Chaetomium thermophilum Prp2 bound to ADP-BeF3− and a poly-U12 RNA reveals a conserved pre-catalytic helicase conformation and only minor shifts of the C-terminal domains. Differences in the hook-loop and a loop of the helix-bundle domain compared with Prp43 evoke divergent conformations of the bound RNA, ultimately impacting the ability to unwind double-stranded RNA.The crystal structure of  via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3− and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3−- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3′ stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised  Since it has no preformed active site, compositional as well as conformational rearrangements ensure the formation of a catalytically active complex. Once the intron has been excised via two subsequent transesterification reactions, the complex is completely disassembled and each component is available for a new round of splicing.In eukaryotes, most precursor messenger RNAs (pre-mRNAs) contain noncoding intron sequences which need to be removed in order to obtain a mature mRNA that can serve as a template for translation. The vast majority of these intervening sequences are removed with the help of the spliceosome  containing residues 286–921 was recombinantly produced in Escherichia coli Rosetta2 (DE3) cells as a GST-tag fusion protein at 16°C using an autoinduction protocol  glycerol, 2 mM MgCl2. ctPrp43(61–764) mutants and ctPfa1(662–742) (ctPfa1-GP) were recombinantly expressed and purified as specified in Tauchert et al. , a tenfold molar excess of ADP (274 µM), a 20-fold molar excess of BeSO4 (548 µM), a 60-fold molar excess of NaF (1.644 mM) and a 2.5-fold molar excess of U12 ssRNA  was incubated for at least 30 min at 4°C prior to crystallization trials. The complex was crystallized using the sitting-drop vapor-diffusion technique by mixing 1 µl complex solution with 1 µl crystallization buffer. Crystals were grown in 100 mM MOPS/Na HEPES pH 7.5, 8% PEG 20 000, 22% PEG MME 550, 20 mM 1,6-hexanediol, 1-butanol, (RS)-1,2-propanediol, 2-propanol, 1,4-butanediol and 1,3-propanediol. Rod-shaped crystals were obtained after two days of incubation at 20°C.A solution consisting of 2 mg ml2.2.v/v) PEG 400 and 5%(v/v) glycerol and flash-cooled in liquid nitrogen for storage. Oscillation images were collected at 100 K on beamline P14 at PETRA III, DESY, Hamburg, Germany using an oscillation range of 0.1° and an exposure time of 0.01 s per image. Data processing was performed using the XDS package .The structure of ctPrp2 in complex with ADP-BeF2.4.5lta; Tauchert et al., 20177 RNA and the ATP analog ADP-BeF3−. The ATP analog was replaced with ADP and dihydrogen phosphate (DHP). The structure was placed into a simulation box with the shape of a dodecahedron. The box was solvated with 24 798 water molecules for Prp2 and 36 616 water molecules for Prp43. Each system was then neutralized by 13 potassium counter-ions. Interactions of protein and RNA were described with the Amber14SB force field , the combination rule for Lennard–Jones interactions was overwritten with the nonbonded interactions suggested by Panteva et al. (2015k = 1000 kJ mol−1 nm−2).Molecular-dynamics (MD) simulations of the release of phosphate from the ATP-binding pocket were set up as follows. In the simulations, the presented Prp2 structure and the structure of Prp43 . The pressure was controlled at 1 bar with the Parrinello–Rahman barostat  extended for RAMD was taken from https://github.com/HITS-MCM/gromacs-ramd  or/and ctPfa1-GP were conducted with a tenfold and a fivefold molar excess, respectively. All ctPrp43 mutants were used at a concentration of 0.5 µM. The ATP consumption per minute (kobs) was calculated usingA430/Δt is the slope of the NADH decrease, ɛ340 is the extinction coefficient of NADH, d is the optical pathlength and c is the protein concentration.The ATPase activities of various ctPrp43 mutants were tested using a nicotinamide adenine dinucleotide (NADH)-dependent coupled enzymatic assay . ctPrp43 mutants were used at a concentration of 0.25 nM, ctPfa1-GP at 1.25 µM and the 3′-overhang dsRNA  at 500 nM. The excitation wavelength was set to 640 nm and the emission was measured at 685 nm. The helicase reaction speed was calculated by determining the initial slope (20–110 s) of each reaction, which represents the maximum reaction velocity . The intersection of the initial reaction slope with the fluorescence signal of the unwound hairpin RNA was used to determine the time it would take to unwind 500 nmol of dsRNA at this initial maximum reaction velocity. The rate of unwinding of one dsRNA (kobs) was calculated by subsequently accounting for the protein concentration used for each measurement. A detailed description of one example of the calculation of the reaction rate constants is provided in b)Supplementary Fig. S1, helix-bundle (HB) and oligonucleotide-binding (OB) domains . The truncated N-terminal extension is only present as ten amino acids, as it has been proven that the helicase core and the C-terminal domains are the key domains for the ATPase function of the DEAH-box family . For U2, only a model of the sugar-phosphate could be built. The ssRNA binds to an RNA-binding tunnel between the helicase core and the C-terminal domains, as previously reported for the spliceosomal DEAH-box ATPases Prp43 and Prp22  and conserved structural features . These interactions are identical among structurally characterized spliceosomal DEAH-box ATPases and ensure a stack of four RNA nucleotides in the ATP-bound state and a stack of five RNA nucleotides in the adenosine nucleotide-free state . The RNA molecule is not involved in any crystal contacts.Seven of the 12 RNA nucleotides were traceable in the electron-density map . The conserved residues comprising the active site bind ADP-BeF3− in a virtually identical manner to that seen in the ctPrp43–ADP-BeF3− complex . The flexibility of the β-hairpin and motif VI have already been discussed by Schmitt et al. . Due to the rotation of the RecA2 domain between the ATP- and the ADP-bound states, the linker connecting the RecA-like domains also displays large differences in ψ and φ angles. Interestingly, motif III (SAT) also exhibits increased conformational variability, which has not been described before. This motif has been proposed to play a role in coupling ATP hydrolysis to unwinding . Interestingly, in one of the ADP-bound structures  it exhibits a conformation that opens a tunnel connecting the γ-phosphate position of the active site to the surface of the protein . This tunnel could potentially represent an exit passage for the resulting inorganic phosphate after ATP hydrolysis that has not yet been described. All other motif III conformations in the ADP-bound state do not allow the formation of a tunnel that connects the active site to the surface. In all ADP-bound crystal structures His421 of the eponymous DEAH-motif (motif II) interacts via a hydrogen bond with the main-chain N atom of Ala451, but only in ctPrp2–ADP-CF1 does this alanine exhibit a conformation that ensures the start of an α-helix at this position . This alternative conformation displaces Ala451, enabling the formation of the channel. In the ATP-bound state this channel is as well occluded by motif III . Here, His421 of motif II interacts with the side chain of Ser450, and Gln621 of motif VI hydrogen-bonds to the side chain of Thr452 . Additionally, the Ala451 main-chain N atom, which interacts with His421 in the ADP-bound state, is now involved in a hydrogen bond to the relay water of the active site  simulations. Because the dissociation of DHP occurs on long time scales, we accelerated the dissociation using random-accelerated MD simulations . In only four out of 30 simulations, DHP exited Prp2 in the opposite direction via the ATP-binding site . A similar pattern was observed in Prp43, where DHP exited through the suggested channel in 18 out of 25 simulations . Here, four simulations also showed an alternative pathway out of the enzyme similar to that in Supplementary Fig. S7a . These findings suggest that the pathway between motifs I and III exhibits the lowest free-energy barrier, whereas other putative pathways would require larger, energetically more unfavorable structural rearrangements. Indeed, visual inspection of the trajectories showed that minor fluctuations of motifs I and III are sufficient to allow the dissociation of DHP . Taken together, the simulations strongly support release of the γ-phosphate prior to dissociation of ADP in Prp2 and Prp43, and this might also be conserved in other family members.We found that among 30 independent RAMD simulations with a successful dissociation event in the case of Prp2, DHP exited 24 times via a pathway between motifs I and III Fig. 2d. In onHP Fig. 2e. Taken3.3.et al., 2017et al., 2019et al., 2017xH-box ATPase crystal structures . In the Prp2 crystal structure the kink is introduced significantly closer to the 5′ end when compared with the Prp43 or Prp22 structures, whereas it roughly overlaps when directly comparing the Prp43 and Prp22 structures . An analysis of the electrostatic potential of the DEAH-box ATPases shows that Prp2 is significantly more positively charged than Prp43 and Prp22 in the region of the RNA kink . This positively charged patch on Prp2 might contribute to a divergent guiding of the RNA backbone into an alternative 5′ conformation with a shifted kink.In all crystal structures of RNA-bound spliceosomal DEAH-box ATPases the 3′ region of the ssRNA exhibits a stacked conformation that is stabilized by conserved interactions with the conserved sequence motifs of both RecA-like domains . The stacked 3′ RNA region also superposes well, but at the first RNA nucleotide