acids extraction. Agarose gel electrophoresis of the resulted nucleic acids showed that dsRNA3 together with the typical pattern of PfHV1 dsRNAs 1 and 2 was most recovered from the 15% fractions . These dP. fici. With the resistance selection and qPCR identification, three derivative subisolates, termed AH1-1-24SatL-OE1 to AH1-1-24SatL-OE3, transformed by RNA3-expression vector were obtained. Expression analysis of dsRNA1 together with RNA3 using qRT-PCR in the three resulting subisolates revealed that the expression levels of RNA3 were significantly up-regulated up to 1.96 to 9.72 folds in these transformed subisolates  of the subisolates were infected with PfHV1 dsRNA1 as detected by agarose gel analysis of their nucleic acids after treated by DNase I  were dual-cultured, and four AH1-1hygV- mycelium discs next to the contact area were randomly selected for RT-PCR analysis of the presence of PfHV1 dsRNAs. This experiment revealed that all the selected subisolates (nominated AH1-1hygV+-1 to -4) were infected by PfHV1 dsRNAs 1 and 2, while not by dsRNA3  and AH1-1y dsRNA3 . WhereasPestalotiopsis strains. DsRNA2 is efficiently transmitted horizontally but not vertically in the same host strain, whereas dsRNA3 could not be transmitted horizontally and vertically.These results suggest that PfHV1 dsRNA1 is efficiently transmitted vertically and horizontally in the host strain, while not easily to other +) and its cured subisolate AH1-1V- (PfHV1-) after culture on PDA in parallel at 4 dpi, no obvious difference was observed in these aspects (C. sinensis var. Fuyun no.6) , they also showed similar morphologies, growth rates, and virulence  . As compirulence . These rP. fici, and their full-length sequences were determined and characterized, suggesting that they belong to a novel hypovirus, tentatively named PfHV1, under the genus Alphahypovirus [in vivo in abundant. The predominant dsRNA form found in infected mycelia appears to be replicative intermediate or replicative form RNA, while the accumulation of plus strand RNA is low due to the RNA silencing antiviral defense response of the fungal host [Cryphonectria parasitica [Sclerotinia sclerotiorum [Valsa ceratosperma [Fusarium graminearum [Phomopsis longicolla [Macrophomina phaseolina [Botrytis cinerea [Rosellinia necatrix [Pestalotiopsis fungus, and the second mycovirus besides of a chrysovirus infecting this fungal genus.In this study, three dsRNA components were detected in ypovirus ,4,30. Hegal host . Other trotiorum ,30,32, Vminearum ,12, Phomngicolla , Macrophaseolina , Botryti cinerea , and Rosnecatrix , and evenecatrix  revealedBetahypovirus and Gammahypovirus. Moreover, a complete papain-like cysteine Pro motif was not detected in the polyprotein of PfHV1, whereas alignment with the polyproteins of other hypoviruses uncovered the presence of two conserved cysteine Pro core residues , with two papain-like cysteine Pro encoded by ORF A and the leading regions of ORF B, respectively, as exemplified by CHV1 ,37,38, wPestalotiopsis spp. strains, like the defective RNA 2 associated with CHV3 [dsRNA 2 is determined as a defective component of dsRNA 1, which is very stable since it could be horizontally transmitted, as well as easily observed in other natural ith CHV3 . Howeverith CHV3 ,43. The ith CHV3 ,45, sincith CHV3 ,46.C. parasitica and S-dsRNA (3.6 kb) of SsHV1 in S. sclerotiorum, both of which encode authentic proteins and contain a poly (A) tail [in vitro translation experiment [Totiviridae and Partitiviridae. We propose these satellite nucleic acids are classified into two classes including those coding dsRNAs with a poly (A) tail (like CHV3 dsRNAs 3 and 4) and those no-coding dsRNAs without a poly (A) tail (like PfHV1 dsRNA3). Additionally, we suggest to broaden the definition of fungal satellites to accommodate these satellite RNAs by including a special type of satellite nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein.dsRNA3 segment is determined to be a SatL-RNA of PfHV1 due to the following reasons: 1) it was co-precipitated with dsRNAs 1 and 2 in the same sucrose fraction of 15% by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs; 2) it codes no structural proteins or RdRp proteins, suggesting its replication depends on the helper virus; 3) no dsRNA3 component was solely observed in any subisolates absent of dsRNA1 after vertical or horizontal transmission. Until now, three satellite or SatL-RNAs have been observed in association with hypoviruses, i.e., dsRNA4 (937 bp in size) and dsRNA3 (dimeric dsRNA4 linking by a poly (A) of CHV3 in (A) tail ,41. Of tperiment ; S-dsRNAperiment . PfHV1 dperiment . Here, dperiment , insteadTotiviridae encode a secreted preprotoxin that is lethal to sensitive cells (virus-free or containing helper virus only), and imparts self-protection against the secreted toxin and confers ecological advantage by killing competing virus- or satellite-free fungi; the satellite RNA associated with SsHV1 most likely enhances the hypovirulence traits of S. sclerotiorum. However, the biological functions of other satellite RNAs remain largely unknown. Here, as compared with the dsRNA3-infected and -free strains of P. fici isolates, i.e., strain AH1-1 subisolates derived from horizontal transmission and those from vertical transmission, no obvious morphologies, growth rates or virulence were observed, suggesting that the SatL-RNA has no impact on these aspects. However, as the satellite-like RNA was overexpressed, it inhibits the replication of PfHV1, suggesting that dsRNA3 negatively regulates the replication of PfHV1. These results suggest that the SatL-RNAs harbored by hypoviruses have a similar biological trait to the satellite RNAs associated with animal and plant viruses. E.g., amplification of satellites by viral RdRp may down-regulate synthesis of viral RNAs and expression of their products in the research of Trichomonas vaginalis virus [in vitro competition assay, the reduction was proposed to be due to the competition of satellite RNA for limited amount of viral replicase with CMV gRNAs [Satellite RNAs have been described in association with several mycoviruses in filamentous fungi and yeast –52, and is virus ; cucumbeis virus ,55. BaseMV gRNAs . FurtherMV gRNAs . SimilarMV gRNAs . Since tP. fici, belonging to an important fungus genus related to agriculture, industry and medicine, a novel hypovirus from P. fici together with its D-RNA and a SatL-RNA, were identified and characterized. To our knowledge, PfHV1 represents the first hypovirus and the second mycovirus isolated from Pestalotiopsis spp., and its SatL-RNA represents a novel class of satellite nucleic acids, which should contribute to our better understanding of the taxonomy, evolution, molecular and biological traits of mycoviruses, as well as the related satellites.In summary, to gain insight into mycoviruses in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Fig(A and B) The secondary structures of 5′- and 3′-UTR of PfHV1 were predicted in RNAfold, respectively. (C) Six AUGs in the 5′-UTR of PfHV1 were found.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigTwo black curves refer to the nucleotides that make up a potential pseudoknot.(TIF)Click here for additional data file.


