This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a  N6-methyladenosine (m6A) mRNA methylation in development , which rely on chemical, enzymatic, and/or antibody-based detection of modified ribonucleosides , 2, 3. Telopment , 6, 7, 8neration , 14, 15,hylation , 10. Furhylation , 18, 19.hylation , make itTandem mass spectrometry (LC-MS/MS) is a highly accurate tool for analysis of modified RNAs, which has so far been applied primarily in two approaches to identify and quantify RNA modifications . The fir13C-methyl]-methionine labeling and mass spectrometry (MS) to assess the turnover of base modifications (13C-dynamods) in newly transcribed RNA. With 13C-dynamods, we trace the proportion of newly methylated ribonucleosides and their decay through time, i.e., methylation turnover, from digested polyadenylated RNA and ncRNA. We first showed that polyadenylated RNA and ncRNA were distinguished by the different turnover frequencies (in h−1) of modified ribonucleosides, which are inherently linked to the different half-lives of mRNA, rRNA, and tRNA . We then applied 13C-dynamods in conjugation with abundance measurements of modified ribonucleosides in polyA+ and ncRNA, which resolved their transcriptional versus posttranscriptional sensitivity in response to actinomycin D and metabolic stresses. Thus, the quantitative nature of 13C-dynamods demonstrates its capacity for sensitive characterization of modified ribonucleosides, their origin and dynamics, in multiple RNA classes of interest.Stable isotope labeling is a well-established method to quantify metabolic activity in cultured cells , 42, 43.and tRNA , 44, 45.13C-methyl]-methionine and analyzed the isotopologues  of modified and unmodified ribonucleosides by tandem mass spectrometry (LC-MS/MS) (A and B). The m + 0 isotopologue  represents the mass of the analyzed molecule where all atoms are present as the most common isotope, whereas the m + 1 isotopologue  indicates the mass shift due to 13C incorporation from the 13C-labeled methionine tracer or from the natural abundance of 13C, 15N, 18O, and 2H stable isotopes. With 13C-dynamods, we measure the isotopologue fractions [m + 1/(m + 1 + m + 0)] of modified and unmodified ribonucleosides, which reflect the amount of 13C enrichment (m + 1) in the measured ribonucleoside relative to the total pool of that ribonucleoside (m + 1 + m + 0) (A). Thus, the isotopologue fractions of each ribonucleoside during [13C-methyl]-methionine labeling are internally controlled for the amount of preexisting ribonucleosides  prior to 13C labeling. Here, we analyzed the isotopologues of N6-methyladenosine (m6A), 7-methylguanosine (m7G), 1-methyladenosine (m1A), N6,N6-dimethyladenosine (m62A), 2′-O-methyladensone (Am), 5-methylcytosine (m5C), and unmodified ribonucleosides from digested polyadenylated (polyA+), large (>200 nt) and small (<200 nt) RNA. We observed increased m + 1 and concomitant decreased m + 0 ion counts in modified ribonucleosides from polyA+ and large RNA after 4 and 24 h of [13C-methyl]-methionine labeling, while the m + 1 fractions  of the unmodified ribonucleosides was unaltered during the labeling period . Thus, the change in the “heavy” (m + 1) isotopologue fraction relative to the unlabeled condition  indicates SAM-dependent RNA methylation of newly synthesized RNA, whose turnover we have examined within and across RNA classes. In contrast to singly methylated ribonucleosides, the m62A modification exhibited an enrichment mostly of the m + 2 isotopologue upon 13C labeling (A), and so the m + 2 fraction was used to assess m62A dynamics in subsequent analyses.S-adenosylmethionine (SAM) is the direct substrate of RNA methylation reactions in eukaryotic cells . To tracC-MS/MS) , A and B+ m + 0) A. Thus, ereafter B. Thus, labeling A, and so6A, m7G, and m5C in polyA+-purified RNA when compared with large (>200 nt) and small (<200 nt) RNA (E), in accordance with the faster life cycle of mRNA in mammalian cells , suggesting that a large portion of the signal for these modifications in the polyA+ fraction might derive from contaminating ribosomal RNA (rRNA). Of note, the isotopologues