position after the stack the path of the RNA differs. In the ctPrp43 structure a kink in the RNA backbone is introduced at this position, mainly by the stacking of U3 with Pro557 and a hydrogen bond to Ser555 . These two residues are part of a loop in the helix-bundle domain, part of the C-terminal domains (Leu554–Gln558). The corresponding loop in ctPrp2 (Leu747–Thr752) displays an alternative conformation with a more extended α-helix due to an insertion. This conformation is stabilized by a network of polar interactions with surrounding residues . Thr752 of this C-terminal loop hydrogen-bonds to Asn548 from the hook-loop motif of the RecA2 domain (Tyr546–Asn548), which itself interacts with Arg811 from the helix-bundle domain. Arg811 also interacts with Glu749, which is stabilized by a hydrogen bond to Ser808. All of these interactions lock the C-terminal loop in a conformation that is incompatible with an RNA kink as seen in the ctPrp43 structure. Instead, a kink in the RNA backbone is introduced at a position closer to the 5′ end. While the conformation of the stacked 3′ RNA region is highly conserved, the divergent conformations of the 5′ RNA region seem to be highly influenced by the C-terminal loop.Prp43 is the only genuine DEAH-box ATPase with published structures in the ATP- and RNA-bound state . While Prp43 and Prp16 share a highly conserved proline that stacks with a base of the RNA in the ctPrp43–ADP-BeF3−–RNA structure, Prp22 has a conserved glutamine at this position. Prp2 is the only spliceosomal DEAH-box ATPase with an insertion in this loop, contributing to its unique conformation . Interestingly, although an insertion is conserved, the type of insertion differs among higher eukaryotes . In fungal proteins the glutamate and threonine of the C-terminal loop are conserved, while in some fungal members the threonine is replaced by a serine, which should still maintain the same interacting properties with the hook-loop as observed in the ctPrp2–ADP-BeF3−–RNA structure. In representatives from animals the glutamate is not present and two asparagine residues are instead conserved.Sequence alignment of the C-terminal loop reveals that it differs among the four spliceosomal DEAH-box ATPases but is conserved in each individual ATPase among different organisms Fig. 5a. While3−–RNA structure is present in Prp2 from all analyzed organisms and suggests that the interplay between these two structural features is conserved in Prp2 .The asparagine of the hook-loop that interacts with the C-terminal loop in the ctPrp2–ADP-BeFSupplementary Fig. S9). In contrast, the C-terminal loops of ctPrp43 and ctPrp22 show the same length of the α-helix as well as a very similar conformation of the loop itself.The conserved insertion in the C-terminal loop, together with the interaction with the hook-loop, leads to a unique conformation of the C-terminal loop in Prp2, which might play a regulatory role  showed a decreased helicase activity, with a kobs of 0.106 min−1 compared with a kobs of 0.178 min−1 for wild-type ctPrp43 . The exchange of the hook-loop  had a more severe effect on the helicase activity, leading to a fourfold lower helicase activity (kobs = 0.044 min−1) compared with ctPrp43. Most strikingly, when both motifs were mutated (ctPrp43-CL2HL2) no helicase activity could be detected, which strongly supports the idea that the motifs act in concert to regulate helicase activity.In order to test the role of the C-terminal loop in the helicase activity and its interplay with the hook-loop, we mutated these two motifs in Prp43 and analyzed the impact on the helicase activity. Therefore, all measurements were performed using a helicase assay previously established for ctPrp43 using double-stranded RNA with a 3′ overhang, a fivefold molar excess of the G-patch motif of ctPfa1 and 1 m43 Fig. 5c. The eSupplementary Fig. S11). All constructs exhibited a similar stimulation pattern, showing stimulation by the ctPfa1 G-patch motif, which was even stronger in the presence of ssRNA.All ctPrp43 mutants were also tested for ATPase activity in order to verify their functional integrity .Among the spliceosomal DEAH-box ATPases, Prp2 plays a special role as to date no dsRNA-unwinding activity could be determined for this ATPase . It additionally interacts with the relay water of the adenosine nucleotide-binding site . Active-site water molecules are structurally highly conserved and specific to each nucleotide-bound state. In the ADP-bound state four water molecules are always coordinated by the active-site magnesium, whereas in the ATP-bound state it coordinates three water molecules . Due to the fact that these water molecules are specific to a certain catalytic state, a sensor serine in RecA2 motif V is able to discriminate between the ADP-bound and ATP-bound states in order to accordingly position the RecA2 domain. The relay water molecule might also function as an active-site component that is sensed by motif III in order to induce the closed conformation of this motif. Conversely, this conformation might be needed to properly position the relay water for its role during ATP hydrolysis. Interestingly, one ADP-bound Prp2 structure . To date, the exact order of events after the generation of ADP and Pi is still unknown, and it is not clear which is released first. For some DEAD-box helicases it has been shown that Pi is released prior to ADP  are identical in all DEAH-box ATPases . In fact, all of these residues are conserved among DEAH-box, NS3/NPH-II and Ski2-like ATPases. Only in the SF2 helicase subfamily of DEAD-box helicases do the key residues from motifs II and VI differ. Here, Gln621 is replaced by a histidine and His421 of the eponymous DEAH motif is replaced by an aspartate, virtually swapping charges. Interestingly, DEAH-box, NS3/NPH-II and Ski2-like ATPases all possess C-terminal domains which restrict the freedom of movement of the RecA domains in comparison to DEAD-box helicases. This difference in domain dynamics might require divergent ATP-hydrolysis mechanisms, which might be orchestrated by different key residues such as those involved in formation of the exit channel. In order to determine the exact order of events during ATP hydrolysis in DEAH-box ATPases, detailed biochemical analyses, as performed for the DEAD-box helicase Dbp5, could provide further insights . In the Prp43 structure the RNA is guided by interaction with a proline and a serine of a loop protruding out of the C-terminal domains . This interaction ensures the kink in the RNA directly after the β-hairpin. Prp2 lacks such an interaction with the RNA at this position, and instead this loop harbors a conserved insertion that ensures an alternative conformation of this loop, which is stabilized by multiple interactions with neighboring residues . One of these interactions is with Asn548, which is part of the hook-loop . This suggests that the different spliceosomal DEAH-box ATPases have different conserved ways of threading the RNA through the tunnel after the stacked 3′ region. Prp43 mainly utilizes a conserved proline and serine of the C-terminal loop and exhibits short residues in the hook-loop that do not intervene in the binding. This explains why mutating the residues of the Prp43 hook-loop to similarly short glycine residues does not have any influence on the helicase activity . Interestingly, in the structure of RNA- and ADP-BeF3−-bound Prp2, the C-terminal loop displays a distinct conformation due to an insertion. Instead of interacting with the RNA, it interacts with a conserved asparagine of the hook-loop and other surrounding residues . This interaction network is likely to be conserved in Prp2 from different species and threads the 5′ RNA region in a completely different manner compared with Prp43 and Prp22 . Despite this difference in the sequence of the C-terminal loop, the hook-loop is consistently conserved among fungi and animals, suggesting a similar conformation and interaction network in animal Prp2s as seen for C. thermophilum Prp2 as a candidate from the fungi .Although recent structural and biochemical evidence suggest that the spliceosomal DEAH-box ATPases might not need unwinding activity to fulfill their functions during splicing, Prp16, Prp22 and Prp43 nevertheless show 22 Fig. 3. On the es Fig. 4a. In thes Fig. 4c. One oes Fig. 4c. This 22 Fig. 3. Intereset al., 2017kobs in the case of ctPrp43-HL2 . This drastic loss in activity of the hook-loop mutant is particularly interesting, as the complete replacement of this motif (YGT) by glycines (GGG) has been shown to have no effect on helicase activity . The properties of the third position of this motif  is likely to have the most significant effect. While threonine and asparagine share the property of being polar, they differ in size. This position of the hook-loop is the closest to the C-terminal loop and in ctPrp2 the larger size of Asn548 allows contact of both motifs, which is not possible in ctPrp43 due to the smaller size of Thr350 . Thus, in ctPrp43-HL2 the larger asparagine replacing the threonine might interfere with the C-terminal loop or the nearby base which stacks with the C-terminal loop proline, thereby altering the threading of the RNA in this position and leading to an impaired helicase capability of this mutant . This indicates that in Prp43 the C-terminal loop is the primary factor in properly positioning the RNA after the 3′ stacked region by introducing the kinked conformation at the beginning of the 5′ region, and the residues of the hook-loop are kept short in order not to intervene in this function. In contrast, in Prp2 the larger size of the asparagine of the hook-loop and the alternative conformation of the C-terminal loop due to an insertion ensure direct interaction of these