This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and  RNA viruses, such as influenza A viruses (IAV) or coronaviruses (CoV), are important pathogens that cause mild to severe respiratory disease in humans. RNA virus spread among humans occurs in seasonal epidemics, outbreaks of emerging viruses, or occasional pandemics . SeasonaAnalysis of IAV strains has shown that human-adaptive mutations occur in nearly all viral proteins, but in particular in the IAV surface glycoproteins hemagglutinin (HA) and neuraminidase (NA); the proteins involved in viral replication polymerase basic 1 and 2 (PB1 and PB2), polymerase acidic (PA) and the nucleoprotein (NP); and the protein involved in modulating host-immune responses, non-structural 1 (NS1) -16. DuriSecondary RNA structures in RNA virus genomes play critical roles in IAV and CoV infection cycles and perform functions in packaging, translation, and viral replication. In addition, RNA structures can be the target of therapeutics . SecondaIn this study, we aimed to gain insight into the evolution of template loops (t-loop), a transient RNA secondary structure that folds effectively “around” the viral RNA polymerase and affects IAV replication efficiency on short viral RNA templates . In the To test the above hypotheses, we performed cell culture assays as well as analyses of IAV, IBV and SARS-CoV-2 sequence data obtained through surveillance. We observe that the sensitivity of H3N2 IAV RNA polymerases to t-loops increased between 1968 and 2017, but that the total free energy (ΔG) of t-loops in the IAV H3N2 genome was linearly increased, and thus the overall stability reduced. Reductions in t-loop stability are also observed in H1N1 IAV following the 1918 and 2009 pandemics. No destabilization of t-loops is observed in the IBV genome, which has been circulating in humans for decades, but an analysis of SARS-CoV-2 isolates reveals destabilization of t-loops as well. Overall, we propose that the ΔG of RNA structures in the RNA genome of emerging respiratory RNA viruses may play a role in the adaption of these viruses to humans.Renilla luciferase control plasmid. In addition, we provided a cellular RNA polymerase I-driven plasmid expressing a 71-nt long template without a t-loop in the first half of the template (NP71.1) and a 71-nt long template containing a t-loop in the first half of the template (NP71.11). These templates were extensively tested previously and used to show that the IFN promoter activity is indicative of poor RNA polymerase activity  and the transfection control plasmids constitutively expressing Renilla luciferase (pTK-Renilla) were described previously [Human embryonic kidney 293T (HEK293T) cells were originally sourced from ITCC and routinely checked for mycoplasma infection. HEK293T cells were maintained in DMEM containing 10 % FBS, glutamate, pyruvate, and high glucose (Gibco). WSN pcDNA3-based protein expression plasmids and pPol-based viral template RNA expression plasmids were described previously -63. The Renilla luciferase. Transfections were performed with lipofectamine 2000 (Invitrogen) to the manufacturer’s specifications. Twenty-four hours later, the medium was aspirated, cells were washed with 1 ml PBS and split into two fractions. Cells were pelleted at 2,500 rpm using a benchtop centrifuge. The pellets from one fraction were resuspended in 30 μl of 1x Laemmli buffer for western blotting and the other fraction was resuspended in 50 μl PBS for the IFN-β promoter activity assay. The assay was done in duplicate using 25 μl of cell suspension in PBS per well in a white 96-well plate format. Next, 25 μl of DualGlo reagent (Promega) was added per well, samples were incubated at RT for 10 min (in dark), and the firefly luciferase readings were taken using a Synergy LX Multimode Microplate Reader (Biotek). Twenty-five μl of Stop-Glo reagent/well was added next, plate was incubated for 10 min at RT (in dark), and the Renilla luciferase readings were taken. Firefly luciferase values were normalized by the Renilla luciferase values. Data analysis was performed in Prism 9.5.0 (Graphpad).Transfections to perform RNP reconstitutions and IFN-β reporter assays in HEK293T cells were essentially performed as described previously . RNP recIAV proteins were detected using rabbit polyclonal antibodies anti-PB1 , and anti-NP  diluted 1:2000 in blocking buffer , 0.1% Tween-20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin  diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit  and IRDye 680 goat anti-rat  were used to detect Western signals with a LI-COR Odyssey scanner.gisaid.org) for 1968 – 2014, and 2017 was downloaded from NCBI  are listed in Sequences were acquired from EpiCoV  was mutated at frequencies of 1.8 x 10per year ,57. Theson rates ,47. Mutaon rates .in silico analysis starts with the RNA polymerase binding to the 3’ end of a viral genome (segment), which places residue 16 within the 20-nt footprint in the active site [Our t-loop analysis was essentially performed as described previously , but witive site ,42. We nive site . With boThe above sliding window was started at the 3’ end of the viral genome and moved in 1-nt intervals along the length of the template RNA. For each window, the RNA sequence upstream of the RNA polymerase footprint, downstream of the RNA polymerase footprint, and between the upstream and downstream sequences was analysed and the stability ΔG of the RNA structures calculated using the ViennaRNA package v.2.5 within Python 3.9 . LocatioH3N2 consensus sequences for PB2, PB1 and PA were generated using EMBOSS Cons and GC content was calculated using Bio.SeqUtils package from BioPython ,67. The Supplement 1Supplement 2Supplement 3


TMPO-AS1, play a crucial role in PCa progression. However, TMPO-AS1’s function in PCa bone metastasis (BM) and its underlying molecular mechanisms are unclear. Herein, we found that the long transcript of TMPO-AS1 (TMPO-AS1L) was upregulated in PCa tissues with bone metastasis, and overexpression of TMPO-AS1L correlated with advanced clinicopathological features and reduced BM-free survival in patients with PCa. Upregulated TMPO-AS1L promoted, whereas downregulated TMPO-AS1L inhibited, the PCa cell bone metastatic capacity in vitro and in vivo. Mechanistically, TMPO-AS1L was demonstrated to act as a scaffold, that strengthened the interaction of casein kinase 2 alpha 1 (CSNK2A1) and DEAD-box helicase 3 X-linked (DDX3X), and activated the Wnt/β-catenin signaling pathway, thus promoting BM of PCa. Moreover, upregulation of TMPO-AS1L in PCa resulted from transcription elongation modulated by general transcription factor IIF subunit 2 (GTF2F2). Collectively, our study provides critical insights into the role of TMPO-AS1L in PCa BM via Wnt/β-catenin signaling, identifying TMPO-AS1L as a candidate marker of PCa bone metastasis prognosis and therapeutic target.The second most common male cancer is prostate cancer (PCa), which has a high tendency for bone metastasis. Long non-coding RNAs, including  Worldwide, the most commonly diagnosed malignancy is prostate cancer (PCa), which is the second most frequently occurring cancer in men . In receCancer development and progression significantly involve the Wnt/β-catenin signaling pathway . When th-MUF promotes EMT via the Wnt signaling pathway [Long non-coding RNAs (lncRNAs) comprise noncoding RNA molecules with a length of 200–100,000 nucleotides. lncRNAs, lacking coding potential, encompass natural antisense transcripts, overlapping transcripts and intronic transcripts . Their c pathway , and inh pathway . Notably pathway , 18, and pathway , 20. HowTMPO-AS1), located on chromosome 12, has been identified as a critical lncRNA in the progression of many cancers [TMPO-AS1 with different exons and lengths (long and short transcripts). We identify that the expression of the long transcript of TMPO-AS1 (TMPO-AS1L) is upregulated significantly in PCa tissues with BM and is associated with poor patient prognosis. Experimentally, we reveal that TMPO-AS1L acts as scaffold to strengthen the interaction between casein kinase 2 alpha 1 (CSNK2A1) and DEAD-box helicase 3 X-linked (DDX3X), and activates the Wnt/β-catenin signaling pathway, which reciprocally promotes PCa BM. Furthermore, general transcription factor IIF subunit 2 (GTF2F2) mediates transcriptional elongation of TMPO-AS1 leading to the upregulation of TMPO-AS1L expression. Clinically, the GTF2F2/TMPO-AS1L/β-catenin axis is verified in PCa tissues. Our study determine the clinical significance and regulatory mechanism of TMPO-AS1L in PCa, providing a candidate therapeutic target for BM in PCa.Thymopoietin (TMPO)-antisense RNA 1  datasets were analyzed. The results showed that the expression of TMPO-AS1 was upregulated in PCa tissues in comparison with that in adjacent normal tissues (ANT)  and bone metastasis-free survival (BMFS)  in the TCGA dataset was too small for meaningful analysis , which revealed that high levels of TMPO-AS1 correlated strongly and positively with metastatic propensity and EMT-associated gene signatures  dataset (GSE21032), which demonstrated significant upregulation of TMPO-AS1 expression in metastatic PCa tissues (M‐PCa) in contrast to that in primary PCa tissues (P‐PCa) and ANT  Fig. . SurvivaFS) Fig. . Neverthsis Fig. . The biores Fig. . This inANT Fig. . PCa mosTMPO-AS1 that differed by their exons and lengths (TMPO-AS1L and TMPO-AS1S)  compared with tissue without bone metastasis (PCa/nBM)  Fig. . Our resBM) Fig. . BesidesMFS Fig. . Meanwhiues Fig. . Consistes Figs.  and S1D,nBM Fig. . We alsones Fig. . TogetheTMPO-AS1L expression in 155 paraffin-embedded PCa tissues , Gleason grade, BM status, and the vital status of the patients  was employed to detect the clinical significance of asm Fig. . TMPO-ASnBM Fig. , rather S1S Fig. . High TMnts Fig. . Notably BM Fig. . High TMMFS Fig. , but notMFS