are analyzed on all ribonucleosides from the same polyA+ pool (C), so the purity of polyA+ enrichment is the same in all cases. This indicates that the relative abundance in mRNAs versus contaminating ncRNA is much higher for m6A, m7G, and m5C compared with m1A, m62A, and Am. Interestingly, we detected m62A in small RNAs from 786O cells, and its kinetics was different when compared with large RNA (E).Quantification of isotopologue fractions showed a faster kinetics for m nt) RNA E, in accan cells . In contractions E, suggesyA+ pool C, so thearge RNA E.13C-methyl]-methionine followed by replacement (chase) with unlabeled methionine and analyzed the isotopologues over time. In accordance with the preceding findings (E), we observed an exponential decay of the m + 1 isotopologue fraction for m6A/m7G but not for Am/m1A/m62A in polyA+ RNA (A). Conversely, the modifications of total/large RNAs exhibited uniformly slow turnover (B), consistent with the high stability of rRNA and tRNA in growing cells  = m(0) + kt) fits well the turnover of m1A/m62A/Am but not m6A/m7G in polyA+ RNA, in which the “U-shaped” curve supports a nonlinear model (m(t) = m(0) e(−kt)) (C and S2A). Conversely, the linear regression fitted well the turnover of ncRNA modifications (B and C). The turnover frequency determined for Am/m1A/m62A in polyA+ was similar to ncRNA modifications (k = 0.031 h−1 on average) and significantly slower than m6A (k = 0.244 h−1) or m7G (k = 0.089 h−1) in polyA+ RNA (A and B). Thus, the turnover of m1A/m62A/Am from polyA+ fractions is incompatible with the exponential turnover of mRNA modifications ) C and S2Aications , B and ClyA+ RNA , A and BA k = 0.2 h−1 or mications , confirm7G was in between that of m62A and m1A/Am in polyA+ RNA (D), and the sensitivity of MS detection was similar across all measured modifications except m5C, as determined from equimolar injections of pure compounds (D). Thus, the distinct turnover of RNA modifications in the polyA+-purified fraction was not explained by differences in normalized abundances alone or analytical sensitivity of ribonucleoside detection. Instead, the modified ribonucleosides were more abundant in large RNA relative to polyA+ RNA by a factor of 17/37/97/9 for Am/m1A/m62A/m7G, respectively, while m6A level was greater in polyA+ RNA by a factor of 2 (E). From the determined turnover frequencies (h−1) of ncRNA (B), we estimated that a 50% contribution of ncRNA signal to the ribonucleoside pool in polyA+-purified RNA would lower the detected turnover of the bona fide m6A mRNA modification, with a turnover frequency of 0.242 h−1, to that of m7G, with a turnover frequency of ∼0.09 h−1 (E). That is, reliable turnover-based detection of a hypothetical mRNA modification that is more abundant by a factor of 17 to 97 in ncRNA relative to mRNA is attainable with a maximal ncRNA contamination of 0.5% (50%/97) to 2.9% (50%/17), but even an optimal performance of available methods to purify polyA+ RNA commonly contains 2 to 3% of contaminating ncRNA (F)  of mlyA+ RNA D, and thompounds D. Thus, tor of 2 E. From tof ncRNA B, we est0.09 h−1 E. That ing ncRNA F , we reasoned that free ribonucleosides derived from metabolic extracts would be derived mainly from endogenous degradation of mRNAs, rather than ncRNAs. Thus, we employed 13C-dynamods to examine the methylation turnover of free ribonucleosides, thereby enriching for mRNA-derived ribonucleosides and decreasing interference from rRNA-derived ribonucleosides. We found a fast kinetics following 13C labeling that was similar for m6A, m7G, and Am, indicating that they are primarily derived from the same RNA type  (F–H). Interestingly, the m62A modification also exhibited a nonlinear, faster kinetics than m1A or 1-methylguanosine (m1G) (F–H), which are known tRNA modifications and were present in the metabolic extracts at high levels (G) , which compromises the quantification under conditions of partial 13C labeling. Thus, while the turnover analysis of the polyA+ fraction could not be used to validate lowly abundant modifications due to rRNA contamination (E), the turnover of Am and m62A in the free