two motifs . These interactions seem to lock Prp2 in a conformation in which it is unable to interact with the RNA in this position in a comparable manner as seen in Prp43 and might therefore impede Prp2 from having helicase activity. In fact, a ctPrp43 mutant with both motifs swapped completely loses its ability to unwind dsRNA . Since the individual exchanges only partially impaired the helicase activity of ctPrp43 and both exchanges together completely abolish its unwinding function, it can be assumed that the interplay of both motifs is required and sufficient to effectively impair the duplex-unwinding capability of a DEAH-box ATPase.In order to verify the importance of the C-terminal loop and its potential interplay with the hook-loop, we swapped these motifs from ctPrp43 with the respective motifs from ctPrp2. By using a fluorescence-based dsRNA-unwinding assay that has been previously established for ctPrp43, we were able to analyze the impact of these mutations on the helicase activity . Here, it is feasible that the hook-loop also interacts with the C-terminal loop as seen for Prp2, but due to the different properties of the C-terminal loop, which closely resembles that in Prp43, the interaction might not totally abolish the helicase function but might instead regulate it in a different manner, which was also the case for ctPrp43-HL2 . Prp22, for example, also exhibits rather short hook-loop residues, but instead of a proline the C-terminal loop harbors a considerably larger glutamine, which could enable an interaction with the hook-loop . Such a divergent interplay could also lead to a different regulation of the helicase activity.While Prp2 and Prp43 might represent two extreme examples, in which in one case the interplay between the C-terminal loop and hook-loop is required to impede function and in the other case interplay is avoided in order to guarantee function, it is imaginable that these two motifs regulate the other two spliceosomal DEAH-box ATPases, Prp16 and Prp22, in a different manner. For example, Prp16 has a highly similar C-terminal loop compared with Prp43, but an even larger polar residue (glutamine) in the hook-loop . While all of them are likely to need to maintain the basic ability to translocate, their targets and biological functions are highly diverse and the divergent modes of interaction with the 5′ region might be key to regulating these different tasks. DHX36, for example, unfolds G-quadruplexes at the 5′ end of an ssRNA with the help of its N-terminal extension, which wraps around the protein in order to reach the RNA-binding tunnel at the 5′ region of the ssRNA (Chen et al., 2018et al., 2017et al., 2019All known DE3−, 6zm2Prp2, complex with RNA and ADP-BeFPDB reference: 10.1107/S2059798321001194/ud5019sup1.pdfSupplementary Figures. DOI:


E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′‐O‐methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT‐containing diribonucleotides with native species in RNA hydrolysates by high‐resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT‐specific iodine‐desulfurization, we disprove the existence of PTs in RNA from  For discovery of novel RNA modifications, a stringent workflow for structure validation is necessary to avoid misinterpretation of artifacts. The comparison of the synthetic standard to the native compound's retention time, full mass spectrum, high‐resolution mass spectrum and metabolic isotope labeling allow for confident identification of RNA modifications. All forms of RNA are initially transcribed with four canonical building blocks, with the transcripts then being enzymatically decorated with any of more than 170 chemical modifications that define the epitranscriptome.E. coli, DndA can be replaced by the desulferase IscS,While PT modifications are new for RNA, they have previously been observed in bacterial DNA.6A) undergoes epimerization and various artifacts arise.7G from the mRNA 5′‐m7GpppN cap, which has been exploited for cap analysis in transcripts.7G for analysis, which He and co‐workers exploited to differentiate m7G in caps from the body of mRNA.The discovery of most RNA modifications, including PT in RNA, has been facilitated by sensitive MS analysis. Although this approach is straight‐forward and new modifications are reported on a regular basis,O‐methylated ribose. Our systematic comparison of RNA hydrolysis protocols highlights the central role of the hydrolysis step and structural validation by high‐resolution MS and other methods in RNA modification discovery experiments as well as in absolute quantification of modified nucleosides.Given the large and growing variety of RNA modifications,E. coli K12 and B7A strains, human embryonic kidney cells (HEK 293), and mouse brain tissue for the presence of PT‐containing diribonucleotides. Total RNA was first fractionated by size‐exclusion chromatography following established protocolsMass spectrometric analysis of DNA phosphorothioation depends on the hydrolytic stability of the PT towards several nucleases, including nuclease P1 (NP1). Nuclease treatment releases PT‐linked dinucleotides from the DNA and is exploited to quantify and characterize the dinucleotide context by LC‐MS.3, 4 FE. coli B7A and S. cerevisiae revealed a discrepancy in the retention times of the dinucleotides relative to the synthetic standards for CpsC and GpsG PT dinucleotides  and metabolic isotope labeling. UHPLC‐HRMS analysis with adapted solvent gradient of the RNA isolated from s Figure . The putE. coli B7A and S. cerevisiae and digested to a mixture of ribonucleosides and diribonucleotides suspected to contain PT. The putative PT‐containing diribonucleotides were isolated by preparative HPLC for HRMS analysis. High‐resolution mass spectra were obtained by orbitrap mass spectrometry for both the synthetic PT diribonucleotide standards and the diribonucleotides isolated from biological samples, which revealed a 2 Da discrepancy , while the native GpsG did not. Metabolic stable isotope labeling of all carbon, nitrogen or sulfur atoms was performed in E. coli K12 using minimal medium M9 containing a single source for 34S, 13C and 15N respectively, and the RNA was purified and analyzed as described earlier.‐methyl, a signal at m/z 646 indicated the presence of a methyl group in the diribonucleotide . The mass increase from m/z 643 to 651 (+8 Da) indicates the presence of two 15N4‐labeled guanine bases, which confirms its nature as a canonical phosphate‐linked GG dinucleotide in HEK cells. The presence of two guanine bases is additionally supported by analysis of the 15N‐RNA extracted from E. coli, which is 10 Da heavier than the starting material with the exocyclic amino group labeled with 15N here in addition. From this data, we conclude that there is no evidence for GpsG in E. coli, S. cerevisiae, human cells or mouse brain tissue. In Figure 15N‐labeled signals indicates the presence of only five nitrogen atoms, whereas CpsC has six. We analyzed the corresponding peak from isotope labeled HEK RNA and confirmed the presence of a methyl group and two pyrimidine ribonucleosides . In human and mouse RNA, five signals were found in targeted CpsC MS analysis using the chromatographic system from Figure m/z of 564 and thus the same ≈1 u mass discrepancy as seen in E. coli and peak 4 of HEK cells in Figure S3. Similarly, the signal vanishes in the presence of L‐methionine‐[2H3]‐methyl and a 3 Da heavier signal at m/z 567 appears, which suggests the presence of a methyl group. The MS spectra from all other peaks from targeted CpsC analysis did not show the expected m/z of 565 for unlabeled RNA, while a signal for methylation can be found in all of them .To ascertain the identities of the observed dinucleotides present in RNA from these organisms, total RNA was extracted from both y Figure  and S3A. earlier.21 Humane Figure .[13b] O‐methylated dinucleotides. For the sake of rigor, we tested the presence of PTs in RNA by exploiting the sensitivity of PTs towards iodine oxidation , S. cerevisiae BY4741, and human A549 cells. Following iodine oxidation, the NP1‐hydrolyzed total RNA was analyzed by UPLC‐MS/MS. As shown in Figures E. coli, S. cerevisiae, mice or humans.The results obtained with multiple mass spectrometric approaches convincingly demonstrate that there are no PT‐containing diribonucleotides in RNA from four model organisms, with the most likely identity of the modified species being 2′‐n Figure . This han Figure . The olin Figure . This isn Figure . This apThe results of these studies cast doubt on the identity of the RNA‐derived molecules as PT‐linked dinucleotides, which initiates a process of predicting and proving the true structure. Here we refer to the workflow depicted in Scheme 15N‐labeled RNA by the absence of one 15N.However, one must also consider the possibility that the observed molecule is an artifact caused by adventitious enzymatic or chemical reactions during cell lysis, RNA purification, RNA processing, or even ionization in the mass spectrometer. Such artifacts are best excluded by analysis of stable isotope labeled nucleic acids. For example, aminations, which occur during some RNA hydrolysis protocols, are identified in m/z 643 that was 2 Da lower in mass than the predicted GpsG, which should have had a signal at m/z 645. Considering that the predicted structure has 21 C atoms, the natural abundance of 13C (1.1 %) would produce an M+1 signal (m/z 644) that is 23 % of parent molecular ion (M) intensity and an M+2 signal (m/z 645) that is 2 % of M. The high sensitivity of triple‐quadrupole instruments can lead to a mistaken identification of M+1 or M+2 signals as M. The most immediately practical candidate dinucleotide structures that could account for this 2 Da difference are 2′‐O‐methylated