Fig. . These rTMPO-AS1L mediates BM in PCa, we constructed a mouse model of BM, in which luciferase-labeled control PC‐3 cells, PC-3 cells with stable overexpression of TMPO-AS1L, and those with stably downregulated TMPO-AS1L were inoculated into the nude mice’s left cardiac ventricle , and decreased mouse BMFS and OS  and the mesenchymal markers VIM (vimentin) and CDH2 (N-cadherin) through western blotting combined with immunofluorescence experiments. Western blotting displayed that TMPO-AS1L upregulation reduced the level of CDH1 and increased levels of VIM and CDH2 in C4-2B cells; by contrast, TMPO-AS1L silencing had the opposite effect on these EMT markers in both PC-3 and C4-2B cells  pathway analysis, which indicated that TMPO-AS1L might regulate the Wnt signaling pathway   for KEGG pathway analysis. The proteins that interact with TMPO-AS1L and regulate Wnt/β-catenin signaling from the KEGG pathway database are shown in Table TMPO-AS1L and CSNK2A1, an RNA pull-down assay followed by western blotting and RNA immunoprecipitation (RIP) assay were further performed, which verified that TMPO-AS1L interacted with CSNK2A1 Fig. . The speK2A1Fig. .TMPO-AS1L/CSNK2A1 complex. Through screening the result of MS, combined with analyzing public databases (Starbase and BioGRID), we identified DDX3X as a suitable candidate  and protein kinases could strongly enhance kinase activity , 28, in ate Fig. , which wS1L Fig. .TMPO-AS1L, CSNK2A1 and DDX3X were constructed and subjected to immunoprecipitation (IP) assays to investigate binding site or domains of the three molecules. It is believed that stem-loop structures are necessary for lncRNAs to interact with proteins [TMPO-AS1L by RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi.), and designed four fragments  that retained stable stem-loop structures as shown in Fig. TMPO-AS1L, interacted with CSNK2A1 and DDX3X, respectively , which would be destabilized by glycogen synthase kinase 3 beta (GSK-3β) [TMPO-AS1L enhanced the interaction between DDX3X and CSNK2A1, further increasing the activity of CSNK2A1 to phosphorylate DVL2, which activated Wnt/β-catenin signaling. Immunofluorescence experiments showed that CSNK2A1 as well as DDX3X, was colocalized with DVL2 in characteristic cytoplasmic puncta when TMPO-AS1L was overexpressed. By contrast, silencing TMPO-AS1L markedly reduced the number of these colocalized puncta, and CSNK2A1, DDX3X and DVL2 showed diffuse staining   and β-cang Figs. . This reTMPO-AS1L in the Wnt/β-catenin signaling and PCa BM. Silencing of DDX3X or CSNK2A1 in TMPO-AS1L overexpressing PCa cells attenuated the effect of TMPO-AS1L on Wnt/β-catenin activation and the phosphorylation of DVL2 , which is a general transcription and elongation factor [TMPO-AS1L , a reversible transcriptional elongation inhibitor, was used to treat lls Fig.  for 3 h,ls Figs.  and S9C.TMPO-AS1 gene, as shown in a schematic diagram , P2 (the transcription start site of TMPO-AS1S), P3 (before the splice site) and P4 . The specific primers corresponding to each site were designed and quantitative ChIP assays were performed under conditions of GTF2F2 overexpression or knockdown. Overexpression of GTF2F2 caused the recruitment of RNA Polymerase II Subunit A (POLR2A), the largest subunit of RNAPII, at P1 and P4, and decreased its distribution at P2 and P3  on a gene can reflect the dynamics of transcript elongation . Thereforam Fig. : P1 , because the sequence of TMPO-AS1S is not long enough to form a functional secondary structure. Nevertheless, we did not detect binding of AGO2 to TMPO-AS1L in PCa cells using RNA pull-down assays combined with MS analysis. Thus, TMPO-AS1L might not function as a ceRNA in PCa. This differs from our own results and those of other published studies about the molecular mechanisms of TMPO-AS1, which could be attributed to the heterogeneity of the tumor model or the context. This might explain why TMPO-AS1 is distributed in the cytoplasm in most tumors, whereas Luo et al. found that TMPO-AS1 was located in the nucleus in esophageal cancer, acting as transcriptional regulator [TMPO-AS1L might interact with various proteins, such as PPIP5K2, PTK2, EIF5, or others. It is possible that TMPO-AS1L simultaneously exerts its biological function through multiple mechanisms in the process of BM or the progression of PCa, which should be further investigated. This reflects the diversity and complexity of the molecular mechanisms of lncRNAs.Although there have been recent improvements in the individualized and systemic treatments of PCa, PCa patients with BM have reduced quality of life and survival because of BM-associated complications , 3. Conss in PCa . Herein, for PCa . Howeverrcinomas . Most stAS1 gene , 40. In egulator . NotablyTMPO-AS1L exerted its biological role in a CSNK2A1/DDX3X-dependent manner. CSNK2A1, the catalytic subunit α of casein kinase II (CK2), is part of the tetramer of the CK2 holoenzyme or can function independently to phosphorylate substrates that have acidic residues lying C-terminal to the phosphorylated serine or threonine [TMPO-AS1L functions in the cytoplasm, only DVLs and β-catenin are possible substrates of CSNK2A1 in our model. As shown by western blotting, TMPO-AS1L upregulated the level of phosphorylated DVL, rather than phosphorylated β-catenin, which inhibits the ‘β-catenin destruction complex’, resulting in inhibition of GSK3β-mediated β-catenin phosphorylation/degradation, promoting its accumulation and translocation into the nucleus [LINC00673-v4 regulated the protein-protein interactions between RBPs and kinases [TMPO-AS1L promoted CSNK2A1 and DDX3X co-localization with DVLs and formed biomolecular condensates in immunofluorescence experiments. Similarly, Cruciat et al. demonstrated that the interaction between DDX3X and CSNK1E induced the recruitment of CSNK1E to DVL2 punctae [TMPO-AS1L/CSNK2A1/DDX3X condensates undergo phase separation, we have provided a new insight that will promote further research. Notably, Guan et al. suggested that LINC00673-v4 enhanced lung adenocarcinoma metastasis via a similar mechanism [LINC00673-v4 expression was significantly lower than that of TMPO-AS1L in PCa, and its expression was no significantly different between PCa/nBM and PCa/BM tissue  profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.The normal prostate epithelial cells (RWPE-1 cells (RRID:CVCL_3791)) and the PCa cell lines (22RV1 (RRID:CVCL_1045), PC-3 (RRID:CVCL_0035), VCaP (RRID:CVCL_2235), DU145 (RRID:CVCL_0105), and LNCaP (RRID:CVCL_0395)) were provided by the Shanghai Chinese Academy of Sciences cell bank . The C4-2B cell line (RRID:CVCL_4784) was provided by the American Type Culture Collection . All cells were grown in their recommended medium supplemented with 10% fetal bovine serum , 100 μg/ml streptomycin and 100 U/ml penicillin G at 37 °C in a humidified 5% CO6 PC-3 cells in 100 μl of phosphate buffered saline (PBS) were inoculated into their left cardiac ventricle. Mice were included in the study if the injection into the left ventricle was successful; otherwise, they were excluded until there were 8 mice in each group. Bioluminescent imaging (BLI) was used to monitor BM, as previously described [The Institutional Animal Care and Use Committee of Sun Yat-sen University approved the mouse experiments . To study BM, male BALB/c-nu mice aged 4–6 weeks old were anesthetized using isoflurane and then 1 × 10escribed . No blinRelated plasmid DNAs were co-transfected into HEK293T cells for 48 h, rinsed two times using precooled PBS, and lysed using cold cell lysis buffer containing a protease inhibitor and phosphatase inhibitor cocktail . Next, antibodies against the target proteins were incubated with the lysates, at 4 °C overnight. Antibody-antigen complexes were captured using 50 μl of anti-HA magnetic beads or anti-His magnetic beads  for 24 h at 4 °C. The beads were then magnetically separated and incubated with HA peptide  or His peptide  for 4 h at 4 °C. After incubation, we obtained the supernatant using magnetic separation. Both the input and Co-IP samples were analyzed by western blotting analysis using anti-HA or anti-His antibodies. Conformation-specific IgG secondary antibodies  were used to remove heavy- or light-chains.TMPO-AS1 full-length sense, antisense, and serial deletion sequences, which were subjected to biotinylation using a Pierce RNA 3′ End Desthiobiotinylation Kit  adhering to the supplier’s guidelines, followed by RNase-free DNase I  treatment and purification using an RNeasy Mini Kit . RNA pull-down assays were conducted using an RNA-Protein Pull-Down Kit . Streptavidin-coated magnetic beads were used to capture the biotinylated RNA, which was added to cell lysates and incubated for 2 h at 4 °C. Subsequently, the beads were subjected to three washes and boiled to retrieve proteins for immunoblotting or mass spectrometry. The proteins on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel were stained using a Pierce Silver Stain Kit  following the supplier’s protocol.In vitro, a MEGAscript T7 Transcription Kit  was used to transcribe t tests , the Mann-Whitney U test , and the Wilcoxon matched-pairs signed rank test . Categorical variables and constituent ratios were compared using the χ2 test. Kaplan–Meier curves and log-rank tests were used to analyze the survival data. Differences with a P < 0.05 (two-tailed) were deemed statistically significant. In figures, statistical differences are shown as follows: N.S. = no significance, *P < 0.05.GraphPad Prism 9.0  and SPSS 19.0  were used to carry out the statistical analyses. The mean ± standard deviation (SD) was used to represent the data. The quantitative experiments were carried out at least thrice independently. One way analysis of variance (ANOVA)  was used to analyze the quantitative data, as were the Kruskal-Wallis test , Supplementary MaterialWestern blotting datas


