Structured RNA elements, programmed RNA conformational changes, and interactions between different RNA domains underlie many modes of regulating gene expression, mandating studies to understand the foundational principles that govern these phenomena. Exploring the structured 3′ untranslated region (UTR) of a viral RNA, we discovered that different contexts of the 3′-UTR confer different abilities to enhance translation of an associated open reading frame. In one context, ribosome-induced conformational changes in a ‘sensor’ RNA domain affect a separate RNA ‘functional’ domain, altering translation efficiency. The structure of the entire 3′-UTR reveals that structurally distinct domains use a spine of continuously stacked bases and a strut-like linker to create a conduit for communication within the higher-order architecture. Thus, this 3′-UTR RNA illustrates how RNA can use programmed conformational changes to sense the translation status of an upstream open reading frame, then create a tuned functional response by communicating that information to other RNA elements. Complex RNA three-dimensional structures undergo functionally important programmed conformational changes. Here, the authors report how two structurally and functionally coupled RNA domains within a viral 3′-UTR sense the ribosome through conformational changes and respond by modulating translation. These elements can be found throughout an mRNA or viral RNA, in both the 5′- and 3′-untranslated regions (UTRs) as well as in the protein coding sequences. In addition, RNA conformational changes, structural cooperation between different RNA domains, or the context of the RNA element potentially could combine to enhance regulatory potential and capacity. However, many underlying principles that allow complex higher-order RNA structure-based regulation remain poorly understood as few examples have been characterized in detail. Viruses, particularly single-stranded positive-sense RNA viruses, contain a variety of structured RNA regulatory elements that interact with and exploit the cellular machinery and thus they provide a wealth of RNAs that serve as models to yield insight into basic principles of diverse and subtle regulatory mechanisms.Eukaryotic regulation of gene expression at the level of translation can be controlled by structured RNA elements within messenger RNAs (mRNAs) that alter translation initiationturnip yellow mosaic virus (TYMV) is an important model for exploring important principles of RNA structure-based translational control9. During infection, TYMV produces two RNAs: a genomic RNA (gRNA) that encodes the movement and polyproteins (MP and PP), and a subgenomic RNA (sgRNA) encoding the coat protein (CP)11  tail they end in the same two-folded RNA domains separated by a short unpaired linker13 11 Fig. . The CP r13 Fig. . Althougr13 Fig. . In the 17 -like structure (TLS) that mimics tRNA to drive aminoacylation  of the viral RNAs. Aminoacylation plays multiple roles, including stabilizing the RNA and promoting binding of eukaryotic elongation factor 1A (eEF1A), which enhances the translation of viral proteins by an unknown mechanism17 Fig. . The TLS21, a structure of the isolated 3′-end acceptor pseudoknot solved by nuclear magnetic resonance22, and a crystal structure of the isolated TLS domain14. The only direct three-dimensional structural analysis of the complete TYMV 3′-UTR is a small-angle x-ray scattering study that yielded low-resolution envelopes of its global structure20, but an atomic-resolution structure of the intact 3′-UTR that could reveal the details of interdomain communication had not been previously solved.Understanding how the two domains of the TYMV 3′-UTR relate and work together requires detailed structural information. To date, structural studies of the TYMV 3′-UTR include chemical probingThe TYMV 3′-UTR’s features make it a useful model system for understanding many aspects of RNA structure-based regulation, including how RNA conformational changes, multi-domain interactions, and the context of folded RNAs combine to regulate function. We used biochemistry, functional assays coupled with mutagenesis, biophysics, and structural approaches to explore this RNA. We discovered that the two-domain architecture allows a mechanism of regulation that depends on the context of the 3′-UTR relative to the ORF. Specifically, the UPD acts as a structure-based ribosome sensor, communicating its conformational status to the TLS to alter the efficiency of downstream functions. By solving the structure of the entire 3′-UTR by x-ray crystallography, we revealed the nature of the communication conduit between the two domains within the higher-order RNA architecture. Overall, these results provide an illustrative model of context-dependent RNA structure-based regulation of function that uses multiple domains, conformational changes, and interdomain communication.We first recapitulated 5′ cap-dependent translation enhancement by the TYMV 3′-UTR using luciferase-encoding reporter RNAs in cell-free translationally competent wheat germ extracts (WGEs). In this system, the presence of a 5′ cap dramatically increased the rate of translation initiation, and the presence of the TYMV 3′-UTR further enhanced translation of the upstream reporter ORF  reporter in WGE, both in the absence (translating) and presence (non-translating) of cycloheximide  map the location and nature of direct contacts between the domains or (2) observe how changes in the conformational stability of one domain affects the structural coupling. Several such mutants were identified, including mutations in the UPD and the interdomain linker (positions U1 to G27) that alter the TLS conformation: some destabilized the TLS while others stabilized it. Hierarchical clustering analysis identified two point mutations within the UPD as representative of these effects  applied to the entire 109-nucleotide-long 3′-UTR RNAule Fig. . Althougcts Fig. . Specificts Fig. . These t30  absorbance to detect mutant-induced changes in structural stability  RNA with and without magnesium shows a change in the values consistent with the RNA adopting its native folded structure in the presence of the metal ions, agreeing with previous studiesWe determined if the different chemical probing patterns and biophysical behavior of mutants U8A and G10C correlate with changes in function. Using in vitro valylation assays with purified valine synthetase enzyme and the entire 3′-UTR, we observed that U8A is aminoacylated at levels at least as well as WT, while G10C has decreased aminoacylation levels Fig. . Mutant 14, and low-resolution reconstruction of the shape of the 3′-UTR had been generated by small-angle X-ray scattering (SAXS)20, the high-resolution structure of the UPD and of the full 3′-UTR RNA were unsolved. Therefore, we solved the structure of the entire TYMV 3′-UTR by x-ray crystallography to a resolution of 3.1 Å  lies in the major groove of Stem 2 (S2), with U5 and U6 pairing with the Hoogsteen edges of A19 and A20, respectively (the latter forms a base triple). The A bases of loop 3  lie in the minor groove of Stem 1 (S1), with two of them (A17 and A18) in a syn conformation. S1 and S2 do not coaxially stack on one another, but are offset to allow L3 to stack on a strand of S2 and L1 on a strand of S1. In addition, a previously unpredicted non-canonical base pair forms between U8 and U24; this pair stacks on Stem 2. Given the observed structural coupling between the UPD and TLS, we expected them to be in direct contact. Surprisingly, the UPD assumes an angle roughly perpendicular to the TLS’ helices with no direct contact between the two domains  and an S-turn motif, with the sugar-phosphate backbone going through two complete inversions in three nucleotides32 . Z-DNA-like elements form precise turns at critical locations in diverse RNA structures and have been found in some of the most stable RNA structures known33. This Z-step conformation was observed in the previous structure of the isolated TLS domain; hence, it is an authentic part of the linker conformation14. S-turns are common but the version seen here is unusual, being unpaired when most such turns are found in paired regions such as the common E-loop motif35. The linker structure appears to be stabilized by extensive base stacking: the non-canonical U8:U24 base pair (not previously predicted36) in the UPD stacks with U25; A26 stacks directly between U25 and the syn-G27:C94 base pair (within the Z-DNA-like element). Communication of the structural status of the UPD to the TLS likely depends on this linker structure. To test this, we used a reporter with the stop codon 39 nucleotides upstream from the UPD, but increased the linker length to 12 nucleotides to disrupt the base stacking and the proximity of the two domains. Translation of this reporter was severely reduced , using chemical probing and functional analysis , it will unfold the structure, which propagates through the spine to destabilize the TLS and depress translation enhancement. Consistent with this, the in-lysate chemical probing data show that during translation, there is increased reactivity of nucleotides in the same regions of the TLS that show increases with the G10C mutation and that are associated with the spine of stacked nucleotides Fig. . In addiRNA structure-based regulation of biological processes is ubiquitous, yet many of the fundamental mechanistic strategies by which changes in RNA conformation result in regulation remain unknown. We used the 3′-UTR from the TYMV as a model to explore how