dinucleotides, with 2′‐O‐methylated ribonucleosides occurring abundantly in most forms of RNA.This strategy was applied here with a structural prediction that starts by considering that the MS analysis detected a signal at We tested this prediction in a series of studies that followed the checklist in Scheme O‐methyl dinucleotide hypothesis as a sample preparation artifact: could 2′‐O‐methyl dinucleotides arise from incomplete hydrolysis of RNA? Indeed, more extensive hydrolysis of native RNA with NP1 for longer than 30 minutes decreases the dinucleotide signal. In contrast, dinucleotide signals from synthetic PT RNA are stable even after 3 hours of NP1 hydrolysis , 5‐methyluridine (m5U) and pseudouridine (Ψ) are released in similar abundances  . To understand why protocol 1 was superior to protocol 2, we repeated the experiment using Benzonase+CIP and NP1+CIP in the presence and absence of PDE1. As shown in the first graph of Figure As shown in Figure s Figure . Howevert Figure , the latO‐methylribonucleosides are recalcitrant to release from RNA during hydrolysis, which raises the question of the identity of the PT mimics as 2′‐O‐methylated dinucleotides that arise due to incomplete RNA hydrolysis. We next defined the structure of the PT mimics. To confirm the predicted structure, we synthesized 2′‐O‐methylated dinucleotides CmC, CmU, UmC and GmG  with a molecule with m/z 664  co‐eluting with a molecule with m/z 583  and fragments that differ by <1 ppm (Table S1). Similar results were obtained by co‐injection with stable isotope labeled RNA from HEK cells .These results show that 2′‐4 Figure  and CmU 3 Figure , S6. TheO‐methylated dinucleotides, we compared MS/MS signal intensities associated with mass transitions corresponding to PT and 2′‐O‐methylated dinucleotides. The CmU and GmG dinucleotides isolated from E. coli, S. cerevisiae, and HeLa cells showed a ≈25‐fold increase in abundance when detected with the mass transitions for the 2′‐O‐methylated dinucleotides rather than the PTs . This observation suggests that the signals detected with the PT transitions likely represent low abundance isotopomers of the dinucleotides with a mass that agrees with the mass of the respective PT dinucleotide. These results prove that 2′‐O‐methylated dinucleotides account for the signals described by Wu et al.To further confirm the identity of the 2′‐O‐Methylation is an abundant modification in both ribosomal RNA (rRNA) and tRNA.O‐methylated dinucleotides in different organisms and different types of RNA. Literature precedent provided guidance on established dinucleotide contexts in tRNA and large and small rRNAs, as indicated by circles in Figure E. coli  should be addressed to the authors.Supporting InformationClick here for additional data file.


Alu retrotransposons belong to the class of short interspersed nuclear elements (SINEs). Alu RNA is abundant in cells and its repetitive structure forms double-stranded RNAs (dsRNA) that activate dsRNA sensors and trigger innate immune responses with significant pathological consequences. Mechanisms to prevent innate immune activation include deamination of adenosines to inosines in dsRNAs, referred to as A-to-I editing, degradation of Alu RNAs by endoribonucleases, and sequestration of Alu RNAs by RNA binding proteins. We have previously demonstrated that widespread loss of Alu RNA A-to-I editing is associated with diverse human diseases including viral  and autoimmune diseases (multiple sclerosis). Here we demonstrate loss of A-to-I editing in leukocytes is also associated with inflammatory bowel diseases. Our structure-function analysis demonstrates that ability to activate innate immune responses resides in the left arm of Alu RNA, requires a 5’-PPP, RIG-I is the major Alu dsRNA sensor, and A-to-I editing disrupts both structure and function. Further, edited Alu RNAs inhibit activity of unedited Alu RNAs. Altering Alu RNA nucleotide sequence increases biological activity. Two classes of Alu RNAs exist, one class stimulates both IRF and NF-kB transcriptional activity and a second class only stimulates IRF transcriptional activity. Thus, Alu RNAs play important roles in human disease but may also have therapeutic potential. Alu elements belong to the class of short interspersed nuclear elements (SINEs), are unique to primates, and arose from a head-to-tail fusion of 7SL RNA –3. Over The majority of Alu elements in human genomes are located in introns and 3’ and 5’ untranslated regions (UTR) of protein-coding genes. Thus, Alu elements are transcribed by RNA pol2 as part of a pre-mRNA but are also transcribed by RNA pol3 as part of their normal life cycle. Alu RNAs are also abundant in cells. Because of their repetitive nucleotide sequence, Alu RNAs have the capacity to form double-stranded RNAs (dsRNA) that can stimulate dsRNA sensors, including TLR3, RIG-I, MDA5, resulting in activation of IRF and NF-kB transcriptional paths and induction of IFNs, interferon-stimulated genes (ISGs), NF-kB regulated genes, pro-inflammatory cytokines and other mediators creating something akin to the host ‘anti-viral’ response –19.Eukaryotic cells possess multiple mechanisms to prevent this unwanted activation of dsRNA sensors by endogenous Alu dsRNA thereby preventing potentially dangerous downstream effects , 20–22. We have previously demonstrated reduced levels of A-to-I editing in leukocytes obtained from people with relapsing remitting multiple sclerosis [MS] and concomitant elevation of levels of Alu dsRNAs and ISGs , 34. We Like MS, causes of persistent activation of the immune response in inflammatory bowel diseases are not well understood . In expeStudies involving patients with COVID and severe influenza infection included HC , COV-M , COV-S  and Flu-S  cohorts (GSE149689) and a separate study with HC (N=7) and COV-S (N=5) cohorts (GSE147507). Studies involving patients with multiple sclerosis [MS] included HC (N=8), MS-E , and MS-N  (GSE126427). Studies involving patients with inflammatory bowel diseases and related syndromes included HC (N=8), ulcerative colitis , Crohn’s , Celiac disease  and irritable bowel syndrome  (GSE126427). Leukocytes and lung biopsies were tissue sources for the GSE149689 and GSE147507 studies, respectively. Leukocytes were also tissue sources for MS and IBD studies. Isolated human dendritic cells  or normal human lung epithelial cells (NHBE)  were infected with SARS-CoV-2 in tissue culture. RNA was harvested after 24 hr. and processed for RNA-sequencing –41.We obtained whole genome RNA-sequencing FASTQ files from the Gene Expression Omnibus and employed the following workflow to identify endogenous RNA A-to-I–editing sites from paired FASTQ sequencing files essentially as previously described. The main identification tool was a python-based package called the SPRINT toolkit  that accAlu DNA sequences were from the GrCh37 (hg19) assembly. We designed unedited Alu DNA templates and changed A nucleotides edited in HC but not disease cohorts to G nucleotides as mimics of A-to-I editing. A SP6 promoter was added to the 5’-end and synthetic double stranded DNA templates were obtained from IDT , 45. RNA2 analysis or Fisher’s exact test were used to determine if nonrandom associations existed between two categorical variables. These data were analyzed using GraphPad Prism.False discovery rates (FDR) were determined to correct for multiple testing using the DESeq R package. Unpaired t tests with Welch’s correction were used to determine statistical significance for nonparametric data. The Kruskal-Wallis test or one-way ANOVA was used to compare two or more independent samples of equal or different sizes. Dunn’s multiple comparison test was used to identify means significant from others. The two-way ANOVA was used to compare mean differences between groups segregated on two independent variables. χWe have previously employed the SPRINT software package to analyze differences in levels of A-to-I editing of endogenous Alu RNAs in viral diseases; mild and severe COVID-19 disease in both leukocytes and lung biopsies and severe influenza  in leukocytes , 35, as We used several approaches to examine levels of A-to-I editing in leukocytes obtained from patients with inflammatory bowel diseases; Cr and UC, and from patients with Ce and IBS, compared to HC. To do so, we first identified the total number of common A-to-I editing sites defined as present in either all samples within the HC cohort or all samples in each disease cohort or that were present in all samples in both cohorts. We determined average proportion of edits/reads at each edited site. We found that the total number of common editing sites was reduced in Cr and UC compared to Ce, IBS and HC  . We screr ADARB1 . Thus, lIRAK4, F11R, SLC12A9, CSAD, LONP2, and NICN1, those with reduced editing sites in UC, Cr and FLU-S, but not COV-S and COV-M, MDM4, CSF3R, TMEM154 and ARPC2, and those with reduced editing sites in UC and Cr but not COV-S, COV-M and FLU-S, SPN, SLC35E2, ARSA, UGGT1, IFNAR2, CTSS, MDM2, EMR2, CTSB, USP4, ZNF611, and LOC493754  from double-stranded DNA templates and tested their ability to activate IRF and NF-kB transcriptional responses using THP-1 reporter cell lines containing a stably integrated luciferase gene under the control of either an interferon stimulated response element (ISRE) activated by IRF transcription factors or a NF-kB response element. We first tested an Alu RNA of the AluSg4 class located in the Activation of RIG-I by viral ssRNAs or dsRNAs is reported to require a triphosphate group at the 5’ end of the RNA, 5’-PPP . Therefo). The B2 RNA we selected lacked the ability to stimulate these transcriptional responses. Thus, both human Alu and mouse SINE RNAs are potent stimulators of IRF and NF-kB stimulated transcriptional activity, and activity of the human Alu RNA resides completely in the left arm.We also tested ability of the mouse B1, B2, B3, and B4 RNAs we synthesized to stimulate IRF and NF-kB transcriptional responses. We found that members of the B1, B3, and B4 classes we selected stimulated transcriptional responses that were similar in magnitude to what we observed with the human AluSg4 RNA . For this experiment, we employed amounts of the complete AluJb RNA that induced maximal IRF and NF-kB driven transcriptional responses. We found that the complete Alu5 RNA had no significant effect on IRF driven transcriptional responses stimulated by AluJb RNA but markedly inhibited NF-kB driven transcriptional responses stimulated by AluJb RNA  can be found in the article/The studies involving human participants were reviewed and approved by Vanderbilt University Medical Center Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.Conceptualization, TA. Methodology, TA, JT, and PC. Formal analysis, TA, JT, RH, and KP. Investigation, JT, PC, RH, and KP. Writing: original draft, TA. Writing: review and editing, TA, JT, RH, KP, and PC. Supervision, TA and JT. Project administration, TA. Funding acquisition, TA. All authors contributed to the article and approved the submitted version.Supported by grants from the National Institutes of Health to TMA  and by a grant from the Pfizer Aspire program to TA. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bacillus coagulans and in the nematode Caenorhabditis elegans. We have updated the data for C. elegans satellites using the latest version of the genome. We analyzed the transcription of satellites in both species in available RNA-seq results and found that they are widely transcribed. Our demonstration that satellite RNAs are transcribed adds a new family of non-coding RNAs. This is a field that requires further investigation and will provide a deeper understanding of gene expression and control.It has been shown in recent years that many repeated sequences in the genome are expressed as RNA transcripts, although the role of such RNAs is poorly understood. Some isolated and tandem repeats (satellites) have been found to be transcribed, such as mammalian Alu sequences and telomeric/centromeric satellites in different species. However, there is no detailed study on the eventual transcription of the interspersed satellites found in many species. Therefore, we decided to study for the first time the transcription of the abundant DNA satellites in the bacterium  Drosophila has been recently published [Drosophila have been reviewed by different authors [C. elegans and the bacterium B. coagulans. We have analyzed these satellites in available RNA-seq results [DNA tandem repeats (satellites) are present in most eukaryotic species, but their amount and composition vary significantly, even in closely related species. Centromere and telomere repeats have been studied in great detail . These rublished ; previou authors ,5. In th authors . Transcr authors . A few o authors . However results ,10,11 anC. elegans [We first determined the distribution of satellites and their families in an updated genome sequence of  elegans . We used elegans . A complC. elegans satellite families with the RNA-seq data [C. elegans. For our study, we have chosen two replicates for each tissue, three for neurons, as described in the results section.We have next aligned the consensus repeat of the main seq data ,10, usinseq data . Sequencseq data  have sevB. coagulans we used the same procedure, with the satellite data previously reported [B. coagulans satellite families in For reported  and the reported . We enclC. elegans genome: Fam_2_35_166. This family has 166 satellites distributed throughout the genome, although it is absent in the X chromosome. This absence suggests a specific function for this family of satellites. Its consensus repeat length of 35 nt is: AAtTgAAAATTTCCGGCAAATCGGCAAaTTGCCGg. The satellites in this family have a highly variable length (4–214 repeats), with an average length of 15.4 repeats. From the results shown in We performed our search for the expression of satellites as described in the previous section. The results obtained are presented in We studied in detail the actual sequence of individual spots in the RNA-seq results. A few examples are given in C. elegans satellite families in a selection of RNA-seq experiments. The table has two parts: in the upper half we compare the expression of a single satellite family in different tissues; in the lower half we compare the expression of different satellites in a single neurons_3 library. The sequence of the consensus repeat of all families is given in In We also compared different satellite families, as shown in the lower half of Once we demonstrated that satellites are transcribed as non-coding RNA molecules, we searched the Rfam database  to deterC. elegans in the Rfam database: it has a length of 784 nt, transcribed from chromosome III: 3,633,005–3,635,788. This RNA contains a sequence of four imperfect satellite repeats with a length of 35–43 nt each. This observation shows that the satellite repeat sequence is also found in a modified form in other locations of the genome. It is not clear which is the relation of these imperfect repeats with the satellite RNAs we have described.Non-coding RNA linc-95 is the only related case that has been thoroughly described for B. coagulans is given in In this case, we used the satellite families previously described . An intrB.coagulans satellite families in published RNA-seq results [In  results . The sea results .C. elegans). Most of the spots we have analyzed coincide in sequence with several repeats of a satellite, which demonstrate that satellite DNAs are transcribed as long fragments; they may cover a whole satellite or at least several repeats. Some examples are given in B. coagulans [C. elegans which has many similarities, with the expected differences between bacteria and eukaryotes. We should first note that satellite repeats possess extreme diversity in their length, monomer size, nucleotide sequence, complexity, genomic distribution, and abundance even in closely related species [Caenorhabditis species are a good example; each of them has a unique distribution of abundant satellites [Our results are limited by the short length of the RNA-seq spots (140 nt in oagulans , so that species . The diftellites .C. elegans [In order to find a role for transcribed DNA satellites, we show in  elegans ,22,23. T elegans ,24. Shor elegans . These pAlternatively, whole satellite RNAs may act as a sponge, as described in circular RNAs ,27, trapIt has also been suggested that RNA, along with RNA-binding proteins, might be mediating chromatin organization . Long saOur results demonstrate for the first time that interspersed DNA satellites are transcribed in different tissues. DNA satellites can no longer be considered a useless feature of the genome. They may be transcribed as small RNAs and play a role in RNA interference. Alternatively, they may have a structural role or act as a sponge to trap other RNAs and proteins. To find out the exact mode of action of these non-coding RNAs, further experimental studies are required; new bioinformatics tools have to be developed, given the repetitive nature of satellite RNAs.As noted many years ago by Mattick and collaborators , the gen


Producing a consistent and positive experience for beef consumers is challenging. The gene expression in muscle at harvest may provide insight into better prediction of United States Department of Agriculture (USDA) quality grade. In this pilot study muscle samples were collected at harvest on sixteen steers with a similar background and identical management. Muscle transcripts were sequenced to determine gene expression. Transcripts related to the extracellular matrix, stem cell differentiation, and focal cell adhesions were differentially expressed in muscle tissue from carcasses with differing USDA quality grades. This confirmed the application of this technique to provide insight into muscle development and fat deposition necessary for better prediction and selection to improve consistency and consumer experience.p < 0.05), but not many DE genes were observed between Choice and Select RNA pools. Exploratory analysis of global muscle tissue transcriptome from Standard and Choice carcasses provided insight into muscle biochemistry, specifically the upregulation of extracellular matrix development and focal adhesion pathways and the downregulation of RNA processing and metabolism in Choice versus Standard. Additional research is needed to explore the function and timing of gene expression changes.Fat deposition is important to carcass value and some palatability characteristics. Carcasses with higher USDA quality grades produce more value for producers and processors in the US system and are more likely to have greater eating satisfaction. Using genomics to identify genes impacting marbling deposition provides insight into muscle biochemistry that may lead to ways to better predict fat deposition, especially marbling and thus quality grade. Hereford steers (16) were managed the same from birth through harvest after 270 days on feed. Samples were obtained for tenderness and transcriptome profiling. As expected, steaks from Choice carcasses had a lower shear force value than steaks from Select carcasses; however, steaks from Standard carcasses were not different from steaks from Choice carcasses. A significant number of differentially expressed (DE) genes was observed in the longissimus lumborum between Choice and Standard carcass RNA pools (1257 genes,  Subcutaneous fat thickness and marbling (intramuscular fat) are integral parts of the United States grading systems