Cell fate is shaped by a unique gene expression program, which reflects the concerted action of multilayered precise regulation. Substantial research attention has been paid to the contribution of RNA biogenesis to cell fate decisions. However, increasing evidence shows that RNA degradation, well known for its function in RNA processing and the surveillance of aberrant transcripts, is broadly engaged in cell fate decisions, such as maternal-to-zygotic transition (MZT), stem cell differentiation, or somatic cell reprogramming. In this review, we first look at the diverse RNA degradation pathways in the cytoplasm and nucleus. Then, we summarize how selective transcript clearance is regulated and integrated into the gene expression regulation network for the establishment, maintenance, and exit from a special cellular state. Beginning with a fertilized egg, numerous identical or distinct cells are continually generated to ensure the proper organization and function of each tissue and organ during the lifespan of multicellular organisms . Deciphevia ectopic expression of a defined cocktail of transcription factors: Oct4, Klf4, Sox2, and Myc  methylation could promote the degradation of specific pluripotency transcripts and facilitate mouse embryonic stem cell differentiation , the initial step of early embryo development in which fertilized egg is reprogrammed into a totipotent embryo, a subset of maternal RNAs must be cleared timely and efficiently apart from transcriptionally awakening the zygotic genome. Impediment in maternal RNA clearance by genetic inactivation of the RNA degradation-associated genes  in mice . A pairw in mice , implyinntiation , whereasntiation . In geneHere, we briefly introduce the RNA degradation factors and describe how they orchestrate the highly regulated RNA degradation pathways. Furthermore, we outline how selective RNA turnover is regulated and becomes an integral part of the gene regulatory network in cell fate decisions.7G) cap will be installed at the 5′ end of nascent RNA (20–25 nucleotides in length), whereas a stretch of non-templated adenosines will be added to the 3′ end (poly(A) tail) (7G cap and poly(A) tail act as versatile platforms to recruit diverse effector proteins and hence affect almost all aspects of RNA metabolism, such as RNA decay and translation efficiency (For the majority of RNA polymerase II (Pol II) transcribed RNAs, a unique 7-methylguanosine (mA) tail) . m7G capficiency . Transcrficiency .During the whole life cycle, RNA is subject to surveillance by the RNA degradation machinery. RNA exonucleases clear the RNAs with exposed free ends in the 5′-to-3′ or 3′-to-5′ direction. In mammals, XRN1 and XRN2, located in the cytoplasm and nucleus, respectively, catalyze the 5′-to-3′ RNA hydrolysis processively . DespiteConversely, 3′-to-5′ RNA degradation is catalyzed by the multi-subunit complex, RNA exosome . The strAs mentioned previously, RNA exonucleases can only attack RNA with exposed free 5′ or 3′ terminus. Thus, the cleavage of the 5′ cap structure (decapping) and/or removal of the 3′ poly(A) tail (deadenylation) is usually a prerequisite for RNA degradation.7G cap and then release 5′ m7GDP and 3′ fragment with monophosphate at the 5′ terminus. DCP2 specifically recognizes m7G-cap or m2,2,7G-cap and catalyzes their cleavage through its NUDIX domain, a motif shared in pyrophosphatases. However, DCP1 functions as a coactivator to enhance the decapping activity of DCP2 and bridges the interaction of the decapping complex with other co-factors  sequences. A biphasic model is proposed for deadenylation: in the initial phase, poly(A)-binding protein PABPC1 facilitates the loading of the PAN2–PAN3 complex to remove the distal part of poly(A) tail through the distributive exonuclease PAN2. In the second, fast phase, the CCR4–NOT complex relays and digests the residual adenosines to a very few adenosines and CAF1 .Although RNA exonucleases are required for complete RNA destruction, decapping and deadenylation are not essential. RNA exonucleolytic decay could be initiated at internal endonucleolytic sites within the RNA body . The RNANon-sense-mediated mRNA decay (NMD) and microRNAs (miRNAs)/small interfering RNA (siRNA)-mediated RNA interference (RNAi) in the cytoplasm may be the best-characterized cases involving the endonuclease activity. NMD is a quality control mechanism employed to eliminate transcripts harboring a premature termination codon (PTC) . If a PTvia the slicing activity of the Ago2 protein  family protein and GW182 to form the functional RNA-induced silencing complex (RISC). Then, it silences its target expression by repressing translation or accelerating mRNA degradation, the latter of which is proved to be the major function of miRNAs in mammalian cells . Mechani protein .via endonucleolytic cleavage of the nascent RNAs, analogous to the Integrator complex  and eRNAs . Recentl complex .Most of our knowledge about RNA degradation pathways is from mRNA decay in the cytoplasm. In mammals, cytoplasmic mRNA degradation is usually initiated by deadenylation. Then, RNAs will be directly cleared through the 3′-to-5′ RNA decay pathway or will be decapped and followed by 5′-to-3′ degradation . In term7G cap, which in turn recruits the polyA tail exosome targeting (PAXT) or nuclear exosome targeting (NEXT) complex through the CBC-ARS2-ZC3H18 axis and serves to target the RNA substrates for degradation by nuclear RNA exosome . Additionally, it is evidenced that RNA degradation is tightly interconnected with RNA processing . In the Homo sapiens and Mus musculus, respectively, or more in other new datasets  tails of many maternal transcripts. Then, these transcripts will be activated translationally to produce more proteins, among which various RNA degradation factors are reported such as ZFP36L2, CNOT6L, CNOT7, BTG4, and PABPN1L . At the  subunit   are first identified at the base of the incipient allantois around embryonic day (E) 7.25 . Dnd1 isecursors . DND1 coecursors . Especiaecursors .Small RNAs of 20–30 nucleotides can be classified into three major classes—miRNAs, endogenous siRNAs (endo–siRNAs), and Piwi-interacting RNAs (piRNAs)—which differ in their biogenesis pathways and associated Argonaute-family proteins . GeneralDgcr8 or Dicer knockout (KO) showed defects in proliferation and differentiation  by the microprocessor, composed of DROSHA and DGCR8, in the nucleus. Subsequently, the resulting pre-miRNAs are exported into the cytoplasm, where they will be further cleaved by DICER into the miRNA duplex and assembled into the RISC complex . Mouse entiation , indicatof mESCs . Upon dintiation   or Argonaute 3 (Ago3) could target and direct the degradation of many maternal mRNAs involved in germ cell development by either direct endonucleolytic cleavage or recruitment of the CCR4–NOT deadenylation complex (piRNAs are 23–31 nucleotides in length and are generated from single-stranded transcripts independent of DICER . In addi complex  (Rouget  complex .endo-siRNAs are generated directly from long double-stranded RNAs by Dicer . They co7G cap and 3′ poly(A) tail modifications, RNAs are extensively decorated at the 3′ terminus or internal sites by other RNA modification enzymes, which have multifaceted roles in RNA metabolism, including RNA decay  tails (shorter than ∼25 nucleotides) . The LSMertility .6A, the most abundant internal modification in mRNAs, can be recognized by diverse readers to mediate different biological activities  modifications and protects them from degradation through the recruitment of the poly(A) tail-binding protein Pabpc1a  preferentially binds to a subset of maternal mRNAs with 5-methylcytosine  and the other PTC-containing isoform (Eif4a2PTC). NMD deficiency stabilized the Eif4a2PTC transcript to produce a truncated protein Eif4a2PTC , which could generate transcripts encoding different proteins or with different 3′ UTR lengths . Longer NEAT1 gene switches from the short isoform NEAT1_1 in ESCs to express the long, full-length isoform NEAT1_2 (By coinciding with the exit of pluripotency toward differentiation for both human ESCs (hESCs) and mESCs, the  NEAT1_2 , a scaff NEAT1_2 . TDP-43  NEAT1_2   during APA in diaphragm MuSCs to circumvent the function of miR-206, thus a high PAX3 level is sustained to support the proliferation of diaphragm MuSCs , it only downregulates the target gene gm MuSCs   is transcriptionally activated in mouse preimplantation development, especially at the two-cell (2C) stage  Jachowi. When LI6A methylation, which can be recognized by the nuclear m6A reader YTHDC1 and then enhances the association between the NEXT complex and LINE1, thereby accelerating LINE1 degradation (Fto, the m6A demethylase, is knocked out, the LINE1 m6A level is elevated, whereas its expression level is reduced accordingly. Moreover, Mice with Fto KO exhibit developmental defects analogous to Zcchc8 KO (Notably, LINE1 can be targeted for degradation by the NEXT complex in mouse ESCs and embryos. The depletion of Zcchc8, the scaffold subunit of the NEXT complex, will lead to LINE1 upregulation and developmental defects in mice . Furtherradation . When Ftcchc8 KO . Mechanicchc8 KO  (Jachowicchc8 KO . CollectInterestingly, it was demonstrated in another two studies that the binding of YTHDC1 to LINE1 recruits Nucleolin/Trim28 or SETDB1/Trim28 complex to facilitate the deposition of H3K9me3 and then silences target gene expression, such as Dux, a master regulator of the 2C-specific transcriptome  Chen et, implyinRNA degradation machineries, composed of RNA exonucleases, endonucleases, and other co-factors, are involved in the processing and maturation of snoRNA, snRNA, and rRNA, among others, and clearing of aberrant mRNAs with PTCs or those without stop codons . Beyond RNA degradation is especially important in cell fate decisions because rapid shifts in the mRNA and protein constitution during the transition between different cell states require activating new gene expression programs, meanwhile silencing the old ones. RNA degradation can independently clear specific pre-existing RNAs associated with the previous cell type , or it cvia the recruitment of the CCR4–NOT deadenylation complex. Although small RNA and NMD can trigger endonucleolytic cleavage of RNA (RNA deadenylation seems to be the initial and rate-limiting step in RNA degradation. RBPs , small Re of RNA , no repoe of RNA , can fune of RNA . Converse of RNA ; whetherDuring the transition between different cellular states, the number of RBPs or miRNAs is dynamically regulated to control RNA degradation . As sign