pool suggests that these modifications indeed partly derive from rapidly decaying RNAs (F), which may include non-ribosomal RNAs. This was supported by the normalized levels of Am and m62A in the free pool being more similar to those of polyA+ than rRNA (H). These results confirm the presence of Am in mRNA  , F–H. Inne (m1G) , F–H, whh levels G 47). W. W6A wasmination E, the tuing RNAs F, which han rRNA H. These  in mRNA  and suggin mRNAs , 50.6A, N6,2-O-dimethyladenosine (m6Am), and m1A (versus ncRNA to metabolic stresses linked to RNA (de)methylation. First, we confirmed that actinomycin (ActD), a pan inhibitor of eukaryotic transcription, completely inhibited the methylation turnover of polyA+ and large RNA (A), in accordance with the cotranscriptional deposition of mRNA and most rRNA modifications (6A (m6A/A) in polyA+ RNA (B), but not of m7G, which supports the role of m6A in global mRNA destabilization, as initially reported (C and D). In contrast, the abundance of modified ribonucleosides in large RNA was unaltered under these forms of stress (E), whereas glutamine deprivation led to increased m6A levels in polyA+ RNA (F), which was in contrast to ActD treatment (B). Thus, the reduced methylation turnover of large RNA simply results from an inhibited transcription of the rRNAs themselves, while the altered abundance of m6A without significant changes in its turnover indicates its posttranscriptional lability to stress conditions via altered (i) decay of m6A-enriched versus m6A-depleted mRNAs or (ii) dynamics of the m6A modification itself. These data show that measurements of methylation turnover and ribonucleoside abundances resolve transcriptional versus posttranscriptional effects on RNA modifications in nonstationary (stimulus-dependent) experiments.The maintenance of methylation reactions requires SAM and serine/glycine, which feed one-carbon metabolism and SAM synthesis . Convers and m1A , 53. We arge RNA A, in accications , 56, 57.lyA+ RNA B, but noreported . Deprivareported , C and Df stress E, wherealyA+ RNA F, which reatment B. Thus, 13C labeling informs on the distinct dynamics of polyA+ and ncRNA modifications and their sensitivity to metabolic perturbations of mammalian cells. 13C-dynamods presents several advances in the application of MS approaches to study RNA turnover and dynamics. First, in contrast to approaches that rely on prior/in-parallel labeling with 15N/13C-enriched nucleosides to distinguish between preexisting and newly modified RNA  (B)   of ribonucleotide (NTP) synthesis  , 43 of v(in h−1) B (43). Manalogs) , 59 or tynthesis , 63, 64.ynthesis , 66. Als13C incorporation into newly methylated RNA does not assess nonmodified RNAs and hence does not capture the removal of RNA modifications. That is, any RNA demethylation activity can only affect the abundances of modified ribonucleosides as a whole (sum of m + 1 and m + 1), not the m + 1 or m + 0 isotopologues individually, which would be required to cause a differential m + 1 fraction. Since RNA transcription and decay rates are constant at metabolic steady state, we propose that the methylation turnover is an accurate readout of RNA turnover, which allowed us to assess the contributions of short-lived (mRNAs) and long-lived (noncoding) RNAs to a pool of modified RNAs and free ribonucleosides. Indeed, this information could not be drawn if labeled ribonucleoside themselves or its precursors were used to trace ribonucleotide synthesis  or transcription. Likewise, the source RNA cannot be pinpointed if the abundance (levels) of modified ribonucleosides is measured in isolation.Reliable study of mRNA modifications by MS analyses of ribonucleosides is challenging due to the high abundance of heavily modified ncRNAs that invariably contaminate the polyA+ fraction , 37. To 1A/m62A) in polyA+-purified RNA as being derived from mRNAs. Based on methylation turnover, we estimated that these modifications in the polyA+ fraction could be detected with a maximal ncRNA contamination of 0.5 to 2.9%, since Am/m1A/m62A levels were found higher in ncRNAs by a factor of 17–37–97, respectively. In contrast, a ∼6% of