and are a large portion of the pricing system. Many certification systems, such as Certified Angus Beef™, are utilizing marbling to reduce variation in tenderness. These schemes are being developed because inconsistency of the tenderness in meat is a major concern in the beef industry due to reduced consumer acceptance . Many reSubcutaneous fat is a major factor in the prediction of yield from a carcass, while marbling has been used to try and guarantee tenderness . As timeSixteen red-faced Hereford steers were fed at Fort Keogh Agricultural Research Station under the standard feedlot Standard Operating Procedures (SOP). These animals were genetically similar and were born within a three-week period and handled from birth through harvest as the same group with a common diet and management. All animals received care according to the “Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, 3rd Edition” and under the supervision of the Fort Keogh Animal Care and Use Committee (ACUC No.: 020104-9). Animals within the same cohort that produced carcasses grading Standard, Select, and Choice at the same age and days on feed were sampled for this study. The animals were harvested after all steers had been fed in the same pen for 270 days in the feedlot. Animals were harvested at a federally inspected facility. At harvest, a muscle sample from the longissimus lumborum (LL) near the posterior end was collected using a Tru-cut biopsy needle , snap-frp-values calculated byThe biopsy samples collected for RNA-seq analysis averaged 165 mg of tissue. A Qiagen RNeasy spin-column kit was used for RNA extraction. Extracted RNA samples had an average concentration of 35.88 ng/uL. RNA quality was assessed using an Agilent 2200 Tapestation and RNA Screentape. The RIN (RNA integrity) values averaged 7.06 with a minimum value of 6.7. The extracted RNA was depleted of ribosomal RNA using an Invitrogen Ribominus kit and then used to create individual cDNA libraries that were then randomly allocated to one of two pools for each quality grade of Standard, Select, and Choice. These pools were created to provide a biological replicate sample and to focus our pilot analysis on genes that were differentially expressed by quality grade and not due to individual animal variation. These pools were then sequenced on an Ion Proton next-generation sequencing platform according to the manufacturer’s instructions. The sequencing reads generated were aligned to the known bovine consensus sequence (Btau 4.2), and a normalized count of reads was generated to determine the expression of each known gene and gene isoforms using Strand NGS RNA-seq software . Differentially expressed genes and transcripts were calculated using a pooled z-test with a test statistic ofp-value. Additionally, fold change between Standard and Choice, as well as up- and downregulation, was determined for a Standard vs. Choice comparison. Gene set enrichment analysis and network and pathway analysis were used to identify genes and gene networks that relate to growth rate, tenderness, and carcass quality. Functional analysis was performed using DAVID bioinformatics software  [A Bonferroni correction was used to account for multiple comparisons and determine a corrected MD, USA) . LibraryMD, USA) ,17. RNA-Shear force was determined using previously published methods . Brieflyn = 16; 6—Choice, 5—Select, 5—Standard). The Generalized Linear Model (GLM) procedure of SAS, Statistical Analysis System  was used to analyze carcass and tenderness data. Lease Square Means (LSMEANS) procedures of SAS were employed due to an unequal number of observations upon which to compare differences between variables. Interaction between aging time and treatment was tested. It was not significant, so the data were analyzed as independent variables.Individual animals were used as the experimental unit  was significantly lower for Standard carcasses but was not different between Choice and Select carcasses. Ribeye area (REA) and kidney, pelvic, and heart fat percentage (KPH) were also significantly different between all three grades, with Choice having the largest REA and highest KPH and Standard the lowest . Fat thiThere was no significant interaction between carcass grade and days of aging for shear force. Therefore, main effect means are presented in p < 0.05) in LL muscle tissue from Choice and Standard carcasses and a similar number  between LL muscle tissue from Choice and Select carcasses. There were also a significant number of DE genes  between LL muscle from Select and Standard carcasses. The complete list of all DE genes with p ≤ 0.01 is attached as p ≤ 0.01) either up- or downregulated in Choice versus Standard quality grades is shown in p ≤ 0.01) either up- or downregulated in Choice versus Select quality grades is shown in In this exploratory study, there were a significant number of differentially expressed (DE) genes  enriched KEGG pathways and gene ontology terms, including cellular components, biological processes, and molecular functions, is shown in Functional analysis was run using DAVID (6.8) bioinformatics software, which revealed differences in the underlying pathways regulating muscle cell growth and proliferation. The complete list of enriched pathways and processes is shown in Many researchers have reported that MyoD, myogenin, Myf5, and MURF 4 ,33, alonAn increase in the genes associated with the extracellular matrix, stem cell differentiation, and focal adhesions would suggest there is still muscle growth occurring. The extracellular matrix is an important part of the development of muscle cells. Focal adhesions are needed to anchor the myofibrillar structure to connective tissue in the muscle to help translate the mechanical action. Flück et al.  reportedThe downregulation of amino acid transport and metabolism could indicate that the muscle in the Choice carcass had reached maximum muscle growth and was in the process of moving consumed nutrients into storage as opposed to using them for growth. Energy consumed that is greater than that needed for growth or maintenance will be stored as fat.As expected, steaks from Choice carcasses had a lower shear value than steaks from Select carcasses; however, there were no differences in differentially expressed genes that could be related to tenderness. Upregulated DE genes observed in samples from Standard versus Choice carcass functions were related to transcriptional regulation, inter- and extracellular signaling, growth, muscle metabolism, and angiogenesis, while downregulated DE genes were related to transcriptional regulation, amino acid transport, and metabolism. Enriched processes, including extracellular matrix, stem cell differentiation, and focal adhesions, suggest continued muscle growth in addition to fat deposition in carcasses of a higher quality grade. This explorative analysis of muscle transcript expression along with the findings of others ,36 may i


Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5′-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at  RNA building blocks include not only the four basic A, U, C and G ribonucleosides/ribonucleotides, but >150 chemically altered residues. RNA modifications have been identified in all types of RNA molecules, including transfer RNA (tRNA), ribosomal (rRNA), and messenger (mRNA), as well as various short and long non-coding RNAs (lncRNAs). Many of these modified residues play important roles in RNA metabolism, including RNA structure formation, stability and dynamics, RNA splicing, polyadenylation, transport, localization, and translatability [recent reviews: ]. They cMost of the naturally occurring RNA modifications are introduced post-transcriptionally by various enzymes, and modification reactions form very complex pathways, leading to hypermodified residues . Not allThe progress in high-throughput sequencing and the advances in methods for the discovery of modified residues at the transcriptome scale have led to the development of the new field of research termed ‘epitranscriptomics’ . Moreovercsb.org)  . Additiocsb.org) .Last but not least, both the backend and the frontend of MODOMICS were re-developed. The current version of MODOMICS is based on the Django Python framework v 3.1. The user interface was completely reconstructed, and the Pathways page was re-implemented in javascript, replacing the obsolete Adobe Flash, thus allowing for rendering the visualization in most browsers. The pathways can now also be represented using a network graph layout in addition to the classical hierarchical graph representation. A completely new application programming interface (API) system  allows the users to interact programmatically with the modification data present in the database. This API returns a response based on the user request formatted as plain text, comma-separated values, or JSON format.MODOMICS was expanded to include a new section that provides associations between RNA modifications and human diseases. The main entry point provides a list of observations linking the disturbance in a process related to RNA modification to a pathological condition in humans. Many of such entries include associations of a mutation in human genes encoding RNA modification enzymes with a disease or, more generally, a pathophysiological condition. Cases of dysregulated expression of RNA modification genes were also included. The knowledge of the impact of RNA modifications on human health and disease expands rapidly, and new studies linking RNA modification status to disease conditions, e.g., cancer, appear almost every day. In addition, entries of the human RNA-modifying enzymes list disease conditions associated with a given enzyme malfunction or misregulation. Considering the fast growth of the epitranscriptomic