the coupled stabilities of two seemingly independent RNA domains could be used to create an RNA conformation-dependent regulatory platform. We propose that one domain (the UPD) acts as a ribosome sensor, communicating information to the other domain (the TLS), which results in subtle changes in stability and subsequent changes in the ability of the 3′-UTR to enhance translation. The structure of the entire 3′-UTR shows that this interdomain communication is through a spine of continuously stacked bases that appears to create a conduit for structural and thus functional coupling.37. In addition, previous studies of the TLS structure suggest that binding of the viral RdRP to the acceptor stem PK could alter interactions within the linker and spine to destabilize the TLS in a different way than would the ribosome interacting with the UPD14, a function likely important on the gRNA. Thus, our data, combined with published results, suggest that the TYMV 3′-UTR architecture creates a multi-domain “regulatory platform” capable of interacting with diverse partners in different contexts and responding in specific ways.Our studies do not directly address the virological purpose or advantage conferred by this 3′-UTR-based regulation strategy, but we propose a model. In the cell, this 3′-UTR could operate within a larger context as part of an RNA-based rheostat that tunes production of CP encoded by the sgRNA to a virologically optimal level, independent of the production of MP and PP Fig. . Specifi39. In general, riboswitches are described as comprising a two-domain architecture: the aptamer domain binds to cognate small-molecule ligands, resulting in stabilization of a specific conformation. The aptamer domain conformation forces an associated expression platform to adopt one of two mutually exclusive conformations to affect production of a gene product involved in the metabolism of the small-ligand effector40. Hence, the riboswitch senses and responds to small-molecule concentrations using RNA structure. The TYMV 3′-UTR has some similar features, comprising a two-domain architecture in which a sensor domain detects the density of ribosomes on the associated ORF  tail but likely mechanistically different? The aforementioned 3′-CITEs found in some other plant viruses bind translation initiation factors or ribosomal subunits and bring these to the 5′ end through long-range communication, aiding the assembly of the translation machinery42. Hence, while some of these viruses may use the mechanism described here, others do not, and it should not be regarded as a universal mechanism. In addition, TLS elements with very different secondary structures are found in other types of plant viruses; these drive aminoacylation with histidine or tyrosine and also have been shown to enhance translation44. However, there is no clear evidence that they are using a two-domain architecture or mechanism of regulation similar to the TYMV 3′-UTR. Specifically, in plant viruses such as tobacco mosaic virus and brome mosaic virus the 3′-UTRs contain multiple UPDs with different arrangements relative to the TLS and the ORF compared to the TYMV48. There likely are multiple mechanisms at play that drive increased rates of protein synthesis from structured 3′-UTRs found in these viruses; however, this does not exclude that RNA interdomain communication plays an essential role in more complex viral 3′-UTRs.It is worth considering if TYMV 3′-UTR-like strategies of regulation based on programmed RNA conformational changes, structural context, and novel interdomain communication may exist in other viral RNAs or mRNAs. There is substantial variation in the 3′-UTRs of tymoviruses both in terms of their structural architectures and requirement for tRNA mimicry and valylation; not all have a domain that mimics a tRNA49 with mechanistic variations. Indeed, the 3′-UTRs of eukaryotic mRNA are often quite long, with potential for the formation of specific RNA structures and thus for the presence of regulation mechanisms based on programmed structural changes, but not necessary for tRNA mimicry. Such conformational changes could be induced by protein binding, miRNA binding, post-transcriptional modifications, or even small-molecule interactions, providing diverse and alternate ways for RNA folding and conformational dynamics to regulate gene expression.The existence of elements like the TYMV 3′-UTR suggests that similar entities could be found in other viruses or even cellular mRNAsEcoRI and XbaI restriction sites in vector pUC19, and the TYMV 3′-UTR and luciferase coding region. Briefly, pUC19 vector (NEB) was cut with restriction enzymes EcoRI and XbaI and gel purified. Thirty-five nanograms of cut plasmid, 35 ng of luciferase-encoding DNA, and 125 ng of TYMV 3′-UTR DNA were added to 2X Gibson assembly mix and Milli-Q water. The reaction was placed at 50 °C for 50 min and then transformed into DH5-α chemically competent cells. All constructs contained the T7 RNA polymerase promoter upstream of the luciferase gene. Sequences were confirmed using the services of GeneWiz and Eton Bioscience.To generate the reporter with the TYMV 3′-UTR (3′-UTR) downstream of the firefly luciferase gene, we used standard molecular cloning methods. The DNA sequence of the 3′-UTR was generated synthetically (Integrated DNA Technologies). DNA encoding the luciferase coding region was generated by PCR from a plasmid template . The DNA fragments were assembled using Gibson assembly methods (NEB) with sequences overlapping the 50 to reduce N + 1 T7 polymerase nucleotide addition. Methylated reverse primers were ordered from GE Dharmacon and IDT. PCR reactions of volume 200–500 µL were conducted, the DNA was purified using the Promega Wizard Kit, and ~1 µg of template was used in T7 in vitro transcription kits mMessage mMachine and MEGAscript (Thermo). RNAs were then purified using the Qiagen RNeasy kit and the concentration was determined using a NanoDrop spectrophotometer (Thermo) at absorbance 260 nm. The quality of the RNA was determined by 8% denaturing PAGE and imaged using ethidium bromide.Templates for in vitro transcription were amplified by PCR using either Phusion polymerase (NEB) or Pfx (Thermo) with primers M13 forward (5′-GTAAAACGACGGCCAGT-3′) and reverse primers to either exclude or include the TYMV 3′-UTR and the various plasmids described above. The reverse primers used when the TYMV 3′-UTR was included were modified to ensure precise 3′ ends (CC and CCA). Specifically, the two penultimate DNA nucleotides were modified with 2′OMe2, 15 µL of wheat germ extract, 33.4 µL of water, and the addition of 6.0 µL of RNA at 100 ng/µL to initiate the experiment. This assay was scaled up twofold to isolate more time points as observed in Figs. ®-Multi Detection system with firefly luciferase reagent (Promega). Results were analyzed using Excel (Microsoft) software with three or greater replicate measurements for each time course.In vitro translation assays were optimized using a WGE translation system (Promega). Typically, a master mix of 60 µL was used containing 2.5 µL of amino acid mix (1:1:1 mixture of provided amino acids), 2.5 µL of 3 M KOAc, pH 7.4, 0.6 µL of 100 mM Mg(OAc)UAA-3′, where the underlined sequence is the stop codon at the start of the UPD. In vitro-transcribed RNAs were resuspended in RNase/DNase free water to 0.5 µM, and Thermus thermophilus valine tRNA synthetase was resuspended to 2.0 µM in 50 mM Tris, pH 8.0, 5 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine HCl (TCEP), 5% (v/v) glycerol. Reactions were set up by the addition of 1 µL 10× reaction buffer ), 1 µL of 2.0 µM Valine tRNA synthetase  enzyme, 0.5 µL 3H-labeled l-valine (60 Ci/mmol), 1 µL of 0.5 µM RNA (folded by a heat-cooling step), and 6.5 µL of RNase/DNase free water. Each reaction was performed in replicates of four (n = 4). Reaction was placed at 35 °C for ~30 min. Reactions were immediately added to an equilibrated and washed vacuum filter apparatus  in 1× wash buffer: 20 mM Bis-Tris, pH 6.5, 10 mM NaCl, 1 mM MgCl2, and trace xylene cyanol for visualization. The vacuum filter was assembled with (bottom to top) thick filter paper (Bio-Rad gel dryer filter paper), two layers of Hybond positively charged membrane , and 0.45 μm Tuffryn membrane filter paper (PALL Life Sciences). Each sample was then rapidly washed five times with 150 μL of 1× wash buffer. The Hybond membranes were then dried, cut, and counted by scintillation (Perkin-Elmer Tri-Carb 2910 TR). Data were analyzed using Excel (Microsoft) software and plotted using KaleidaGraph (Synergy Software).RNAs for use in in vitro valylation assays contain a 5′ leader sequence 5′-GGACACUUCCACThermus thermophilus HB8, TEx18A06), which was cloned into a pET11a bacterial expression vector51 and expressed in BL21 DE3 cells. Six liters of LB broth were inoculated and cultures grew at 37 °C until optical density at 600 nm (OD600) was 1.5. Expression was induced by the addition of 1 mM isopropyl β-d-1-thiogalactopyranoside at 37 °C for 4 h. Cells were then centrifuged at 5000 r.p.m. for 12 min at 6 °C and pellets were collected and stored at −80 °C. Cell pellets were resuspended in 50 mM K2HPO4/KH2PO4, pH 6.0, 5 mM MgCl2, 10 mM DTT, and one Roche protease inhibitor tablet (EDTA-free) in a total volume of 50 mL. The resuspension was sonicated for 2 min  and centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was collected and heated to 70 °C for 30 min and centrifuged at 30,000 × g for 30 min at 4 °C. Supernatant was collected and added to 20 mL of Toyopearl Buffer A and loaded onto an