ncRNA contamination would be required to fully account for the slower m7G turnover detected in polyA+ RNA, since m7G levels were found only approximately nine times higher in ncRNAs. Since the m7G turnover reflected that of a bona fide mRNA modification, its slower kinetics in polyA+-purified RNA suggests a temporal delay relative to m6A turnover. A further evidence for the distinct turnover of m7G relative to m6A in mRNAs is the response to metabolic stresses, where the m7G turnover changed in large RNA but not polyA+ RNA following 4 h of 13C labeling (C and D). In this respect, while m6A is cotranscriptionally deposited in mRNA    and largin vitro , but we e.g., a stress condition, may affect the afferent  or efferent  turnover parts of methylated RNAs. Combined with abundance measurements, this allowed us to assess transcriptional versus nontranscriptional sensitivity of modified RNAs to stress conditions. This was evidenced by the reduced methylation turnover of ncRNAs following serine deprivation and ActD treatment, wherein the abundance of modifications was unaltered in the former  but m6A levels decreased in the latter . The observed inhibited turnover of methylated ncRNAs under serine deprivation is in line with the expected inhibition of mTOR activity and thereby of rRNA biogenesis (6A levels in polyA+-purified RNA without significantly changing (not increasing) its turnover. These data suggest that m6A is posttranscriptionally sensitive to glutamine levels through either (i) an altered decay of m6A-enriched versus m6A-depleted RNAs or (ii) inhibition of m6A demethylation itself. The demethylation of m6A is a stepwise conversion into adenosine through the formation of N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) intermediates  and preexisting methylated (m + 0 fraction) RNAs differently are exposed differently to RNA degradation, e.g., through nuclear/cytosolic compartmentalization.A nonstationary condition, ogenesis . In contmediates , whose pthylases , 53, it ependent . With exOur study demonstrates how quantification of methylation turnover and abundance can be used to examine the presence of RNA modifications in RNA classes, their temporal dynamics, and sensitivity to stress conditions. These insights open new directions to be further explored by MS and orthogonal approaches to obtain information on particular RNAs.2 in high-glucose DMEM medium  supplemented with 10% fetal bovine serum (FBS) . For 13C labeling experiments, 786O cells were grown in high-glucose DMEM medium without glutamine, methionine, and cystine  supplemented with 10% dialyzed FBS  to minimize contributions of unlabeled amino acids typically present in regular FBS, 2 mM glutamine, 0.1 mM cystine, and 0.2 mM [13C-methyl]-methionine (CK Isotopes Limited). Cells were maintained at 50 to 60% confluence (or 30–40% in chase experiments) and washed with PBS before switching to 13C-labeled medium (or regular medium in chase) for the indicated time periods.786O cells were obtained from the Crick Cell Services and cultured at 37 °C with 5% COvia two rounds of polyA tail hybridization with Oligo-dT magnetic Dynabeads .At the conclusion of metabolic labeling, the medium was aspirated and extraction of total RNA was performed with the mirVana isolation kit according to manufacturer’s instructions . Large (>200 nt) RNAs were purified by adding 1/3 volume of 100% ethanol to the aqueous phase recovered from the organic extraction before loading into the filter cartridge of the mirVana Kit. Small (<200 nt) RNAs were purified by collecting the total filtrate, addition of 2/3 volume of 100% ethanol, and loading into the filter cartridge. Polyadenylated RNA was purified from total/large RNA 2, and 10 mM NaCH3COO pH 5.3 and incubated for 2 h at 37 °C. Subsequently, NH4HCO3 (100 mM) and 5 units of alkaline phosphatase (CIP)  were added and the sample incubated for 2 h  at 37 °C. Formic acid was added at 0.1% v/v in a final volume of 50 μl, samples were filtered  and 15 to 20 μl analyzed in duplicate by LC-MS. Ribonucleosides were resolved with a C18 reverse-phase column  and eluted with a gradient of 0.1% v/v