field, this section of MODOMICS does not intend to provide a comprehensive and up-to-date view of all studies that link epitranscriptomics to health conditions. Instead, we aim to highlight the association between RNA modification pathways and disease. MODOMICS indicates the modifications linked to specific disease conditions, rather than broadly focusing on disease and comprehensively listing all kinds of modification disturbance observed. We invite the scientific community to contact us to expand the catalog of epitranscriptomics-disease links in MODOMICS.172 new modified residues were added to the database. Previously, the catalog of modifications in MODOMICS included mostly ribonucleosides and only a few ribonucleotides associated with modifications of the 5′ and 3′ termini, e.g., various caps. For the current release, this catalog was expanded to include nucleotide 5′-monophosphate counterparts of all ribonucleosides. This expansion enabled MODOMICS to be linked to the ligand (LIG) entries in the RCSB Protein Data Bank database and establish a relationship between modified residues in MODOMICS and the experimentally determined RNA structures in the RCSB database that contain modified residues. It must be emphasized that for one nucleoside residue, multiple nucleotide residues can exist that differ by the number and position of phosphates at 5′, 3′ and 2′ positions, e.g., G versus GMP, GDP, GTP, 2′3′-cyclic phosphate, etc. Only some of the possible nucleotide residues are relevant for consideration in MODOMICS. Currently, MODOMICS contains 180 nucleotide residues, 152 nucleoside residues and three bases (modified bases are exclusively in the queuosine pathway).P), topological polar surface area (TPSA), and a number of hydrogen bond donors and acceptors (HBD and HBA) were added. Each chemical structure is now available for download in multiple formats  as a two-dimensional flat structure (2D) or three-dimensional low-energy conformer (3D), generated using Open Babel .The page for an individual residue includes new and updated links to the external resources. The most important new additions are the RCSB Protein Data Bank database , Human Metabolome Database , PubChemMODOMICS now offers a structural comparison of modified residues. Search options include the exact search (to find structures exactly matching the provided structure), the substructure search (to find structures containing the provided fragment), and similarity search (to find structures similar to the provided one). The search structure can be provided by pasting the SMILES code  or by drawing the structure in a molecular editor . The seaThe MODOMICS collection of functionally characterized proteins involved in RNA modification is under constant development. 40 new proteins were added to the previous collection. Among new proteins that were added in this release are SARS Cov-2 NSP proteins involved in the capping of viral RNA  and a coThus far, MODOMICS has been mainly focused on individual modification sites and reactions studied by biochemical approaches. The advances of epitranscriptomics provide new datasets and databases that address the presence of modified residues across different transcriptomes. MODOMICS will continue to be focused on the biochemistry and biochemical pathways of RNA modifications and does not intend to become a primary resource for storing epitranscriptome-wide RNA modification data. On the other hand, MODOMICS will certainly need to store sequence information about experimentally validated sites of modified residues in a much larger group of transcripts. There are multiple projects and databases devoted to mapping various modifications on the transcriptome scale, and the evidence in the field is of variable quality. Hence, for the next release of MODOMICS, we plan to establish links to primary databases storing information about the sites of modifications. In this context, we plan to include the annotation of the individual modification sites, to distinguish information obtained from different primary sources, so the users could identify and select the data only from the sources they trust, e.g., obtained with two independent experimental approaches. Further, we plan to extend information related to the structural context of RNA modifications, to include non-natural modifications of RNA molecules present in experimentally determined 3D structures, update the visualization options, and refurbish the website to keep up with changing trends in web design. We also intend to renew data structures, make MODOMICS more compatible with other databases and web servers, facilitate automated data exchange, and introduce the ability to search sequences with strings that include modification codes.https://iimcb.genesilico.pl/modomics/.The data are accessible freely for research purposes at


Immune checkpoint inhibitors (ICIs) have elicited durable antitumor responses in multiple types of cancers. However, ICIs could also induce potential toxicities that involve all organs, including renal system. In this study, we aimed to conduct a comprehensive description of the ICIs‐induced renal toxicities and the potential effects of chemotherapy.025 and IC025 are, respectively, 95% confidence interval lower end of ROR and IC. If the value of ROR025 exceeding one or IC025 higher than zero, then a signal was considered statistically significant.We conducted a pharmacovigilance study based on US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database between 01 January 2014 and 30 June 2019. Disproportionality analysis was used to assess the association between ICIs and renal adverse events (AEs), including reporting odds ratio (ROR) and information component (IC). RORA total of 30,602,758 reports were extracted from the database, with 4578 reports for ICIs‐associated renal AEs. Renal AEs were more frequently reported in anti‐PD‐1/PD‐L1 versus anti‐CTLA‐4 monotherapy group . Similarly, renal AEs were more commonly reported in ICIs polytherapy other than monotherapy group . Notably, ICIs plus chemotherapy strategies reported more renal toxicities compared to sole ICIs regimens , whereas exhibited lower fatality outcome rates. Importantly, acute kidney injury  and renal failure  were the top two most commonly reported ICIs‐associated renal AEs, and also observed with the top two highest level of fatality outcome rates.A spectrum of renal AEs was detected in ICIs regimens and could be reinforced by ICIs combination. Compared to sole ICIs regimens, ICIs plus chemotherapy strategy reported more renal toxicities but lower fatality outcome rates. With the increasing popularity of ICIs especially combination strategies, it is vital important for clinicians to guarantee balance between durable clinical effects and potential renal toxicities in latest immunotherapy strategies. Our study shows a high reporting frequency of renal AEs induced by ICI monotherapy  and reinforced by combination of ICI regimens. Notably, ICIs plus chemotherapy were more frequently associated with renal toxicities but lower fatality outcome rates compared to sole ICIs regimens. In conclusion, it is vital critical for clinicians to fully recognize ICIs‐associated renal toxicities, seek balance between durable clinical effects and potential fatal toxicities in latest immunotherapy strategies. FAERS database is a free available post‐marketing surveillance database managed by FDA, which contains millions of spontaneous adverse event reports submitted by individual patients, lawyers, physicians, drug companies, hospitals, etc.For each records, variables like age, gender, outcomes, drug names, and preferred term  were extracted from the database. PT is a middle level of Medical Dictionary for Regulatory Activities (MedDRA) (Version 22.0 English),ICIs in our study include anti‐PD‐1 antibodies (nivolumab (opdivo), cemiplimab (libtayo), and pembrolizumab (keytruda)), anti‐PD‐L1 antibodies (atezolizumab (tecentriq), avelumab (bavencio), and durvalumab (imfinzi)), and anti‐CTLA‐4 antibodies (ipilimumab (yervoy) and tremelimumab). To our knowledge, combination with chemotherapy drugs could also affect ICIs clinical effect and toxicities. Thus combined chemotherapy drugs (40 drugs) were also considered in our studies. Both standard and original drug names were used to identify class‐specific ICIs and chemotherapy drugs in FAERS database. Details for these standard and original drug names could be seen in supplementary material Tables .2.2025 and IC025 are, respectively, 95% confidence interval lower end of ROR and IC. The value of ROR025 exceeding one or IC025 exceeding zero was deemed statistically significant to detect a signal. Drug‐ADR combinations with at least three reports were considered in our study. Disproportionality analysis was also used to compare renal toxicities on different regimens: male versus female, younger group (age <60) versus older group (age >=60), anti‐PD‐1/anti‐PD‐L1 versus anti‐CTLA‐4 monotherapy, ICIs monotherapy (anti‐PD‐1/anti‐PD‐L1 and anti‐CTLA‐4) versus ICIs combination (anti‐PD‐1/anti‐PD‐L1 plus anti‐CTLA‐4), and ICIs alone versus ICIs plus chemotherapy. Only ROR was calculated to compare renal toxicities among different regimens. Venn diagram was used to explore cancer types and complications in more frequently reported AEs and provide valuable indications. The overlapping of some areas can show the common characters of different subgroups.Disproportionality analysis was used in our study to assess whether suspected target renal AEs were differentially reported in ICIs compared to other drugs among the full database.In order to get more stable signals, shrinkage transformation model was conducted based on disproportionality analysis. Related statistical formula is as followsNObserved