equilibrated Toyopearl-Butyl column in Toyopearl Buffer A: 50 mM K2HPO4/KH2PO4, pH 6.0, 0.8 M ammonium sulfate, 5 mM MgCl2, and 1 mM DTT. Fractions were collected using an FPLC system  by eluting samples using Toyopearl Buffer B: 50 mM K2HPO4/KH2PO4, pH 6.0, 5 mM MgCl2, and 1 mM DTT. Eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dialyzed into 50 mM Tris, pH 7.2, 5 mM MgCl2, and 1 mM DTT at 4 °C overnight. The dialyzed sample was purified using a Hi-TRAP Q ion exchange column  with the column equilibrated in Q Buffer A: 50 mM Tris, pH 7.2, 5 mM MgCl2, and 1 mM DTT. The sample was eluted using Q Buffer B: 50 mM Tris, pH 7.2, 5 mM MgCl2, 1 M NaCl, and 1 mM DTT. Eluted fractions were analyzed by SDS-PAGE and dialyzed into 50 mM Tris, pH 8.0, 5 mM MgCl2, 1 mM TCEP, and 5% glycerol at 4 °C overnight. Dialyzed sample was collected, the concentration was determined using a NanoDrop spectrophotometer (Thermo), and the sample was stored at −80 °C.ValRS expression and purification plasmid was obtained from Riken DNA Bank (https://primerize.stanford.edu/53). Primers and primer plates to generate the WT DNA template and each of the 109 TYMV 3′-UTR point mutations were ordered from IDT and resuspended in water to a concentration of 100 µM. PCR was completed using Phusion polymerase (NEB). DNA templates were purified using Agencourt AMPure XP magnetic beads (Beckman) and resuspended in sterile water. Purified DNA templates were then in vitro transcribed using T7 RNA polymerase (NEB) and RNAs were purified using magnetic beads from a Thermo Poly(A)Purist™ MAG Kit. DNA and RNA concentrations were determined using a NanoDrop spectrophotometer (Thermo) and quality was checked by agarose gel electrophoresis.DNA templates to generate RNA for one- and two-dimensional chemical mapping experiments were generated by a tiling PCR strategy using the Primerize work flow , 0.5% dimethyl sulfate (DMS), 42 mg/mL N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) or 2% glyoxal, and incubated at room temperature for 15–30 min. Reactions were quenched using either 2-mercaptoethanol or 2-(N-morpholino)ethanesulfonic acid sodium salt (MES) pH 6.0. Chemically modified RNAs were isolated using a Poly(A)Purist™ MAG Kit (Thermo) and reverse transcribed using SuperScript III reverse transcriptase (Thermo) at 42–45 °C for 40–60 min using a fluorescently labeled primer (IDT): 5′-/5-6FAM/AAAAAAAAAAAAAAAAAAAAGTTGTTGTTGTTGTTTCTTT-3′.Chemical mapping procedures were carried out as in ref 56 and the RNAstructure suite  with MatLab (MathWorks). For further information on Primerize, one- and two-dimensional chemical mapping procedures see:Labeled DNA products were eluted in HiDi formamide spiked with Gene Scan ROX 350 size standard (Thermo). Samples were run on an Applied Biosystems 3500 XL capillary electrophoresis system and the data were analyzed using HiTRACEhttps://ribokit.github.io/HiTRACE/) andHiTRACE RiboKit (https://daslab.stanford.edu/resources/).Das lab website at Stanford University .Difference mapping and secondary structure diagram coloring was completed using MatLab and HiTRACE RiboKit: RiboPaint (https://software.broadinstitute.org/GENE-E/) and Morpheus (https://software.broadinstitute.org/morpheus/).Hierarchical clustering analysis with the completely processed data from the two-dimensional chemical mapping experiments was completed using: GENE-E  or Excel (Microsoft). The data were clustered based on chemical perturbation differences from the WT RNA across the TYMV 3′-UTR (nucleotides 1–109), the TLS domain only (28–109), and the UPD with the spacer sequence 5′-UUAG-3′ (nucleotides 1–27) using the Euclidean distance and one minus Pearson's correlation metrics (data not shown) for the NMIA, DMS, and CMCT datasets.57. DNA templates for SV-AUC and melting curves only contained a 5′ hammerhead ribozyme sequence and were amplified using M13F and modified reverse primers containing 2′OMe modification (see translation assay procedures). An example template that was used for biophysical analysis:DNA templates encoding the desired RNA sequences were ordered as gBlocks from IDT, cloned into pUC19, and sequenced. DNA was amplified for transcription by PCR and Phusion polymerase (NEB). DNA templates to generate RNA for crystallography contained 5′- and 3′-flanking self-cleaving ribozyme sequences that were removed during purificationTAATACGACTCACTATAGGGAGATCGAGAACTTACTGATGAGTCCGTGAGGACGAAACGGTACCCGGTACCGTCTAAGTTCTCGATCTTTAAAATCGTTAGCTCGCCAGTTAGCGAGGTCTGTCCCCACACGACAGATAATCGGGTGCAACTCCCGCCCCTCTTCCGAGGGTCATCGGAACCA-3′5′-2, 30 mM Tris, pH 8.0, 10 mM DTT, 0.1% spermidine, 0.1% Triton X-100, and T7 RNA polymerase, as well as 1–5 µL RNasin RNase inhibitor (Promega). Transcription reactions were incubated overnight at 37 °C, and then inorganic pyrophosphates were pelleted by centrifugation. The supernatant was subjected to ethanol precipitation and RNA was purified by 8% or 10% denaturing polyacrylamide gel electrophoresis. Bands containing the desired RNA were visualized by UV shadowing and excised, and RNA was passively eluted overnight at 4 °C into ~40 mL of diethylpyrocarbonate (DEPC)-treated milli-Q filtered water (Millipore). RNA was concentrated using Amicon Ultra concentrators (Millipore). RNA quality was checked by 8% or 10% denaturing PAGE and stained with ethidium bromide for visualization.The bold sequence is the T7 promoter, the underlined sequence is the hammerhead ribozyme, and the italic sequence is the 3′-UTR. Transcription reactions were conducted using 200–500 µL volume PCR reactions to generate DNA template in 2.5 or 5 mL in vitro transcriptions. Transcription reactions contained: 8 mM each NTP, 60 mM MgClf/f0, and radius of hydration, using the specific volume 0.53 cm3/g . SV-AUC software tools were available at: http://www.jphilo.mailway.com/.In vitro-transcribed RNAs were resuspended to 2.0 absorbance at 260 nm in 5 mM sodium cacodylate buffer, pH 6.5, in DEPC-treated water and heated to 95 °C for 3–5 min, and then slow-cooled to room temperature and diluted twofold in 2× AUC buffer . The resulting 1× AUC RNA (~1.0 OD at A260) and a buffer blank, prepared identically in the absence of RNA, were then loaded in the AUC cells. AUC was performed in a Beckman XLA centrifuge using a 4-position An60Ti rotor centrifuged at 35,000 r.p.m. for 16 h at 20 °C and 180 scans were collected at absorbance 260 nm. The WT and mutant RNAs were collected in replicates of three. Scans for each replicate were analyzed using DCDT+, SEDNTRP, and SVEDBERG to determine sedimentation coefficient, diffusion coefficient, RNAs for crystallization were prepared as described above. The sequence used for in vitro transcription was:TAATACGACTCACTATAGGGAGAAAGATCGAGAACTTACTGATGAGTCCGAGAGGACGAAACGGTACCCGGTACCGTCtaagttctcgatctttaaaatcgttagctcgccagttagcgaggtctgcGAAAgcagataatcgggtgcaactcccgccctttctccgagggtcatcggaaccaGAGGTGCTTGTATATAACCTCCACGATGGTGCACCTTGGGCAACACCACCACTCGCTTCGGCTTGTGGTGGTGGCAAATCATCTACATTAGGATCCGTATCGG-3′5′-GGCTATCGAATTCChilo iridescent virus ribozyme62, which was used to generate homogenous 3′ ends. The ribozyme-cleaved RNA was purified on by 10% denaturing PAGE and re-folded at 65 °C for 3 min at 2–5 mg/mL in a buffer containing 2.5 mM MgCl2 and 10 mM HEPES-KOH, pH 7.5, and then allowed to equilibrate to room temperature before a final concentration of 0.5 mM spermidine was added to the RNA. Crystal Screens I and II and Natrix I and II screens from Hampton Research were used in initial screens at 4 °C, 16 °C, 20 °C, and 24 °C. Crystals were optimized using custom screens. The RNAs used for the final structure determination were crystallized in 50 mM sodium cacodylate, pH 6.5, 2.5 M ammonium sulfate, 20 mM MgCl2, 1.0 mM spermine, and 2 mM hexamine cobalt chloride. Crystals were flash frozen in liquid nitrogen for x-ray diffraction with the addition of 20–30% glycerol in the same crystallization buffer. Diffraction data were collected at Advanced Light Source Beamline 4.2.2 using “shutterless” collection at 100°K; 180° datasets with “oscillation” images of 0.2°. Data were indexed, integrated, and scaled using X-ray diffraction and spectroscopy64. Pointless from the CCP4 suite65 determined the space group was I222 and Phenix66 was used to determine a molecular replacement solution based on these parameters and a search model consisting of residues 1–26 and 32–82 of the TYMV TLS lacking ligands (PDB ID: 4P5J)14. Iterative rounds of model building and refinement using COOT68 and Phenix66 were used to generate a complete model of the RNA. Crystal diffraction data, phasing, and refinement statistics are contained in Supplementary Table The bold sequence is T7 promoter, the italicized sequence is 5′ hammerhead ribozyme, the lowercase sequence is the RNA of interest with capitalized bases indicating those that were changed for crystallographic reasons, and the underlined sequence represents a slightly modified ribozyme sequence derived from the 20 using the program Primus and then further analyzed using Gnom and Gasbor. Projected scattering profiles were generated by using a model of the TYMV 3′-UTR PDB file merged with the PDB ID: 4P5J in the absence and presence of the UPD, in the crystallized state, or with manual movements of the UPD relative to the TLS domain. The predicted scattering data were plotted and compared to the experimental data using CRYSOL70.SAXS scattering profiles were previously collectedStructural figures were generated using PyMol .Further information on research design is available in the Supplementary informationReporting SummarySource Data