Polypharmacy is associated with poorer self-rated health (SRH). However, whether polypharmacy has an impact on the SRH progression is unknown. This study investigates the association of polypharmacy with SRH change in 1428 participants of the Berlin Initiative Study aged 70 years and older over four years. Polypharmacy was defined as the intake of ≥5 medications. Descriptive statistics of SRH-change categories stratified by polypharmacy status were reported. The association of polypharmacy with being in SRH change categories was assessed using multinomial regression analysis. At baseline, mean age was 79.1 (6.1) years, 54.0% were females, and prevalence of polypharmacy was 47.1%. Participants with polypharmacy were older and had more comorbidities compared to those without polypharmacy. Over four years, five SRH-change categories were identified. After covariate adjustment, individuals with polypharmacy had higher odds of being in the stable moderate category , stable low category , decline category , and improvement category  compared to being in the stable high category independent of the number of comorbidities. Reducing polypharmacy could be an impactful strategy to foster favorable SRH progression in old age. In 2021, the proportion of individuals in Germany older than 65 years reached 22.1% of the entire population and is projected to further increase in the future .In treating multiple comorbidities, the adherence to evidence-based guidelines of disease management usually involves the concurrent prescription of multiple medications ,5. The rDespite the concurrent intake of multiple medications being a widespread phenomenon, there is still no agreement on a definition of polypharmacy . The mosSelf-rated health (SRH) is one of the patient-reported outcomes (PRO) which represents a complex subjective measure of the individual’s overall health status and encompasses biological and psychosocial aspects of health ,12. SRH Several studies reported an association between polypharmacy and poor SRH, however, only in a cross-sectional design ,19. SRH Thus, the aim of this study was to (1) identify categories of SRH-change over the period of four years and describe how they differ between older individuals with and without polypharmacy and (2) assess the association between polypharmacy and different SRH-change categories. Allgemeine Ortskrankenkasse (AOK)—Nordost statutory health insurance fund. Dialysis patients and kidney transplant patients at baseline were excluded. This study utilized data from the Berlin Initiative Study (BIS). The BIS is a population-based prospective cohort of 2069 community-dwelling older individuals. Participant recruitment took place between November 2009 and July 2011 with four subsequent biennial follow-up visits. The concept and design of the BIS are described elsewhere . To partFor the current analysis, data from the BIS baseline visit (2009–2011) and the BIS second follow-up visit (2014–2015) (hereinafter referred to as the SRH follow-up visit) were used. The inclusion criteria were answering the SRH question at both visits and provision of information about the polypharmacy status at the BIS baseline visit. Of the 2069 participants taking part in the BIS baseline visit, eight participants were excluded due to missing SRH, and three participants were excluded due to missing information about their polypharmacy status. Between both study visits, 361 participants died, and 268 were lost to follow-up. At the SRH follow-up visit, one participant was excluded due to missing SRH, yielding a final sample of 1428 participants . Medication was assessed by asking participants to bring their medications and medication lists to the study visit in addition to self-reporting their medication intake. Prescription and over-the-counter (OTC) medications were recorded and entered into the standardized questionnaire linked to a drug database in which drug information including the prescription requirement as well as the anatomic therapeutic chemical (ATC) code were automatically assigned. Polypharmacy status was then recorded as a binary variable, where participants regularly taking five or more medications concurrently were regarded as individuals with polypharmacy, whereas those regularly taking less than five medications or none were regarded as individuals without polypharmacy . Participants were asked to rate their health by answering the question “In general, how do you rate your health condition?” with five possible responses: very good, good, moderate, poor, and very poor. This measure has been widely used in previous studies ,24. As ostable high category when they reported their SRH as good during both visits; (2) stable moderate category when they reported their SRH as moderate during both visits; (3) stable low category when they reported their health as poor during both visits; (4) decline category when their SRH-changed from moderate to poor or from good to moderate or poor between visits; and lastly, (5) improvement category when their SRH-changed from moderate to good or from poor to moderate or good between visits.SRH progression was assessed by observing the change in SRH between both visits, through which five SRH-change categories were identified. Participants were assigned to the (1) During the baseline assessment of the participants, data on demographics, lifestyle factors, comorbidities, and medications were gathered by way of a standardized questionnaire in addition to anthropometric and clinical assessments. The collected data were augmented by the AOK claims data in which all comorbidities were listed according to the Tenth Revision of the International Classification of Diseases (ICD-10), which corroborated self-reported data. Data about the following sociodemographic variables were collected: age, gender, monthly income as a categorical variable ); general and vocational education as low, intermediate, or high according to the Comparative Analysis of Social Mobility in Industrial Nations (CASMIN) scale ; and las2).Lifestyle factors were recorded as categorical variables: frequency of alcohol consumption ; physical activity ; and body mass index (BMI)  polypharmacy status as well as (2) polypharmacy status and SRH-change category were described by using means and standard deviations or medians and interquartile ranges for continuous variables. For categorical variables, absolute and relative frequencies were reported. To address the first research question, descriptive statistics were used to summarize the baseline characteristics of the SRH-change categories stratified by polypharmacy status. To address the second research question, multinomial regression analysis was used to compute crude and adjusted odds ratios (OR) and 95% confidence intervals (95% CI) for the association between polypharmacy and the SRH-change categories. The multinomial regression model was adjusted for a knowledge-based set of variables consisting of gender, income, CASMIN, partner status, physical activity, frequency of alcohol consumption, BMI, and CCI categories . In ordeAs a sensitivity analysis, the main analysis was stratified by gender to investigate its potential effect on the association between polypharmacy and SRH-change categories. All analyses were conducted using the R statistical software . 2. One-third (34.7%) of the participants were living with five or more comorbidities. The prevalence of polypharmacy was 47.1% (N = 672). In participants without polypharmacy, 68.7, 27.1, and 4.2% rated their health as good, moderate, and poor, respectively, whereas in participants with polypharmacy, 37.8, 49.4, and 12.8% rated their health as good, moderate, and poor, respectively. Participants without versus with polypharmacy were more frequently regular alcohol consumers (22.0 vs. 19.3%), more likely to be physically active more than five times a week (36.4 vs. 23.5%), and had less often a BMI ≥30 kg/m2 (21.3 vs. 33.2%). Regarding CCI, 18.0% of the participants without polypharmacy were living with five or more comorbidities compared to 53.6% of the participants with polypharmacy. When comparing individuals who were excluded to study participants  years. The majority of participants were in the stable high category (33.5%), followed by the decline category (28.6%), the stable moderate category (20.1%), the improvement category (13.7%), and lastly, the stable low category (4.1%). Almost half of the individuals without polypharmacy were in the stable high category (46.2%) compared to only about one-fifth (19.3%) of the individuals with polypharmacy. Furthermore, within the individual categories, participants with polypharmacy were less often females, were less physically active, had higher BMI values, and had a higher number of comorbidities. The adjusted multinomial regression model showed that polypharmacy was associated with increased odds of belonging to other categories than to the stable high category, with odds ratios ranging from 1.9 to 3.6. The polypharmacy prevalence reported in our study (47.1%) was comparable to the combined polypharmacy and hyperpolypharmacy prevalence of 48% reported in the ESTHER study conducted