is the number of observed target drug AEs records, NDrug is the number of any target drugs‐associated AEs records, NEvent is the number of target AEs records, and NTotal is the total number of any AEs records in any drugs.https://fis.fda.gov/extensions/FPD‐QDE‐FAERS/FPD‐QDE‐FAERS.html.All analyses were conducted using the software SAS 9.4 (SAS Institute) and R (version 3.4.3), and the results were calculated and checked by two different group members (Fangyuan Hu & Yinghong Zhai). All the data in the analysis can be available at 33.1A total of 30,602,758 reports were extracted from the database (from 01 January 2014 to 30 June 2019), with 4578 reports for ICIs‐associated renal AEs. Baseline of renal AEs for ICIs and control group is presented in Table 3.2025: 1.37) and pembrolizumab+ipilimumab (ROR025: 1.05) group. In ICIs plus chemotherapy strategies, signals were detected in atezolizumab (ROR025: 1.00), ipilimumab+nivolumab (ROR025: 1.29), and durvalumab+tremelimumab (ROR025: 1.71). Renal toxicities were more frequently reported in patients treated with anti‐PD‐1/PD‐L1 versus those treated with anti‐CTLA‐4 (ROR025: 1.52), for those treated with ICIs polytherapy versus ICIs monotherapy (ROR025: 1.10), and for those treated with ICIs plus chemotherapy versus sole ICIs (ROR025: 1.17) strategies  and renal AEs is shown in Table t Tables , other dt Tables .3.3025: 3.19) than nivolumab (ROR025: 1.66) and ipilimumab (ROR025: 1.04) monotherapy group  and renal failure  account 35.02% of all ICIs‐associated renal AEs records, we further explored difference in potential cancer types and complications in death cases for this two AEs.Different cancers were rarely overlapping in both ICIs with or without chemotherapy groups Figures  and S2. 4In addition to durable antitumor responses and remarkable clinical effects in multiple types of cancers, ICIs could induce system toxicities like renal toxicities, and their outcomes had not been systematically characterized. To our knowledge, our study was the first and the biggest study that gives a comprehensive description of the ICIs‐induced renal toxicities. We also took chemotherapy into consideration. Given the increasing administration of ICIs in the recent years, it is vital important to extensively characterize renal toxicities for clinical oncologists. There were mainly five findings observed in our research.First, renal AEs reporting frequency differed between anti‐PD‐1/PD‐L1 and anti‐CTLA‐4 immunotherapies. It seems like renal toxicities were more frequently reported in patients treated with anti‐PD‐1/PD‐L1 versus those treated with anti‐CTLA‐4. In addition, a total of 129 class‐specific signals were significant in anti‐PD‐1/PD‐L1 classes compared to 18 signals in anti‐CTLA‐4. Notably, similar trend was also observed in neurologic toxicitiesSecond, combination of ICIs regimens would reinforce renal toxicities. In our analysis, renal toxicities were more commonly reported in patients treated with ICIs polytherapy than those treated with ICIs monotherapy. Interestingly, similar results were also detected in further class‐specific AEs. For instance, compared to nivolumab and ipilimumab monotherapy, acute kidney injury was more frequently reported in nivolumab+ipilimumab group. Importantly, trend of ICIs combination was observed in some novel clinical trials, and directly observed treatment‐related grade 3 or higher adverse events were more common in combination group than those in monotherapy group.Third, ICIs plus chemotherapy were more frequently associated with renal toxicities compared to sole ICIs regimens. Similar results were also observed in some class‐specific renal AEs like acute kidney injury. Notably, some latest studies indicated that more toxicities were observed in ICIs plus chemotherapy group instead of sole ICIs group, which caters to our findings.Fourth, a spectrum of renal AEs was detected in different ICIs, with the outcomes differed. A total of 201 and 38 signals were retrospectively detected in ICIs with chemotherapy and without chemotherapy groups, with most to be found for the first time. Interestingly, in most renal AEs, a lower fatality outcome rates were observed in patients treated with ICIs regimens without chemotherapy compared to those with chemotherapy, which suggested that compared to ICIs, ICIs plus chemotherapy may bring more remarkable clinical advantages. Similarly, novel trials has proved that ICIs plus chemotherapy has significantly longer overall survival and progression‐free survival versus chemotherapyFifth, acute kidney injury and renal failure were observed to be the top two most frequently reported renal AEs. Importantly, these two AEs were also considered to report the highest level of fatality outcome rate. In further analysis, most overlapping death cases for this two AEs were occurred in NSCLC and malignant melanoma. That is reasonable since increasing latest ICIs strategies were applied in this two cancers.Our study had some limitations. First, this is an observational, retrospective real‐world study and the incidence of these renal AEs cannot be determined. Second, too much missing data were occurred in some important variables like age and gender, thus further analysis was not conducted on these variables. Third, detail demographic variables and clinical information were not included in FAERS database which could help to conduct further clinical evaluations. Fourth, cancer types were not reported to the database, thus class‐specific drug to cancer conclusions were not available. Last but not least, some PTs have similar clinical meanings, which may cause unnecessary confusions, while PT is also considered as a standard level that truly represent the most significant differences.5A spectrum of renal AEs was detected in different ICIs, especially in anti‐PD‐1/PD‐L1 agents and could be reinforced by combination of ICIs regimens. Notably, compared to sole ICIs regimens, ICIs plus chemotherapy were more frequently associated with renal toxicities but lower fatality outcome rates. With the increasing administration of ICIs especially combination strategies, it is vital important for clinicians to recognize ICIs‐associated renal toxicities in latest immunotherapy strategies.The authors declare no potential conflict of interest.Concept and design: Jia He, Fangyuan Hu, and Lei Yuan designed the study. Fangyuan Hu, Xiaofei Ye, Yinghong Zhai, and Lei Yuan controlled the quality of data and performed the statistical analysis. Fangyuan Hu, Yinghong Zhai, Jinhai Sun, Jinfang Xu, and Xiaojing Guo managed and checked all the data. Jia He, Fangyuan Hu, Zhen Lin, and Yinghong Zhai contributed to the manuscript preparation, editing, and review. Fangyuan Hu, Yinghong Zhai, Xiaofei Ye, Lei Yuan, and Jizhou Liang took part in the manuscript revision. All authors read, checked, and approved the final manuscript.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Supplementary MaterialClick here for additional data file.


A simple mRNA vaccine was shown to protect mice against tuberculosis more than 15 years ago. Like COVID-19, tuberculosis is a respiratory infection killing over a million people per year. It too presents a global emergency. Can the stunning success of RNA vaccination against COVID-19 be replicated for TB? The speed of development, deployment and high efficacy of the new RNA vaccines against COVID-19 is stunning!The emerging disease was rapidly recognized as presenting a global health crisis and provided the necessary incentives for this major breakthrough for vaccinology . Early pTuberculosis, like COVID-19, will also kill over a million people this year and, notably, it does so every year , despiteMycobacterium tuberculosis, purified, and the naked in-vitro-transcribed (ivt) mRNA was injected four times into the skin of the mice at 3-week intervals. When challenged with virulent M. tuberculosis infection four weeks later the mice were significantly protected. The protection was less than that obtained with BCG, the only anti-TB vaccine that is currently available for human use. The finding has not been followed up. Perhaps on the basis that development of practical ivt RNA vaccines would be too difficult and expensive, attention shifted to DNA vaccines.The demonstration in 2004 of a protective effect of RNA vaccination against tuberculosis in mice was one of the first proofs of concept for RNA vaccines . In thatThe subsequent research, development and application of RNA (and DNA) vaccines in general has tended to focus upon potentially lucrative targets such as therapies for allergies and cancers. Nevertheless, some progress has been made towards protective RNA vaccines against some other, mainly viral, infectious diseases , and somM. tuberculosis has not been discounted; current understanding of the requirements for generating protective immunity is limited [in vivo stability and antigen production. The approach is otherwise similar to DNA vaccinology; templates for multiple antigens can be used and it avoids the complications introduced by vectors such as whole bacteria or viruses that contain components that inhibit the required immune responses. Currently, mRNA is expensive to make, but manufacturing costs will surely fall in response to demand. Furthermore, pure mRNA is inherently highly stable, for example when freeze-dried in a sealed vial, so future vaccines may not require a cold chain for delivery if adequate heat-stable adjuvanting can be accomplished [The view that adequate protection against bacterial infections such as TB will require delivery of a complexity of diverse antigens by virulence-attenuated vectors such as live BCG or attenuated  limited  and mult limited , and mus limited ,12 and a limited ,10. The  limited ,12, thenmplished . Multipl