Identification of molecular markers for early detection or prediction of metastasis is crucial for both management of HCC patient postoperative treatment and identify new therapeutic targets to inhibit HCC progression and metastasis. In the current study, we investigated the clinical correlation between Pin1, RhoA and RhoC and their association with HCC metastasis.Using a randomized study design of primary HCC samples from 139 patients, we determined messenger RNA expression of Pin1, RhoA and RhoC and their prognostic value.p < 0.001) and RhoC , and their co-overexpressions correlated with metastatic HCC (p = 0.000012) and poor recurrence-free survival of HCC patients (p < 0.00001), which showed better prognostic significance than either Pin1, RhoA or RhoC overexpression alone. Co-overexpressions of Pin1 + RhoA/RhoC were also an independent factor for predicting development of metastasis after curative resection in our multivariate regression model (p < 0.001).Our findings demonstrated for the first time the clinical correlation of Pin1 in HCC metastasis. Pin1, RhoA and RhoC transcript levels were significantly higher in HCC specimens when compared with the paired adjacent non-tumorous liver. Pin1 overexpression was closely correlated with that of RhoA  contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide, especially in Eastern Asian regions including Hong Kong and China where hepatitis B is more prevalent, while cases of HCC are also slightly increasing in low-incidence areas such as the United States and Canada . Despitecis–trans isomerization of specific proteins that are phosphorylated at Ser/Thr-Pro motifs [The identification and characterization of a peptidyl-prolyl cis/trans isomerase, Pin1, has led to the discovery of a new postphosphorylation regulatory mechanism in cell signaling pathways through promoting the o motifs –5. Such o motifs . Our groo motifs –10.The aim of this study was to investigate the clinical correlation between Pin1 and HCC metastasis. We also examined the potential interaction between Pin1 and two members of the Rho subfamily of the Ras superfamily of homologious genes, RhoA and RhoC, which has been implicated in tumorigenesis, tumor progression and metastasis of HCC –15 by deFresh tumor specimens were randomly obtained from 139 patients (79 patients with paired tumor/non-tumor specimens and 60 patients with tumor specimens only) who underwent surgical resection of primary HCC at the Department of Surgery, Queen Mary Hospital, The University of Hong Kong, between 1998 and 2007. One hundred and four were men and thirty-five were women. Patients’ age ranged from 15 to 88 years, with a mean age of 58.6 years. The patient characteristics were summarized in Additional file Total RNA was extracted from tumors and their corresponding non-tumorous liver samples (if available) from HCC patients using Trizol reagent and RNA miniprep extraction kit according to the manufacture’s instruction (Life Technologies), and 500 ng each RNA sample was used to prepare cDNA using PrimeScript™ RT Master Mix (TaKaRa).Real-time PCR was performed in a final volume of 15 μl containing 1.5 μl RT transcript, 0.2 μM of each primer, 1X ROX reference dye and 7.5 μl of FastStart Universal SYBR Green Master (ROX) (Roche). The primers sequences were as follows: Pin1-FP: AAGATGGCGGACGAGGAG, Pin1-RP: CACTCAGTGCGGAGGATGAT; RhoA-FP: GTGCCCACAGTGTTTGAG, RhoA-RP: AGGGCTGTCGATGGAAAAA; RhoC-FP: GCAGGGCAGGAAGACTAT, RhoC-RP: GTTGGGGCAGAAGTGCTT; actin-FP: 5’- CGAGCATCCCCCAAAGTT-3’, actin-RP: 5’-GCACGAAGGCTCATCATT-3’. Real-timePCR was carried out using the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) at 95°C for 10 min, followed by 40 cycles at 95 °C for 15 sec and at 56 °C for 1 min. The expression of actin was used to normalize that of the target genes. Each assay was done in triplicate, the average was calculated, and the expression level of targets mRNA was expressed as fold to actin..P < 0.05 was considered statistically significant.Data analysis was performed using SigmaStat 3.5  or SPSS 16.0 . The Rank Sum test was used to analyze differences between experimental groups of clinical specimens. Spearman’s correlation test was applied to determine correlations. Chi-square test was used to compare categorized data. Disease-free survival (DFS) was calculated by the Kaplan-Meier method, and differences in survival rate were compared using the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. To study the role of Pin1, RhoA and RhoC in HCC carcinogenesis and progression, we first examined their transcript levels in 79 pairs of tumors and the adjacent non-tumorous liver of HCC patients by quantitative polymerase chain reaction, and expressed as the fold when compared with transcript level in the adjacent non-tumorous liver , yet its association with metastasis has not been demonstrated in HCC thus far. On the other hand, RhoA and RhoC overexpressions have been correlated with HCC progression and metastasis in many clinical studies –16. In tThis study demonstrates for the first time the correlation of Pin1, RhoA and RhoC with the development of metastasis in HCC patients. We found that HCC patients with overexpression of Pin1, RhoA or RhoC showed increased rate of metastasis, when compared with those with down-regulation counterparts. For example, around 40% of HCC patients with overexpression of Pin1, RhoA or RhoC developed distant metastasis, whereas the percentage decreased to around 10% in patients with down-regulation of Pin1, RhoA or RhoC, suggesting that their down-regulations impaired the metastatic processes. In addition, we further demonstrated that 46% of the patients with co-overexpressions of Pin1+RhoA/RhoC developed distant metastasis, while less than 10% patients with no such co-overexpressions developed metastasis. Similar trends were also observed in the recurrence-free survival analyses of HCC patients, in which patients with downregulations of Pin1, RhoA or RhoC showed a trend of better prognosis for recurrence-free survival when compared with those with overexpressions, and those with co-overexpressions of Pin1+RhoA/RhoC demonstrated a significantly poor recurrence-free survival than those with no co-overexpressions. Hence, our results clearly demonstrated the correlations of Pin1 with RhoA and RhoC as well as their involvement in the metastatic process, and their co-overexpressions associated with metastatic HCC, which further potentiates the development of combined inhibitors against Pin1/RhoA/RhoC in order to combat metastasis in HCC patients.in vitro through upregulating the transcription factor Ets-1, resulting in overexpression of RhoC mRNA and proteins [in vitro [in vitro and in vivo.There are two possible linkages between Pin1 and RhoA/RhoC. Our previous study on Pin1 demonstrated a preferential Pin1 overexpression in HBV-related tumors , whereasproteins , 18. In proteins . Hence win vitro , as wellIn this study, we showed that the combination of biomarkers demonstrated a better prognostic value than either one of Pin1, RhoA or RhoC, suggesting that these factors act together in the cancer metastatic process and thus effect of downregulation of one factor could be compensated by the others. Therefore, it will be more comprehensive to monitor a combination of factors in this instance. Indeed, there are increasing evidences demonstrating the efficacy of combination of molecularly-associated biomarkers. For example, combination of osteopontin and its receptor CD44v6 improves the sensitivity and predictive range for predicting tumor recurrence and survival in non-small cell lung cancer patients , combinaTo summarize, this study demonstrated for the first time the clinical correlation of Pin1 in HCC metastasis. Pin1 was significantly correlated with the transcript levels of RhoA and RhoC, and their co-overexpressions correlated with metastatic HCC and recurrence of HCC patients, suggesting that Pin1, RhoA and RhoC not only could be used as potential biomarker for predicting development of metastasis after curative resection, but also as potential therapeutic targets to inhibit HCC metastasis.Additional file 1:Table S1. Patient characteristics in this study. (PDF 30 kb)Additional file 2:Table S2. Pin1, RhoA and RhoC expressions (fold to actin) in all HCC samples and the patient’s clinicopathological parameters. (XLSX 25 kb)Additional file 3:Figure S1. Comparison of Pin1, RhoA and RhoC overexpressions (Tumor/non-tumor) in HCC patients with or without HBsAg. (TIF 2557 kb)