in Saarland, Germany, despite the younger age range of that sample (mean age 70 years) . It was We were able to identify five SRH-change categories; stable high, stable moderate, and stable low in addition to a decline category and an improvement category. Previous studies investigating SRH in older adults identified only three change categories , with the majority of the participants showing no change in SRH over the follow-up period, which is in agreement with the results of our study when the three stable categories are combined ,31,32. TIn our study, the highest number of participants was in the stable high category, reflecting the notion of successful aging by maintaining a high level of SRH as described by Rowe and Kahn . These p2 and the lowest frequency of physical activity was found in these categories as well. Physical activity was found to affect SRH in older adults positively [Conversely, the stable moderate and stable low categories were characterized by a high prevalence of comorbidities and polypharmacy at baseline in our study. Both were found to be associated with moderate-to-poor SRH . The higsitively . Therefositively .The decline category is a complex one reflecting the combination of senescence and pathological SRH decline with advancing age. SRH decline could be attributed to the tendency to develop more comorbidities as well as increased functional impairment . TherefoDespite having a high number of comorbidities at baseline, individuals in the improvement category were most likely functionally independent and more physically active . PossiblDespite SRH improvement being favorable per se, polypharmacy was associated with elevated odds of being in the improvement category. A possible explanation could be that individuals in the improvement category had poorer SRH at baseline in comparison to participants in the stable high category. This is in line with the findings that polypharmacy is associated with worse SRH . MoreoveStudies addressing the association between polypharmacy and SRH-change categories are scarce. Compared to the existing literature, our results that polypharmacy is associated with the belonging to SRH-change categories other than the stable high category is in accordance with the results of previous studies that assessed the association between polypharmacy and HRQoL ,45. ThisRegarding gender differences, females had higher odds of being in the decline category in comparison to males. This could be explained in part by females’ tendency to consider non-life threatening chronic conditions such as hypertension as well as non-health-related life events in their assessment of health . ConversThere are several pathways through which polypharmacy might negatively influence the progression of SRH. The mere consumption of several medications and experiencing their side effects could lead older adults to perceive their health as poor . MoreoveNon-adherence could also contribute to the negative effect of polypharmacy on SRH. Non-adherence refers to deviations from the prescribed treatment, including overuse, underuse, or incorrect use of medications. It could be attributed to regimen complexity, forgetfulness, or side effects of the medications . It was Our study benefited from multiple strengths. First, this study included a large cohort of 1428 community-dwelling older adults and longitudinal data with a follow-up period of four years. Second, we included all regularly taken medications including OTC and did not restrict them only to prescription medications. Third, the data utilized for this study were extracted from primary BIS data complemented by secondary claims data. This study was not without limitations. First, as the information about medication was self-reported, it was prone to recall bias. However, we tried to minimize bias through asking participants to bring their medication lists and packages to thoroughly assess their medication intake. Second, the polypharmacy definition we used was based on a numerical threshold and did not address medication appropriateness. However, this enabled us to compare our results to other studies, as the definition we used is the most commonly used definition of polypharmacy. Third, we did not include cognitive ability as an inclusion criterion because cognitive impairment was assessed only during the SRH follow-up visit. However, there were no major differences between those with and without cognitive impairment in the distribution among the SRH-change categories . Fourth,Although polypharmacy is recognized as a public health challenge in older individuals, its effect on SRH-change independent of comorbidities is widely unrecognized. Our finding should prompt physicians to implement strategies to alleviate polypharmacy, such as medication reconciliation and possible deprescription as well as utilization of non-pharmacological approaches to control factors contributing to the development of multimorbidity . Simplification of drug regimens and the development of single-pill combinations for the most commonly occurring comorbidities in older adults could be an additional strategy to increase adherence and subsequently decrease potential drug–drug interactions and improve SRH. To empower this vulnerable group, the aforementioned strategies should be implemented while keeping individual preferences of older adults at the forefront of the decision-making process. This could then lead not only to positive changes to their physical but also to their psychosocial wellbeing.Additional factors other than the number of medications could affect the association between polypharmacy and the SRH-change categories, such as the type and appropriateness of medications. In this respect, future research should address the improvement and decline categories in greater detail while taking gender differences into consideration.


Introduction: The Corail femoral stem has excellent long-term survivorship in total hip arthroplasty (THA). However, there remains a paucity of information on the specific performance of the high offset collarless stem in relation to subsidence, loosening, offset, and failure rates.Methods: Retrospective data were collected on all consecutive high offset collarless Corail stems implanted at a single centre in the UK. Data included patient demographics, femoral Dorr classification, radiographic analysis for radiolucent lines, and stem subsidence. The postoperative femoral offset was measured against the native offset of the contralateral hip. Any early failures, re-operations, or requirements for revision surgery were recorded.2. The mean length of follow-up was 84.5 (12-130) months. Subsidence was recorded on 113 (69.7%) stems. The mean amount of total stem subsidence in the whole cohort was 1.62mm (0 to 3.9mm). There was no correlation between the amount of subsidence and the preoperative Dorr classification, age, sex, BMI, or indication for surgery. Radiolucent lines were exclusively seen in stems paired with a large-diameter 36-mm femoral head. The high offset stem accurately reproduced native femoral offset; the mean difference in offset was -1.21mm (-24mm to +21mm). There were no early failures, re-operations, or revision surgeries.Results: We identified 162 stems for inclusion in the study. Ninety-five patients were male. The mean age was 60.5 (40 to 78) years, and the mean BMI was 29.8 (21 to 50) kg/mConclusion: The use of a high offset stem can accurately reproduce native femoral offset when chosen for THA. The high offset collarless Corail stem does not result in early failures in THA, and we support its use. Total hip arthroplasty (THA) is a well-established treatment for end-stage osteoarthritis (OA) of the hip. Cementless fixation of the femoral component allows for a strong biological fixation between the stem and bone, permitting the longevity of the implants and protecting against aseptic loosening . One of The Corail stem has an excellent survivorship rate of 91.82% at 15 years on the National Joint Registry (NJR) for England, Wales, Northern Ireland, and the Isle of Man [There has been an increase in the use of KA collared Corail prostheses due to the perceived advantages it has in providing early axial and rotational stability, protection against early postoperative stem subsidence, and the dispersion of medial calcar forces, resulting in a reduced incidence of radiolucent lines -4. AlthoCurrently, the NJR does not differentiate between each Corail stem sub-type in the annual report, and the majority of published data indicating the success of the Corail stem are based on the KA collared standard offset stem . There iThe primary aim of this study was to evaluate the early radiographic outcomes of the high offset collarless Corail cementless stem. We evaluated the stem size, the presence of radiolucent lines, stem subsidence, the ability of the stem to recreate the native femoral offset, and all-cause stem survivorship in a large consecutive cohort. We hypothesised that the KHO collarless high offset Corail stem would be better at reproducing the native femoral offset when appropriately sized and would not result in excessive stem subsidence or early implant failures.A retrospective review of all high offset collarless Corail stems implanted over a 10-year period between March 2010 and March 2020 was performed .A minimum of one-year follow-up was necessary for inclusion in the study to ensure an appropriate time period was available to accurately identify early stem subsidence on serial radiographs. All patients had complete data sets available for analysis.All patients had equivalent standard anteroposterior (AP) pelvic radiographs taken both preoperatively and during subsequent postoperative follow-up periods. The focus film distance was 100cm, centred on the symphysis pubis, with the position of the lower limbs internally rotated by 15 degrees to