formic acid (solvent A) and 80% acetonitrile in 0.1% formic acid (solvent B) at a flow rate of 0.2 ml/min and 40 °C: 100% solvent A for 3 min, 12% solvent B for 12 min, and 100% solvent B for 2 min after which the column was re-equilibrated with 100% solvent A for 3 min . The ribonucleoside separation was performed using U3000 HPLC (Thermo Scientific) and the detection by a TSQ Quantiva Triple Quadrupole mass spectrometer  controlled by the Xcalibur software version 4.0.27 (Thermo Scientific). The HPLC was coupled to the TSQ Quantiva using a HESI (heated electrospray) ion source (Thermo Scientific) operating in positive ionization mode with the following parameters: capillary voltage, 3500 V; sheath gas flow, 7.35 l/min; gas temperature, 325C. The first and third quadrupoles (Q1 and Q3) were stringently fixed to 0.2 units of resolution and set to detect the mass of the precursor ribonucleoside ion (Q1) and of the base and ribose product ions (Q3). The ribonucleosides were identified by comparison of the retention time and detected mass transitions to commercially available standards. The collision energies were experimentally defined based on the fragmentation pattern of each ribonucleoside standard and chosen based on the maximum intensity of the base product; the ribose ring was used only as a qualifying transition. The retention time, mass transitions (m/z), and collision energies of each ribonucleoside were: adenosine, ∼4.1 min, 268.1 -> 136.1 m/z, 20 V; guanosine, ∼5.4 min, 284.1 -> 135.0 m/z, 35.5 V; cytidine, ∼1.4 min, 244.2 -> 112.05 m/z, 12 V; m6A, ∼8.7 min, 282.1 -> 150.1 m/z, 20 V; m1A, ∼1.8 min, 282.1 -> 150.1 m/z, 20 V, m7G, ∼2.3 min, 298.05 -> 166.1 m/z, 20 V; m5C, ∼1.8 min, 258.2 -> 126.1, m/z, 13 V; m62A, ∼11.9 min, 296.2 -> 164.1 m/z, 22 V. Each mass transition above corresponded to the m + 0 isotopologue and increased by one (m + 1), two (m + 2), and three (m + 3) units for detection of the other isotopologues, e.g., m62A: 297.2 -> 165.1 (m + 1), 298.2 -> 166.1 (m + 2), 299.2 -> 167.1 (m + 3). The dwell time for each transition was 30 ms for a duty cycle of 930 ms (31 transitions), and 8 to 20 data points per chromatographic peak were obtained for “short” and “long” peaks, respectively. A mix of ribonucleoside standards containing 0.5, 1, 5, 10, 50, 100, 500 fmol, 1, 5, 10, 50, or 100 pmol of each ribonucleoside was run in parallel after the biological samples for absolute quantifications, a subset of which is shown in Fig. S2D. Data were recorded using the Xcalibur 3.0.63 software (Thermo Fisher Scientific) and analyzed using Skyline (version 19.1) (Purified RNA (100–250 ng) was digested into ribonucleosides using one unit of nuclease P1  in 25 μl of buffer 25 mM NaCl, 2.5 mM ZnClon 19.1)  (Supplemon 19.1) .13C-methyl]-methionine, metabolic activity quenched by adding ice-cold PBS. Metabolites were extracted by addition of 600 μl ice-cold 1:1 (vol/vol) methanol/water to the cell pellets, samples were transferred to a chilled microcentrifuge tube containing 300 μl chloroform and 600 μl methanol . Samples were sonicated in a water bath for 8 min at 4 °C and centrifuged  for 10 min at 4 °C. The supernatant containing the extract was transferred to a new tube for evaporation in a speed-vacuum centrifuge, resuspended in 3:3:1 (vol/vol/vol) methanol/water/chloroform  to phase separate polar metabolites (upper aqueous phase) from nonpolar metabolites (lower organic phase), and centrifuged. The aqueous phase was transferred to a new tube for evaporation in a speed-vacuum centrifuge and resuspended in 100 μl water for LC-MS acquisition. LC-MS analysis was performed using a Dionex UltiMate LC system (Thermo Fisher Scientific) with a ZIC-pHILIC column , as described previously (m/z using a Q Exactive Orbitrap instrument (Thermo Fisher Scientific) with heated electrospray ionization and polarity switching mode at a resolution of 70,000 (at 200 m/z). MS parameters were as follows: spray voltage 3.5 kV for positive mode and 3.2 kV for negative mode; probe temperature, 320 °C; sheath gas, 30 arbitrary units; auxiliary gas, five arbitrary units. Parallel reaction monitoring (PRM) was used at a resolution of 17,500 to confirm the identification of metabolites; collision energies were set individually in HCD  mode. Data were recorded using the Xcalibur 3.0.63 software and analyzed using Tracefinder 4.1 (ThermoFisher Scientific) according to the manufacturer’s workflows.At the end of cell culture with [eviously . A 15 mi62A) relative to the total ion counts of the m + 0 plus m + 1 isotopologues. The kinetics of isotopologue fractions and goodness of fit were determined using the Curve Fitting toolbox of Matlab R2020a (MathWorks) either by a linear regression [f(x) = p1∗x + p2] or an exponential fit: a one-term function in the chase experiments , and a two-term function to fit the isotopologue fractions of free ribonucleosides .The isotopologue fractions were defined as the total ion counts of the m + 1 isotopologue (except for m + 2 in mThe mass spectrometry.raw data and Skyline files pertaining This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3′-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5′-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3′-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3′-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3′-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an  Caliciviridae family. It is a major cause of acute gastroenteritis in humans. Its genome is approximately 7.5 knt long and typically encodes three open reading frames (ORFs). The 5′ untranslated region (UTR) (4–5 nt long) exists upstream of the ORF1. ORF1 encodes the viral nonstructural viral proteins , while ORF2 and ORF3 encode the major (VP1) and minor structural protein (VP2), respectively. A 3′ UTR (30–60 nt in length) exists downstream of the ORF3 and polyadenylated at its 3′ end halted (A) .Fig.FigCalicCaliciviridae  is also synthesized in all members of the in vitro or cell-based systems has been reported  RdRp was demonstrated for the first time in an in vitro . After the template RNA was incubated with the RdRp, RNA complementary to the template RNA was synthesized, forming a double-stranded RNA (dsRNA) with the template RNA. The dsRNA then remained after incubation with an S1 ribonuclease, to digest the single-stranded RNA into small nucleotides. Subsequently, the synthesis of the dsRNA is then used as an index that the RdRp synthesizes the complementary RNA in the following experiments.We used a nonradioactive labeling method to detect RNA synthesis by RdRp Escherichia coli cells, purified, and analyzed by SDS-PAGE. The mobility of the major band was calculated as 57 kDa according to the molecular weight markers , and the band was detected by anti-RdRp and anti-His antibodies . Finally, the purity of the RdRp was calculated as 96% .The HuNV GII. P3 RdRp was tagged with six histidines at its N-terminus, expressed in A). All the template RNAs used here carry GGG and CCC sequences at their 5′- and 3′-ends, respectively, to efficiently synthesize template RNAs in vitro and form stable dsRNAs. When increasing amounts of RdRp were incubated with 100S RNA, almost no RNA was synthesized . However, when increasing amounts of RdRp were incubated with 100AS RNA, dsRNA was synthesized in increasing amounts . To confirm that these synthesized RNAs were dsRNAs, the RNAs were treated with S1 ribonucleases. The amounts of the synthesized dsRNA were then increased according to the amounts of the RdRp . The efficient activity of S1 ribonucleases was confirmed by the fact that the marker ssRNAs (100S and 100AS) were digested well, but the dsRNA (ds100) markers were resisted with S1 ribonuclease treatment . These results therefore indicate that the RdRp specifically recognizes the 3′-end of the antisense viral genome RNA and indicate also that this region is important to initiate the synthesis of the positive-strand RNA by the RdRp.The 5′ terminal nt 1 to 100 of viral genomic RNA of the HuNV GII. P3 strain (100S [sense]) or its complementary sequence (100AS [antisense]) was then used as template RNAs see A. All thA). The sequence of nt 74 to 100 was highly conserved among the norovirus genogroups I, II, IV, and VII (B). To determine whether