Klebsiella phage KP34. The enzyme is unique among known bacteriophage RNA polymerases in that it recognizes two unrelated promoter sequences, which provided clues for the evolution of phage single-subunit RNA polymerases. As the first representative enzyme from the “phiKMV-like viruses” cluster, its use in run-off RNA synthesis was investigated. RNA-Seq analysis revealed that the KP34 RNA polymerase does not possess the undesired self-templated RNA terminus extension known for T7 RNA polymerase and is suitable to synthesize RNAs with structured 3′ termini such as sgRNAs. A KP34 RNA polymerase Y603F mutant is engineered to incorporate deoxy- and 2′-fluoro ribonucleotide into RNA.We have characterized the single subunit RNA polymerase from  In vitro run-off RNA synthesis catalyzed by phage-encoded single-subunit RNA polymerases (ssRNAP) is a fundamental technology for RNA research. The most widely used enzyme in this technique is the RNA polymerase (RNAP) encoded by bacteriophage T7 due to its high efficiency and extensive characterization  Polymerase, RNA 5′ Pyrophosphohydrolase (RppH), T4 DNA ligase, NTPs, dNTPs, and RNA purification kits were from New England BioLabs . RNase inhibitor was from Thermo Fisher Scientific . PrimeSTAR Max DNA Polymerase and Premix Taq DNA Polymerase, SMARTScribe and ProtoScript II Reverse Transcriptase are were from TAKARA . DNA purification kit was from Axygen . Ni-NTA resin was from Qiagen . Preparative Superdex S200 for gel filtration was from GE Healthcare . Radiolabeled nucleotides were from PerkinElmer . 2′-F-dNTPs were from TriLink .Oligonucleotides were obtained from TsingKe . DNase I, restriction endonucleases, E. coli BL21(DE3). The bacteria were cultured in LB medium containing 50 μg/ml kanamycin at 37°C until they reached an A600 of ∼1.2. The gene for KP34 RNAP was induced by the addition of 0.5 mM IPTG at 28°C and incubation continued for 3 h. Cells were harvested, resuspended and sonicated in lysis buffer . Resuspended cells were lysed via sonication on ice using 60% probe amplitude for 5 min . Cell debris was pelleted by centrifugation for 50 min at 12000 rpm, 4°C. His-tagged KP34 RNAP was purified with Ni-NTA-agarose column. The lysate was run over a 2 ml (bead volume) Ni-NTA gravity column pre-equilibrated with lysis buffer. The column was washed with 10 column volumes of wash buffer . KP34 RNAP was eluted off the column by 4 column volumes of elution buffer . The RNAP was further purified by gel filtration chromatography on a 200 ml preparative Superdex S200 column. The column was pre-equilibrated with a 400–500 ml balance buffer . Then a 1–2 ml sample which was concentrated by Millipore Amicon Ultra-15  was added to the column. Fractions were eluted off the column by 200–300 ml balance buffer. Fractions containing the KP34 RNAP were concentrated again and dialyzed twice against a storage buffer . KP34 RNAP mutations were introduced through PCR mutagenesis using PrimeSTAR Max DNA Polymerase, and mutant RNAPs were purified following the same procedure as that for the wild-type enzyme. The T7 RNAP gene was cloned into plasmid pQE-82L through homologous DNA assembly using ClonExpress® II One Step Cloning Kit from Vazyme . The plasmid pQE-82L-T7 RNAP was transformed into BL21(DE3). N-terminal His-tagged T7 RNAP was purified following the same procedure as mentioned above for KP34 RNAP. His-tagged Syn5 RNAP was produced from a pET24 vector harboring the N-terminal His-tagged Syn5 RNAP gene between the NdeI and NotI sites and purified according to previous report . KP34 genomic DNA was prepared according to a previous report . The Plas report . Oligonus report .GGG-3′ or 5′-ATGTTACAGGAGTAGGG-3′) insertion between the BamHI and XbaI sites was linearized by EcoRI treatment. For BamHI and XbaI sites, and transcription templates were then PCR amplified from the plasmids carrying each of the three promoters, respectively. Sequences of all PCR primers and synthetic DNA oligonucleotides mentioned above were listed in TM PCR Cleanup Kit (Axygen), and DNA concentrations were determined by NanoPhotometer® (Implen).Transcription templates for 2, 4 mM of each of the 4 NTPs , 40 U/μl RNaseOUTTM Recombinant Ribonuclease Inhibitor, 0.04 U/μl E. coli inorganic pyrophosphatase, DNA template , and RNA polymerase . Reactions with KP34 and T7 RNAP were carried out at 37°C for 1 h and reactions with Syn5 RNAP were carried out at 24°C for 1 h. For the assays shown in 2O and 5 μl 3X RNA loading dye . Then, 7 μl of each sample was loaded onto 12% TBE native gels or 2% TAE agarose gels. DNA templates and RNA products were separated by electrophoresis and visualized by staining with ethidium bromide. The transcripts were purified with Monarch® RNA Cleanup Kit (New England BioLabs) and their concentration was determined using NanoPhotometer®(Implen). The reaction mixtures for 2, 2 mM spermidine, 10 mM DTT, 200 μM CTP, GTP and UTP, 10 μM [α-32P]ATP, 1.5 U/ml RNaseOUTTM recombinant ribonuclease inhibitor, 0.5 μM DNA templates, and 100 nM KP34 or T7 RNAP that were incubated at 37°C for 30 min before adding one unit of DNase I to each reaction mixture, and then incubated for an additional 20 min at 37°C. Reactions were then terminated by the addition of 4 μl of 3X RNA loading dye containing 95% formamide, 40 mM EDTA, 0.025% bromophenol blue and 0.025% Xylene cyanol FF. Samples were then heated at 90°C for 1 min, and 4 μl of each sample was loaded onto a 25% TBE-Urea denaturing gel. After electrophoresis, gels were dried and the radioactivity was analyzed using a Fuji BAS 1000 Bioimaging Analyzer.For the assays shown in In vitro transcription products were treated with DNase I for 20 min at 37°C to remove the DNA templates and then were purified with Monarch® RNA Cleanup Kit (New England BioLabs). Transcripts were incubated with RNA 5′ Pyrophosphohydrolase (RppH) at 37°C for 30 min to remove pyrophosphate from the 5′ end of triphosphorylated RNAs and to leave 5′ monophosphate. 5′ monophosphorylated RNAs were purified with Monarch® RNA Cleanup Kit (New England BioLabs), and the cDNA library was prepared by the NEBNext Small RNA Library Prep Set for Illumina (New England BioLabs). The cDNA library was sequenced on an Illumina HiSeq 2500 sequencing platform. The raw data in FASTQ format was obtained from the SeqHealth company .® RNA Cleanup Kit (New England BioLabs) and then incubated with E. coli Poly(A) Polymerase at 37°C for 30 min to add adenosines to the 3′ end of RNA. The products were purified again. RNAs were then reverse transcribed to cDNA.Transcription reactions (10 μl) were incubated at 37°C for 20 min with DNase I to remove the DNA templates. Transcripts were purified with a Monarch+ RNA) was mixed with 1 μl 3′ SMART CDS Primer II A (12 μM) to 4.5 μl total volume. The sample RNA/primer was denatured for 5 min at 65°C and was then spun briefly and put promptly on ice. The following components were added (5.5 μl): 2 μl 5X First-Strand Buffer, 0.25 μl DTT (100 mM), 1 μl dNTP Mix (10 mM), 1 μl SMARTer II A Oligonucleotide (12 μM), 0.25 μl RNase Inhibitor (40 U/μl), and 1 μl SMARTScribe Reverse Transcriptase (100 U/μl). Reaction mixtures were incubated at 42°C for 1 h. cDNA amplification was done by 12.5 μl Premix Taq DNA Polymerase (TAKARA) using 0.2 μM 5′ universal prime (UPM) and 0.2 μM 3′ gene specific primer (GSP1) in a 25 μl reaction volume. 3′-RACE: 100 ng poly A+ RNA, 5 μM 2 μl VN-oligo(dT)18(50 μM) was mixed with 1 mM dNTP to 10 μl total volume. Sample RNA/primer was denatured for 5 min at 65°C, then spun briefly and put promptly on ice. The following components were added (10 μl):4 μl 5X ProtoScript II Buffer, 2 μl 0.1 M DTT, 1 μl ProtoScript II Reverse Transcriptase (200 U/μl), 0.2 μl RNase Inhibitor (40 U/μl), and 2.8 μl deionized H2O. The 20 μl cDNA synthesis reaction was incubated at 42°C for 1 h. cDNA was then amplified by 12.5 μl Premix Taq DNA Polymerase (TAKARA) using 0.2 μM 5′ gene specific primer (GSP2) and 0.2 μM 3′ VN primer in a 25 μl reaction volume.5′-RACE: 1–3.5 μl RNA ; the reaction was incubated at 16°C for 30 min. Plasmids containing inserted sequences were amplified and sent for sequencing.PCR products were purified by AxyPrepKlebsiella phage KP34 is distantly related to the T7-, SP6- and P60-like viruses of the short-tailed phage Autographivirinae subfamily  also initiates KP34 RNAP transcription, while with much less efficiency and precision, as judged by the amount and heterogeneity of run-off products  by the two RNAPs. In gel analysis of an sgRNA synthesis, as expected, a product larger than that of the desired size was not observed for KP34 RNAP while T7 and Syn5 RNAP products contain species significantly larger in size, indicating RNA 3′ undesired extension by these RNAPs and further formation of complex structures by the extended RNA . Furtherin vitro and in vivo  with the BioProject accession code PRJNA555705.RNA sequencing data have been deposited in the Sequence Read Archive (SRA) (XL and BZ designed and performed the experiments and wrote the manuscript. HW and HX analyzed the data. FH guided protein expression and purification. YY, BY, RC, and ZD-K provided reagents and contributed to manuscript revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson–Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of “normal” aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated.  