demonstrate the greater trochanters in profile.Patients’ demographics, BMI, and indications for surgery were recorded. Preoperative radiographs were used to classify the femora as per the Dorr classification .Intraoperative data collected included femoral stem size, femoral head size, bearing surfaces used, and brand of acetabulum shell implanted.Serial postoperative radiographs were analysed for stem subsidence and the presence of radiolucent lines. A comparison to the patient's native offset was made if a contralateral THA was not present. Any complications, re-operations, or requirements for revision surgery were recorded.Stem subsidence was evaluated on serial postoperative radiographs using the Engh and Massin method , while pClassification and radiographic measurements were performed by two independent orthopaedic surgeons. Any discrepancies were resolved by the senior author. All patients had radiographs available for analysis preoperatively and at day one, six weeks, six months, and one year postoperatively. Further radiographs were available at three (n=2), five (n = 89), and 10 years (n = 71) postoperatively for patients who had reached those follow-up points. The NJR data and hospital records were checked to evaluate the data on any stems that may have undergone revision surgery.Statistical analysis The data were checked for normal distribution with the D'Agostino-Pearson normality test. The unpaired t-test was used to identify if there was a significant difference between groups. Spearman's correlation coefficient was calculated to determine the correlation between independent factors with both stem subsidence and radiolucent lines.All analyses were completed on GraphPad Prism version 8.4.2 . Statistical significance was assumed at p<0.05.Surgical techniqueAll THAs included in the study were implanted through a posterior approach. The acetabular shell used was almost exclusively the Pinnacle shell , except in nine cases where a Gryption shell  was used in patients when there was an intraoperative concern with the stability of the Pinnacle trial shell.Bearing surfaces were ceramic-on-ceramic in all but two cases, which were ceramic-on-polyethylene.Although preoperative templating allowed for a presumption of stem size, all stems were selected based on careful intraoperative surgical technique. The high offset Corail stem was selected for patients with a high neck-shaft valgus angle, based on clinical judgement, in order to recreate the native offset and hip centre. The femoral canal was prepared by increasing the size of the broach sequentially until an optimum fit was achieved with longitudinal and rotational stability. All patients were encouraged to fully weight-bear from day one post-surgery.We identified 162 high offset Corail stems that were suitable for inclusion in the study. This included 95 male and 67 female patients. The mean age at the time of surgery was 60.5 (40 to 78) years, with a mean BMI of 29.8 (21 to 50) kg/m2. The mean length of follow-up in the cohort was 84.5 months (12-130 months).The indication for surgery was OA of the hip in 124 (76.5%) patients; 22 (13.6%) patients had avascular necrosis (AVN) of the femoral head; nine (5.5%) patients underwent THA for a fractured neck of femur (NOF); six (3.7%) patients had sequelae of developmental dysplasia of the hip; and one (0.6%) patient had a non-union following internal fixation for a NOF fracture.We classified 30 as type A femora, 99 as type B, and 33 as type C femora as per the Dorr classification.There were five stems judged to be undersized on the postoperative radiographs, and the remaining 147 stems were considered to be appropriately sized.Subsidence of the femoral stem was only seen early; we recorded the majority of subsidence on the six-week radiograph, with a small degree occurring up to the six-month radiograph. Stem subsidence wasn’t observed after six months postoperatively. A degree of subsidence was recorded on 113 (69.7%) stems. The mean amount of total stem subsidence in the whole cohort was 1.62mm (0 to 3.9mm). Subsidence was seen in all of the undersized stems, although due to the small number of undersized stems, we were unable to determine its significance.There was no significant difference identified between the amount of stem subsidence observed relative to the preoperative femoral Dorr classification  , sex (rs =0.12), femoral head size, or indication for surgery Figure .Specifically, we did not see any significant subsidence in the stems used for patients with NOF fractures. However, it is important to emphasise that the nine patients treated for NOF fractures were all young with type A or type B femora. The senior author would typically use a cemented stem for older patients with NOF fractures.When comparing the native femoral offset to the femoral offset created by the high offset collarless stem, the mean difference in offset was -1.21mm (-24mm to +21mm)  stems, exclusively seen in zones one and seven. All stems that demonstrated radiolucent lines were combined with a 36-mm femoral head; no stems with 28- or 32-mm femoral heads showed any evidence of radiolucent lines. The radiolucent lines did not progress on the five-year radiographs and appear to have no effect on clinical outcomes or stem survival. In addition to radiolucent lines, we analysed the postoperative radiographs for evidence of calcar resorption. Calcar resorption wasn’t observed in any radiographs in this study.No patients in the study cohort underwent any re-operations, and to date, no stems have required revision surgery.This study of 162 consecutive femoral stems demonstrates that the KHO Corail high offset collarless stem does not result in excessive early stem subsidence and accurately recreates native offset in THA. Although there is excellent data on the Corail stem, there is currently a paucity of data available on this specific stem design . ConcernOur study required a minimum follow-up period of 12 months, with a mean follow-up of 84.5 months. Failure of cementless stems generally occurs early, with peri-prosthetic fractures, dislocations, and early migration of implants secondary to poor fixation being the main reasons for early revision -15. LateCollarless stems are advantageous due to the more even surface loading of the stem, resulting in complete osseointegration . CollareThe use of a collared stem relies on an accurate femoral neck cut and the seating of the collar on the femoral calcar. Poor calcar-collar contact could potentially increase the risk of proximal femoral stress shielding and iatrogenic fracture and result in leg length discrepancies -21. The collarless designs are also dependent on meticulous surgical technique; the sizing of the femoral stem in our study was conclusively chosen based on intraoperative trailing. If a collarless stem is undersized, it is likely that early subsidence could become problematic. The precise amount of radiographic stem subsidence required to become clinically significant is unknown ,22. LessWe didn’t observe any clinical consequences of stem subsidence in our study. Subsidence is only seen in the early postoperative period; this is supported by multiple studies, with subsidence rarely seen after six months -25. ThisMagill et al.  reviewedLarge femoral heads increase torque in retroversion; this could also theoretically explain why radiolucent lines were exclusively seen in stems coupled with large-diameter 36-mm femoral heads. Collared stems allow for greater early torsional stability when compared to collarless stems, which may be protective against this finding and may explain why previous studies have identified fewer radiolucent lines with collared stems . HoweverIt is important to consider the risk of early iatrogenic peri-prosthetic fractures when using any cementless stems. Lamb et al.'s  registryOne interesting finding from this study was the ability of the high offset Corail stem to accurately recreate native femoral offset. The neck-shaft angle of standard stem designs varies across manufacturers. Insufficient femoral offset can result in instability, limping, abnormal abductor function, and gait, with an increase in the requirements for walking aids. To the best of our knowledge, no previous study has investigated the ability of the high offset Corail stem to recreate native femoral offset. We found that the high offset stem was very accurate in recreating offset, with the mean difference in offset being only -1.21mm. It is the senior author's opinion that the high offset stem is often more suitable than the standard offset stem in the predominantly Caucasian population that we treat in the UK. This study was not designed as a comparative study to the standard offset stem, so this is simply an observation within our study cohort.This study does have limitations; the retrospective nature of the study means that not all variables can be controlled. We tried to decrease variables by limiting the study to a single surgeon within a single centre and including all consecutive stems for analysis. This study reports radiographic outcomes and stem survivorship; we have not included patient-reported outcome scores in our analysis. We have, however, addressed the aims of our study by showing that early failures are not a concern when using the high offset collarless cementless Corail stem design.In conclusion, this study demonstrates that the high offset collarless Corail stem does not result in early failures in THA, and we support its use. Furthermore, preoperative femoral Dorr classification, BMI, or early postoperative weight bearing do not affect early stem subsidence. In addition, it is important to accurately size collarless stems, as undersizing could result in stem subsidence, and this study demonstrates that the use of a high offset stem can accurately reproduce native femoral offset when chosen for THA.