this stem-loop (nt 74–87) was important for initiating RNA synthesis by the RdRp, a deletion mutant of 100AS RNA that lacks the 31 nt (nt 70–100) at the 3′-terminus of the 100AS RNA was constructed . Its complementary template RNA was also constructed as a control (designated as Δ31S RNA). When Δ31AS RNA and Δ31S RNA were used as template RNAs, the RdRp did not synthesize the complementary RNA from either template RNAs . To remove the single-stranded template RNA to confirm the synthesis of the dsRNA, the reaction products shown on the upper column in Figure 4A were treated with S1 ribonucleases. Absent or faint RNAs were detected . These results therefore indicate that nt 70 to 100 of 100AS RNA containing the predicted stem-loop structure is necessary for initiating the complementary positive-strand genomic RNA synthesis by the RdRp.We examined the secondary structure in the 3′-end regions of the antisense genomic RNA of HuNV GII.P3 strain. We found that nt 74 to 87 (located at the 3′-terminus) in the 100AS RNA forms a predicted stem-loop structure see A. The se and VII B. To detB). These results therefore confirmed the importance of the 31 nt region at the 3′-end of antisense genomic RNA for the RdRp activity.To confirm that the 31 nt RNA, carrying the nt 70 to 100 of 100AS RNA . The result showed that the RdRp synthesized dsRNAs derived from Δ6AS and Δ9AS, respectively, to the same extent as that of the 100AS RNA, whereas the synthesis of dsRNA from the Δ12AS RNA template was much less efficient (C), indicating that the nt 90 to 100 is not important for initiating dsRNA synthesis.Similarly, to narrow down the regions in nt 70 to 100 of the 100AS RNA responsible for initiating RNA synthesis by the RdRp, the following deletion mutants were constructed: Δ6AS, Δ9AS, and Δ12AS RNA, in which nt 95 to 100, nt 92 to 100, or nt 89 to 100 were deleted, respectively C. The refficient C, indicastem-loop) AS RNA, in which the stem-loop region (nt 74–87) was deleted (D); (ii) sw (switch) AS RNA, in which the stem regions, nt 75 to 78 and nt 83 to 86, were switched together to preserve the stem structure (E); and (iii) lp (loop) AS RNA, in which nt 79 to 82 (GUUA) at the top loop of the stem loop in the 100AS RNA was changed to the complementary sequence (CAAU) (F). When the ΔslAS and swAS RNAs were used as templates for RdRp, small amount of the dsRNAs was synthesized . Alternatively, dsRNA was synthesized as well with lpAS RNA . These results, therefore, also indicate that the stem-loop region is important, and the stem region (nt 74–78 and nt 83–87) is more important than the top-loop region (nt 79–82) (see To know the importance of the stem-loop region (nt 74–87), three kinds of mutant template RNAs were also constructed: (i) Δsl  structure E; and (ie (CAAU) F. When t–82) see . All the32Phosphate at the 5′-end. When 100AS RNA was incubated with the RdRp, the amount of the RNA-RdRp complex increased as the amount of the RdRp increased . However, when Δ31AS RNA lacking in the 31 nt of the 3′-end of the 100AS RNA was incubated with increasing amounts of the RdRp, Δ31AS RNA formed a complex with the RdRp much less efficiently than 100AS RNA even at the highest concentration . These results indicate that the 31 nt is recognized by the RdRp with high affinity and implies that this recognition is required for the initiation of genome positive-strand RNA synthesis.The direct interaction of 100AS RNA with the RdRp was examined by the electrophoretic mobility shift assay (EMSA) technique using RNAs labeled with B). When the 32P-labeled 100AS RNA was incubated with increasing amounts of the same nonlabeled 100AS RNA, the amount of the labeled RNA-RdRp complex decreased in correspondence with the increase in nonlabeled 100AS RNA. The amount of the shifted RNA-RdRp complex decreased to 13.5% . In contrast, when 32P-labeled 100AS RNA was mixed with increasing amounts of nonlabeled Δ31AS RNA lacking in nt 70 to 100 at the 3′-end of 100AS RNA, the 32P-labeled 100AS RNA-RdRp complex competed with nonlabeled Δ31AS RNA, but the efficiency of competition with nonlabeled Δ31AS RNA (decreased to 35.8%) was