Ageing is a complex and unavoidable process that can be defined as the functional decline after a period of maturity. In this respect, maturity is understood as the endpoint of development and the condition of maximal functional performance capability. Aging is a dynamic process, as is maturity—biological systems in the form of cells and multicellular organisms do not reach a defined condition of maturity. By comparison, development results in states of maximal functional performance capability of subcellular signaling pathways or individual tissue functions, whereas other, adjacent pathways or tissues are already in decline. For example, humans reach their maximal sprint velocity at the age of 23 years , but their maximal endurance capability ten years later , when they have already lost maximal speed. When the maximal creative and intellectual performance of the human mind is reached, is currently not precisely defined . This heFrequently, these hallmarks are interrelated, for example, telomere attrition as one hallmark leads to genomic instability, another hallmark. This association is typical for complex systems, such as biological organisms that are dynamically interrelated as though in a four-dimensional network. Because aging predisposes to age-associated disease and is the primary risk factor for diseases like cancer, dementia, and osteoporosis, it is of utmost importance for an aging society to know how to prevent and treat age-associated diseases. To study the aging process and the underlying biological pathways in humans, one can either investigate exceptional human longevity, centenarians who have a deceleration of morbidity, or the opposite situation, where children age as if in time lapse and suffer from accelerated morbidity. These childhood syndromes, termed progerias, are of segmental nature, in that they display some symptoms of aging without mirroring the whole spectrum of age-associated disease. These “caricatures of aging” are also denominated as premature aging, in which aging occurs before entering the state of maturity. This also implies that the deleterious forces that drive the aging processes in these children are stronger than the opposing forces of growth and development to maturity. Therefore, it is very interesting to understand these forces, the pathomechanisms of premature aging, as they will help to understand the pathomechanism of the aging process itself. Premature aging syndromes serve as model diseases to study aging, although not all of them have been validated as showing a pathomechanism that also drives the “normal” aging process, and therefore numerous unresolved questions remain to be answered. The progeria of the adult, Werner syndrome, is also characterized by an accelerated, segmental aging process that starts with a lack of the pubertal growth spurt. In the second and third decade it develops with a loss of subcutaneous fat and signs of skin aging, cataracts, hair loss, atherosclerosis, and osteoporosis . Werner In this review we would like to address if, and how, the nucleolus and RNA polymerase I transcription might contribute to the segmental aging pathologies of childhood progerias.One question is whether the nucleolus, the site of ribosomal transcription and synthesis, is involved in the aging pathomechanisms or even a driver of age-associated physiological decline. The nucleolus is a non-membrane-bound organelle that organizes around the sites of rDNA transcription by RNA polymerase I (RNA Pol I) and the co-transcriptional pre-ribosomal assembly and processing . The nucIn yeast cells, it has been shown that the redistribution of the silence information regulator, Sir2, from the telomeres to an AGE locus prolongs lifespan. The nucleolus has been identified as the AGE locus  and the Caenorhabditis elegans [The size of the nucleolus, during early microscopy, was recognized as, and is still valid as, a prognostic factor in tumor pathology and an indicator of the activity of the ribosomal synthesis machinery . The nuc elegans . This im elegans . The met elegans ,17. TherC. elegans by the Antebi group [ncl-1 in C. elegans led to enlarged nucleoli in several tissues [ncl-1 blocked the lifespan-extending effects of the manipulations of a genetic caloric-restriction model on the TOR pathway, the insulin/insulin-like growth factor pathway, the germline-less mutants, the mitochondrial effectors, the translation-initiation factors, and the ribosomal S6kinase. Overexpression of ncl-1 reduced nucleolar size and increased lifespan. All lifespan-extending models exhibited small nucleoli and the size of the nucleoli in wild-type C. elegans was a predictor of life expectancy. Reduced nucleolar size was associated with a reduced ribosomal RNA and protein content, suggesting that the downregulation of ribosomal synthesis is an indicator of long life. Finally, the authors analyzed nucleolar size in long-lived Drosophila melanogaster and observed reduced nucleolar size in these insects as well as in muscle biopsies of dietary-restricted, exercising humans. This study, thereby, identified nucleolin and its nucleolar function as a central player in lifespan regulation and is in agreement with the metabolic theory of aging which proposes that interventions that dampen high-energy demanding processes, including ribosomal biogenesis and translation, prolong the lifespan. Moreover, the findings that small nucleoli are related to longevity [A study in bi group  identifibi group . Knockdo tissues . Knockdoongevity  and largongevity , could, The HGPS, frequently termed childhood progeria, is a severe developmental and degenerative syndrome that is characterized by a typical facial appearance (bird-like facies) with micrognathia, alopecia, and prominent veins on the forehead. Hypoplasia of the claviculae, generalized osteoporosis with pathological fractures, and the lifespan-limiting general atherosclerosis are hallmarks of this syndrome. Life expectancy is severely reduced because of cardiovascular insults, including stroke and heart infarction in the second and third decades (mean life expectancy of 14.6 years) . HGPS isC. elegans and mice [Considering progerin and if it influences the proteome of HGPS cells, Buchwalter and Hetzer discovered that nucleolar dysregulation might be involved in the pathophysiology of premature aging . By labeand mice ,30. Askiand mice  and a heand mice  in elderand mice , might band mice , might iand mice . The CS is a rare autosomal recessive disease that can be caused by six different genes, which are all involved in nucleotide excision repair (NER) of UV lesions . It is cThe CS phenotype in humans is induced by mutations in the Cockayne syndrome B protein (CSB) (70%), the Cockayne syndrome A protein (CSA) (20%), the subunits of the transcription/DNA repair factor TFIIH, XPB and XPD, and XPG and XPF, and two NER proteins. All of these proteins play central roles in the repair of helix-distorting lesions in DNA provoked by UV light. The total failure of this DNA repair pathway results in the cancer-prone skin disease XP that is characterized by the development of different cancers on UV-exposed skin. Early diagnosis and consequent UV protection reduces skin-cancer risk and allows almost normal development of XP children . The CS cases that do not suffer from a combination with XP are cancer free, indicating that developmental impairment and premature aging are not caused by non-repaired mutagenic UV lesions, although CS patients are frequently highly UV sensitive. The same type of UV sensitivity can also be found in UV-sensitivity syndrome (UVsS), a rarely diagnosed hypersensitivity of the skin to UV light ,45. UVsSPreviously, Iben et al. demonstrated that the TFIIH and the CS proteins, XPB and XPD, are co-immunoprecipitated with RNA Pol I and the RNA Pol I-specific initiation complex containing transcription-initiation factor IB (TIF-IB) indicating a role for the TFIIH in rRNA synthesis. Moreover, co-localization studies of the TFIIH with RNA Pol I and TIF-IB in the nucleolus, at the site of active transcription by RNA Pol I, confirmed this hypothesis. By using electron microscopy and immunogold labelling, the authors demonstrated localization of the TFIIH in the dense fibrillar component (DFC), a nucleolar structure in which pre-rRNA is synthesized. The TFIIH depletion impaired in vitro RNA Pol I transcription activity that could be rescued by adding highly-purified TFIIH in a concentration-dependent manner. These results indicated that the TFIIH is required for productive ribosomal RNA synthesis . In 2002, Hoogstaten et al. demonstrated, in a series of elegant experiments, that the TFIIH resides in the nucleolus in a transcription-dependent manner . In thisIn addition to the fact that TFIIH plays an essential role in transcription by RNA Pol I, Assfalg et al. further characterized the function of TFIIH in rDNA transcription. They demonstrated that TFIIH is bound to the initiation complex, including selectivity factor 1(Sl1), upstream binding factor (UBF), and transcription initiation factor IA TIF-IA with RNA Pol I. Furthermore, by analyzing chromatin immunoprecipitation (ChIP), TFIIH was found to bind to the gene-internal regions of the rDNA, indicating functions of the transcription factor beyond initiation of the RNA Pol I transcription. To study the interaction of TFIIH with the rDNA after transcription initiation, an assay called template-immunoprecipitation assay (TIP) was developed. Here, in vitro transcription and chromatin immunoprecipitation are combined, allowing kinetic analysis of DNA-protein interactions. The rDNA template was added to nuclear extracts with the first nucleotides, allowing synchronization by the formation of the initiation complex. By the addition of the missing nucleotides, single-round transcription was initiated and stopped by the crosslinking agent formaldehyde. On subsequent precipitation of TFIIH and the polymerase, the temporal interaction of TFIIH with the rDNA was analyzed. Immediately after transcription initiation, proteins of the initiation complex, including TIF-IA, leave the rDNA, whereas TFIIH and RNA Pol I are associated longer with the template. Immunoprecipitations after the transcription cycle showed that TFIIH and RNA Pol I are in a complex. These experiments indicated that TFIIH might act as an elongation factor that productively interacts with RNA Pol I. The mutated XPB and XPD subunits of TFIIH, as found in CS cells, reduced the affinity of TFIIH for rDNA and decreased the productivity of the transcription by RNA Pol I, implying qualitative dysfunction of TFIIH in the XPB- and XPD-mutated CS cells. Because the RNA Pol I transcription activity accounts for up to 70% of the total transcription in growing cells, a disturbance in the RNA Pol I transcription action might dysregulate ribosomal biogenesis and hence reduce cell growth. This cellular pathology might