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.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   Eukaryotic RNA viruses employ different mechanisms to maximize the coding capacity of their genomes and produce the required stoichiometry of translated products. One such mechanism is programmed ribosomal frameshifting (PRF), which occurs when translating ribosomes encounter an impediment while associated with a slippery sequence and slip backward or forward into an alternate reading frame that bypasses the frame 0 termination codon . The moscis-acting elements are considered the primary determinants for efficiency  is composed of a heptanucleotide slippery sequence, a spacer region and a structured frameshift stimulating element (FSE) . The slificiency , other fficiency , cellulaficiency ,4,18,19 ficiency , concentficiency , as wellFSEs with diverse structural features have been reported for a number of different viruses . Structucis- (and/or trans-) acting interactions and how these interactions contribute to active and inactive FSE conformations and frameshifting efficiency. An additional overlooked issue is how the frameshifted ribosome can resume translation through an FSE structure sufficiently stable to have caused the ribosome to pause. Problematically, FSE have been mainly studied following excision and integration into reporter constructs, which eliminates any contributions of distal sequences that might positively or negatively affect frameshifting efficiency RNAs are a recent grouping of subviral RNAs with monopartite, plus-strand genomes of 2.7 to 4.5 kb. ulaRNAs code for at least two proteins; a 5′ proximal product (ORF1) that corresponds to a replication-required protein in related viruses, and the viral RdRp , which iPhylogenetic analysis of ulaRNAs suggest that they separate into three classes : Class 1An unusual member of the dicot-infecting Class 2 ulaRNAs is citrus yellow vein-associated virus . Unlike other Class 2 ulaRNAs, CY1 is missing two large fragments that together with additional alterations eliminate ORF5 . Using shttps://rna2drawer.app/) (The frameshifting efficiency of full-length CY1 assayed in wheat germ extracts (WGE) is unusually high for a plant virus at 29%, nearly 6-fold higher than that of umbravirus pea enation mosaic virus 2 (PEMV2) . The CY1er.app/)  were useEscherichia coli cells. The presence of the desired mutations was confirmed through Sanger sequencing (Eurofins Genomics).Plasmid pET17b-CYVaV, which contains the entire length of WT CYVaV genomic RNA (gRNA) complementary DNA (Accession number JX101610) downstream of a T7 promoter , was usein vitro transcription using bacteriophage T7 RNA polymerase and pET17-b CY1 plasmids linearized with HindIII. Synthesized RNA was quantified using a DeNovix DS-II FX spectrophotometer and the quality of RNA was verified on a 1% TBE native agarose gel. Uncapped in vitro transcribed RNA (0.5 pmol) was translated in 10 μl of WGE with 35S methionine as per the manufacturer's (Promega) recommendations. Briefly, RNA was denatured at 65°C for 5 minutes, followed by snap cooling in ice. The translation mixture was incubated at 25°C for 45 minutes and then resolved in a 10% SDS–PAGE gel. After drying, the gel was exposed to a phosphorus screen and scanned by an Amersham Typhoon fluorescent image 166 analyzer. %PRF was determined by quantifying radiolabeled bands using ImageJ software , followed by phenol-chloroform extraction and 3 M sodium acetate ethanol precipitation. Five micrograms of total RNA were subjected to 1.5% agarose electrophoresis, transferred to nitrocellulose and probed with α32P labeled oligonucleotides complementary positions 248–262, 390–429, 1029–1068, 1974–2013, 2202–2239, 2464–2502 and 2655–2692 nt. The blot was exposed to a phosphorous screen and scanned in an Amersham Typhoon fluorescent image 166 analyzer. The band's intensity was quantified using ImageJ software  (All RNA structures were drawn using RNAcanvas (er.app/) .Figure in vitro SHAPE structure probing data. As with umbraviruses . The small H3 stem-loop is highly conserved in Class 2 ulaRNAs with 11 of 19 identical residues and four additional residues conserved in all dicot-infecting Class 2 ulaRNAs  reduced frameshifting by 29 and 42%, respectively. Inclusion of both sets of alterations (m8C), which should reform the stem, enhanced frameshifting to WT levels. This result supports the presence of the long-distance pairing that forms the Bridge in the secondary structure of CY1 and its importance for efficient frameshifting.Tombusviridae, whether -1 PRF or ribosome stop codon readthrough, requires a long-distance interaction between the apical loop or interior bulge loop of the recoding stimulating e with sequences in the: (i) apical loop of the 3′ terminal hairpin; (ii) apical loop of the 3′ penultimate hairpin or (iii) spacer sequence linking the two hairpins  were designated as [X], [Y] and [Z], respectively Figure . All ClaSince the single mutations still permit four (PK1a) or five (PK1b) paired bases, the experiment was repeated with the addition of a second mutation that should further weaken the putative interactions. m10A with two mutations in [X] that should affect formation of both PK1a and PK1b Figure , decreasin vivo. Transcripts containing these mutations were transfected into Arabidopsis thaliana protoplasts and levels of CY1 minus strands were assessed 24 h later using RNA gel blots  reduced translation of p21 by at least 75% when levels should have been significantly higher than WT due to reduced frameshifting. Compensatory mutations (m12C and m13C) increased translation of p21 to greater than WT levels . This indicates that the specific [Z] alterations were not directly responsible for reduced translation. Rather, the reduction in translation may have resulted from a portion of the H3 loop sequence being newly accessible to a detrimental alternative pairing that disrupts the functioning of the 3′ CITE or other elements necessary for efficient translation. Why translation was enhanced by the compensatory mutations is not currently understood.Residue C650 in H3 apical loops of all Class 2 ulaRNAs is shared by both PK2a and PK2b  is composed of identical H4 apical loop sequences (5′GUCGAC) Figure  and B. Cin vitro synthesized transcripts subjected to SHAPE. Altering the central G in the PK4 H5 sequence to a C (m17A) reduced frameshifting by 45% , and most of the H4 apical loop PK3 pairing sequence, are unreactive with the SHAPE reagent, suggesting that they may also have alternative unidentified pairing partners. PK4 sequences in H2 and H5 are strongly reactive with the SHAPE reagent, and thus are proposed to be unstructured in the alternative FSE in CY1. Interestingly, the H2 PK4 sequences for several Class 2 ulaRNAs (in red) are not available for pairing, supporting the possibility that PK4 does not form in this structure.If the conserved, alternative structure for H1 forms Figure , right, in vitro synthesized transcripts contain the inactive structure. This is similar to the ribosomal readthrough element in carmovirus TCV, whose inactive structure dominated in transcripts and in vivo  and PK4 (m17B) significantly reduce translation of p21 whereas many mutations in C650 and its partner sequences significantly enhance p21 translation? Previous analysis of the CY1 3′ CITE revealed that a number of alterations in the CITE lower stem significantly affected frameshifting . As shown in cis-acting interactions are needed to destabilize the CY1 FSE before a frameshifted ribosome can proceed with translation. None of the long-distance interactions  would have been discernable if the local FSE structure had been studied in a reporter construct or via optical tweezers, which would have significantly affected any drawn conclusions. Our results strongly support the gathering consensus  are involved in transitioning between inactive and active structures. More importantly, the discovery of PK3 and PK4 suggests that onsensus ,22,30 thgkad744_Supplemental_FileClick here for additional data file.


Methods to achieve high star ratings for the High-Risk Medication (HRM) measure are thought to result in unintended consequences and to compromise several member experience measures that ultimately put at risk the plan sponsor’s Medicare Part D Centers for Medicare Medicaid (CMS) star rating.To determine if HRM scores are associated with relevant member experience measure scores.This is a cross-sectional analysis utilizing CMS 2013 and 2014 plan star ratings reports (2011 and 2012 benefit year data) for Medicare Advantage prescription drug (MA-PD) plans and prescription drug plans (PDPs). Medicare contracts with complete data for all measures of interest in 2013 and 2014 star ratings reports were included (N = 443). Bivariate linear regressions were performed for each of 2 independent variables: (1) 2014 HRM score and (2) 2013 to 2014 change in HRM score. Dependent variables were the 2014 scores for “Getting Needed Prescription Drugs,” “Complaints about Drug Plan,” “Rating of Drug Plan,” and “Members Choosing to Leave the Plan.”The bivariate linear regressions demonstrated weak positive associations between the 2014 HRM score and each of the 2014 member experience measures that explained 0.5% to 4% (R2) of variance of these measures. The bivariate regressions for the 2013 to 2014 change in the HRM score and 2014 member experience measures of interest demonstrated associations accounting for 1% to 8% of variance (R2). The greatest associations were observed between each independent variable and the 2014 “Getting Needed Prescription Drugs” score with correlation coefficients of 0.21 and 0.29.HRM star ratings and change in HRM star ratings are weakly correlated with member experience measures in concurrent measurement periods. Plan sponsors may be more aggressive in HRM utilization management, since it is unlikely to negatively impact CMS summary star ratings.