explain the growth and neurological retardation observed in CS patients .Nonnekens et al. studied TFIIH and CSB in the RNA Pol I transcription activity in the cells of mouse models of CS and the related progeroid disorder trichothiodystrophy (TTD). Using ChIP analysis, the authors demonstrated that TFIIH binds the entire rDNA coding sequence, supporting the notion that TFIIH is involved in the elongation step of the RNA Pol I transcription activity. The coverage of the rDNA by the polymerase itself is reduced in cells from the CSB mutant and the TFIIH/TTD mice, indicating that CSB and TFIIH are necessary for the recruitment of the enzyme. Moreover, the authors discovered a maturation defect in the multistep process of rRNA cleavage in the TTD cells. This could be confirmed in the brain tissue of TTD mice and suggests that when the transcription process is disturbed, the co-transcriptional maturation and assembly of the ribosomal subunits might also be affected . However, 90% of CS cases are caused by mutations in CSA or CSB. Interestingly, Bradsher et al. detected a co-localization of CSB, TFIIH and RNA Pol I in the nucleolus, indicating the involvement of CSB in RNA Pol I transcription. They confirmed this hypothesis by using an in vitro transcription system, including rDNA template, initiation factors, and RNA Pol I. The addition of purified recombinant CSB enhanced RNA Pol I transcription by approximately 10-times. Furthermore, they detected a reduced rate of new rRNA synthesis in the CSB-mutated CS cells as compared with the same cell line rescued with the wild-type CSB gene. CSB catenates a complex of RNA Pol I and TFIIH that is reduced in the CSB-mutated CS cells as compared with the rescued cell line with the wild-type CSB gene. Moreover, they isolated a complex of CSB, TFIIH and RNA Pol I that contained the CS protein XPG . Yuan et al. investigated the hypothesis that the CSB is an activator of RNA Pol I transcription and is recruited by the transcription terminator factor 1 (TTF1), which is a master regulator of rDNA promoter activity . The CSBSchmitz et al. demonstrated that components of the NER pathway, including the CS proteins, XPG and XPF, are required for de-methylation and activation of the rDNA promoter. In the XPG-Cockayne syndrome cells, the number of active rDNA promoters was severely reduced because of hypermethylation and could be rescued by XPG-expression. An XPG mutant protein lacking endonuclease activity was unable to reduce the hypermethylation and inactivation of the rDNA promoters, indicating that the enzymatic activity of XPG is required for the regulation of rDNA transcription . The rol+ and an increase of nicotinamide. Reassessing this chain of events in C. elegans, the authors demonstrated that inhibition of RNA Pol I transcription activity and stabilization of G4 quadruplex structures lead to accelerated aging and a shortened lifespan that could partially be rescued by a NAD+ precursor. Therefore, premature aging in CS is described as a consequence of a failure in the resolution of secondary DNA structures that are predominantly localized in the rDNA.In a series of knockdown experiments, Scheibye-Knudsen et al. demonstrated that there is a close association between ribosomal transcription by RNA Pol I and mitochondrial function . Loss ofIn considering the cellular consequences of a disturbance in RNA Pol I transcription, as described in the publications above, Alupei et al. investigated ribosomal function and cellular signaling in CS-patient cells in comparison to reconstituted and UVsS cells . The autThe nucleolus and ribosomal transcription deserve greater attention by researchers interested in aging mechanisms. The concept that ribosomal transcription is a process that is unaffected by the functional decline characterizing all cellular and organismal pathways is challenged by the results reviewed in the present manuscript. Whereas, the level of nucleolar activity strongly correlates with the lifespan of model organisms and humans, the quality of transcription by RNA Pol I and the influence on aging is an upcoming issue. The quality of ribosomal performance as a driver of the aging processes was discussed in the early 1960s by L.E. Orgel, who proposed that errors in the translation process would lead to further errors when the ribosomal proteins themselves are affected, a self-perpetuating effect . This “e


Aedes aegypti encodes seven PIWI genes, four of which are somatically expressed. This somatic piRNA pathway generates piRNAs from viral RNA during infection with cytoplasmic RNA viruses through ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Yet, additional insights into the molecular mechanisms mediating non-canonical piRNA production are lacking. TUDOR-domain containing (Tudor) proteins facilitate piRNA biogenesis in Drosophila melanogaster and other model organisms. We thus hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all A. aegypti Tudor genes. Knockdown of the Tudor genes AAEL012437, Vreteno, Yb, SMN and AAEL008101-RB resulted in significantly reduced viral piRNA levels, with AAEL012437-depletion having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from viral RNA.PIWI-interacting RNAs (piRNAs) comprise a class of small RNAs best known for suppressing transposable elements in germline tissues. The vector mosquito  In animals, three distinct small RNA-mediated silencing pathways exist: the micro (mi)RNA, small interfering (si)RNA and PIWI-interacting (pi)RNA pathways . In all Drosophila melanogaster germline, single-stranded precursors are produced from genomic piRNA clusters that contain remnants of transposable elements viruses that cause debilitating diseases such as dengue, chikungunya and Zika  in aedine mosquitoes and cell lines, suggesting that two independent small RNA pathways contribute to antiviral immunity in these insects  are predominantly produced in a ping-pong amplification loop involving the PIWI proteins Ago3 and Piwi5  proteins serve important functions in piRNA biogenesis, including the prevention of non-specific degradation of piRNA substrates, facilitating PIWI protein interactions, and aiding in small RNA loading onto specific PIWI proteins  is largely uncharacterized, we decided to focus our attention on this protein, which we named Veneno (Ven). Ven-depletion dramatically reduces piRNA production from both viral RNA strands, while ping-pong dependent piRNA production from endogenous sources (Ty3-gypsy transposons and histone H4 mRNA) is only mildly affected. Ven resides in cytoplasmic foci, reminiscent of piRNA processing granules in Drosophila and interacts directly with Ago3 through canonical TUDOR domain-mediated sDMA recognition. In addition, Ven associates with orthologs of Drosophila piRNA pathway components Vasa (AAEL004978) and Yb (AAEL001939)  , which iA. aegypti Tudor genes, we combined HHpred homology detection with Jackhmmer iterative searches  at a32P-labeled DNA oligonucleotides. For real-time quantitative polymerase chain reaction (RT-qPCR) analyses, DNaseI-treated RNA was reverse transcribed and PCR amplified in the presence of SYBR green. See For small RNA northern blotting, RNA was size separated on polyacrylamide gels and cross-linked to nylon membranes using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride . Small RTotal RNA from Aag2 cells transfected with dsRNA targeting either Veneno or Firefly Luciferase was used to generate small RNA deep sequencing libraries in parallel. For each condition, three transfections and library preparations were performed. For small RNA-IP libraries, PIWI proteins were immunoprecipitated from SINV-infected Aag2 cell lysates. Purified PIWI protein–piRNA complexes were washed and small RNAs were extracted using proteinase K-treatment. Deep sequencing was performed using Illumina’s Truseq technology, as described in . See SupFluorescent imaging was performed on paraformaldehyde-fixed Aag2 cells that were permeabilized and counterstained using Hoechst solution. Confocal images were taken using an Olympus FV1000 microscope. Images used for the quantification of GFP-signal granularity were taken using a Zeiss Axio Imager Z1 with ApoTome technology. See GFP- and RFP-tagged transgenes were immunoprecipitated using GFP- and RFP-TRAP beads (Chromotek), respectively, according to the manufacturer’s instructions. V5-tagged transgenes were purified using V5-agarose beads (Sigma). For Ago3 and Piwi5 IP experiments, antibodies targeting endogenous proteins were added to lysates at 1:10 dilution and incubated for 4 h at 4°C, followed by overnight binding to Protein A/G PLUS agarose beads (Santa Cruz). Protein extracts were resolved on polyacrylamide gels, blotted to nitrocellulose membranes, and probed with the indicated antibodies. Details on generation of antibodies, experimental procedures, and antibody dilutions can be found in the Supplementary Data.For MS analysis, precipitated proteins were washed extensively and subjected to on-bead trypsin digestion as described previously . SubsequAag2 cell lysate was separated on a 10–45% sucrose gradient by ultracentrifugation. Subsequently, protein was precipitated in acetone and trichloroacetic acid and RNA was extracted using acid phenol/chloroform. For more details, see Supplementary Data.A. aegypti also involves members of this protein family. To faithfully identify all A. aegypti Tudor genes and their corresponding fruit fly orthologs, we used a homology-based prediction approach combining HHPred and Jackhmmer algorithms  lack clear one-to-one orthology with Drosophila counterparts, suggesting that these genes emerged as a result of duplication events that occurred in the Culicidae family (mosquitoes). Conversely, CG15042 and Krimper in D. melanogaster likely resulted from a duplication in the Drosophilidae. Alternatively, the genes without an ortholog may have been lost from the Drosophila lineage, or diversified to an extent that they are no longer recognized as orthologs in the multiple sequence alignment. The AAEL008101 gene encodes two splice variants, of which only AAEL008101-PB is expressed in Aag2 cells , namely AAEL001939, which we refer to as Yb. In cases where there is clear one-to-one orthology, A. aegypti genes will be named after their Drosophila ortholog throughout this study.While the bootstrap values suggest relatively low phylogenetic signal in the TUDOR domains themselves, the majority of g2 cells . Lastly,A. aegypti Tudor proteins along with AAEL004290 in a functional knockdown screen for vpiRNA biogenesis. AAEL004290 is the ortholog of Eggless, a histone methyltransferase involved in the piRNA pathway in D. melanogaster that is predicted to contain TUDOR domains . WeWe have previously shown that biogenesis of genic piRNAs from histone H4 mRNA depends on amplification by Ago3 and Piwi5 . InteresSmall RNA northern blotting is suitable for the detection of only a handful of highly abundant piRNAs. For a more comprehensive analysis of small RNA populations upon Ven knockdown KD), we prepared small RNA deep sequencing libraries from Aag2 cells infected with SINV. Depletion of Ven resulted in a strong reduction of vpiRNA production from both strands (75% and 80% reduction of (+) and (−) strand-derived vpiRNAs, respectively), whereas viral siRNA levels were unaffected Figure  and B. A, we prepThe effect of Ven-KD on piRNA production from transposable elements is relatively minor compared to the changes in vpiRNA production, especially for those derived from the (+) strand (25 and 55% reduction of piRNAs derived from (+) and (−) strand, respectively; Aedes aegypti reference genome assembly AaegL5 . ,29. Aeder Figure , suggestr Figure . Removalr Figure  and F. Ir Figure . Ven conr Figure , which lSimilarly, a glutamine (Q)-rich sequence directly downstream of the MYND-type Zn-finger is not sufficient for Ven-accumulation, as removal of the MYND domain alone (C234) disrupts Ven-body formation. Upon removal of the C-terminal (N670) or both TUDOR domains (N399), the distinct subcellular localization of Ven is largely retained, suggesting that TUDOR domains do not play a major role in granule formation Figure  and H. TA. aegypti  resulting in the strongest depletion of vpiRNAs. Thus far, the direct ortholog of Ven in D. melanogaster (CG9684) has not been studied extensively. In a systematic analysis of all Drosophila Tudor proteins, germline-specific knockdown of CG9684 did not affect steady-state levels of transposon transcripts or female fertility rate   . A. aegyA. aegypti and D. melanogaster, ping-pong dependent piRNA biogenesis depends on Tudor proteins recruiting PIWI proteins to perinuclear granules. Yet, whereas the biogenesis machinery in both insects adheres to the same principles, the mechanisms that assemble the ping-pong amplification complex in these granules differ substantially. In Drosophila, the Tudor protein Krimper interacts directly with both partners in the ping-pong loop (Ago3 and Aub) and recruits them to the nuage  to Ven-bodies. Additionally, whereas sDMA modifications are dispensable for Krimper-Ago3 interaction, Ven binds Ago3 in an sDMA-dependent manner in A. aegypti. Lastly, while the ping-pong amplification machinery is specific to the germline in D. melanogaster, the proteins involved in the multi-protein complex described in this study are expressed ubiquitously in A. aegypti.Both in he nuage ,55. In Ade novo under Ven-KD conditions. This is in contrast to endogenous piRNAs and their precursors , which are pre-existent prior to the establishment of Ven-KD. Hence, de novo vpiRNA production may be especially sensitive to perturbed Ven expression.Knockdown of Ven greatly affects production of piRNAs derived from exogenous viral RNA, while only a modest reduction is seen for genic histone H4 mRNA- and transposon-derived secondary piRNA levels. The apparent stability of histone H4 mRNA-derived piRNAs is especially surprising, as their production has previously been shown to depend on ping-pong amplification involving the PIWI proteins Ago3 and Piwi5 . SimilarA. aegypti  are expressed in somatic tissues (A sophisticated network of accessory proteins that guides diverse RNA substrates into distinct piRISC complexes may be of particular importance in  members ,62, of w tissues . Moreove tissues . Tudor pSmall RNA sequencing data have been deposited in the NCBI sequence read archive under SRA accession SRP127210. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009997.Supplementary DataClick here for additional data file.


The technical variables tested were 1) heating of the serum samples at 60 °C for 120 minutes prior to RNA extraction  Total RNA was extracted from paired non-heated and heated serum samples using a miRCURY RNA Isolation kit for Biofluids (Exiqon) or a SeraMir Exosome RNA Purification kit (Systems Biosciences), and the levels of 384 miRNAs were quantified by RT-qPCR using Human microRNA Array A (Applied Biosystems) or Human Panel I, V4.M (Exiqon). 22.1Single donor human serum samples (n = 4) were purchased from Innovative Research  and divided into two equal-volume aliquots. One aliquot per serum sample was heated at 60 °C for 120 min.2.2Total RNA, including miRNAs, was isolated from the heated and non-heated serum samples using a miRCURY RNA Isolation kit for Biofluids  or a SeraMir Exosome RNA Purification kit . Two hundred μL of serum were used to extract RNA using the miRCURY RNA Isolation kit. MS2 RNA carrier  was added to the lysis solution to enhance RNA isolation efficiency. An additional 250 μL aliquot of the same serum samples was used to extract RNA using a SeraMir Exosome RNA Purification kit.2.3The Exiqon RT-qPCR procedure was similar to that described previously 2.4For the Applied Biosystems RT-qPCR assays, RNA was reverse-transcribed using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and Human Pool A MegaPlex RT primers (Applied Biosystems). The RT product was pre-amplified (12 cycles) with MegaPlex PreAmp primers (Applied Biosystems) and used as a template in the qPCR assays in combination with TaqMan Low Density Human microRNA Array A (Applied Biosystems) and Taqman Universal Master Mix, no AmpErase UNG (Applied Biosystems). The arrays were run in an ABI 7900HT Real-Time PCR System equipped with a TLDA block (Applied Biosystems). The qPCR cycling conditions were 94.5 °C for 10 min and 40 cycles (97 °C for 30 sec and 59.7 °C for 1 min).2.5The amplification and melting curves were analyzed using Sequence Detection Systems (SDS) software, version 2.4.1 (Applied Biosystems). The Ct values of the four biological replicates per experimental condition were averaged prior to calculating the number of miRNAs detected and the distribution of their Ct values.The views expressed in this manuscript do not necessarily reflect those of the U.S. Food and Drug Administration. The mention of any manufacturers or trade names is only for clarity and does not constitute endorsement.


The software successfully annotates such sites and results in no false positives for data with no sequencing or assembly errors.RNA editing sites. This software should be especially useful for researchers generating large numbers of plastome sequences for taxa with high levels of RNA editing.Compared to manual annotation, ReFernment offers greater speed and accuracy for annotating  These relatively small genomes are a major source of data for phylogenetic analyses. As of September 2018, more than 2700 plastome sequences from green plants have been published (Amborella Baill. (Hein et al., https://github.com/TARobison/ReFernment.Although RNA editing appears to occur at a lower rate in angiosperms than in other clades, 138 RNA editing sites were detected in the plastome of ReFernment operates by refining existing annotations. Thus, the software uses an annotation generated by programs such as DOGMA, CpGAVAS, Verdant, or AGORA (Wyman et al., ReFernment operates under the assumption that only U‐to‐C or C‐to‐U RNA editing is occurring in the plastome (Takenaka et al., The utility of ReFernment is simple: it saves users time in the final stages of annotation. Manually accounting for RNA edits generally takes hours for a typical fern or hornwort plastid genome, but with ReFernment, this process takes less than a minute. There are currently efforts to publish some 1000 additional fern plastomes in the coming years, and hopefully similar efforts are underway for hornworts, meaning many thousands of hours can be saved by the implementation of this simple program. ReFernment not only saves the researcher time, but also provides consistent methodology for the annotation of RNA editing. In many cases, RNA editing sites are not annotated in plastid sequences and only conceptual translations are provided. This not only results in confusion in how to annotate such sites consistently, but also makes it difficult for researchers interested in the evolution of such sites to readily identify them.ReFernment offers easy and rapid annotation of RNA‐edited sites and automatic conceptual translation of amino acid sequences, streamlining the process of GenBank submission and saving the user valuable time.