lower than that with the nonlabeled 100AS RNA (decreased to 13.5%). These results thus indicate that the 100AS RNA and the RdRp interact specifically and imply that the 31 nt RNA (31AS RNA) is required for this interaction.To confirm that this interaction was specific, two nonlabeled RNAs, 100AS RNA and Δ31AS RNA, were used as competitors in EMSA B. When tA and B, respectively); EMSA was used to examine the interaction of the 31AS RNA with the RdRp. The 32P-labeled 31AS RNA was incubated with increasing amounts of the RdRp. However, no 31AS RNA-RdRp complex was detected even at the highest concentration (600 pmol) of the RdRp . Also, when the nonlabeled 31AS RNA was used as a competitor for the complex of 100AS RNA and RdRp, the competition was less efficient than when using the nonlabeled 100AS RNA as a competitor . It is possible that the 31AS RNA was too short to form the stable complex with the RdRp efficiently. Therefore, to determine whether the 31AS RNA region interacted with the RdRp, the interaction of the RdRp with add31AS-S RNA . However, the Δ31S RNA itself (nt 31–100 of the positive-sense RNA) did not have an activity to synthesize the complementary RNA by the RdRp . It also did not form a complex with the RdRp . Finally, these results show that 31AS RNA is required for interacting with the RdRp and required for initiating RNA synthesis as well.Similarly, since the 31AS RNA  was exain vitro assay detected RdRp inhibitory effects by compounds. We tested 2′-C-methylcytidine (2′CM) and its ribonucleotide triphosphate form, 2′-C-methylcytidine triphosphate (2′CM-CTP), as known inhibitors of the HuNV RdRp. When increasing concentrations of 2′CM were added to the reaction mixture, no inhibitory effect on the RNA synthesis was observed  of 2′CM-CTP was calculated as 57 ± 20 μM.We examined whether our We determined here that the HuNV RdRp initiates the template-specific initiation of RNA synthesis, and that the RdRp interacts with the 3′-end region of the antisense genomic RNA.A). Similarly, the introduction of deletions and mutations in the 31 nt region revealed that the predicted stem-loop region and the region close to the stem sequences are required as a template for RNA synthesis by the RdRp. There are reports on the activity of the HuNV RdRp in vitro, and the template RNAs used in these reports are those corresponding to partial 3′-terminal genomic or sense/antisense subgenomic sequences of HuNV  RNA from the 100 nt of the 3′-end region of the antisense genomic template RNA (100AS RNA), but did not synthesize the antisense RNA from the 100 nt of the 5′-end region of the genomic template RNA (100S RNA) A. Simila of HuNV , 7, 9 or of HuNV . The HuNA and B). The add31AS-S RNA, which is composed of the connection between the 5′-end of 31AS RNA and the 3′-end of Δ31S RNA (shown in C). These results also show that the nt 70 to 100 in 100AS RNA (31AS RNA) is required for interaction with the RdRp. As shown in Figure 4B, the add31AS-S RNA was active as a template for the RdRp. Finally, these results imply that the 3′-end (the nt 70–100) of the antisense genomic RNA is required for recognition and initiation by the RdRp for synthesis of positive-sense genomic RNA.Second, we also demonstrated the interaction of HuNV RdRp with antisense genomic RNA by using EMSA. Our results showed that the RdRp forms a complex with 100AS RNA, and the specific interaction was shown by competition with the same nonlabeled 100AS RNA. The deletion of nt 70 to 100 in 100AS (Δ31AS RNA) also caused a loss in the interaction with the RdRp in EMSA, and the competition with Δ31AS RNA was less efficient than that with 100AS for the 100AS RNA-RdRp binding. These results indicate that the nt 70 to 100 (31 nt) was required for the interaction with the RdRp , A and Bshown in , formed shown in C. These et al. have reported predicted secondary structures at three locations: the 5′-end, 3′-end, and the upstream of the subgenomic RNA in the whole HuNV and murine norovirus (MNV) genomic and antigenomic RNAs. They also showed that all of these regions are important for MNV genome replication   , 16, 17.B). This result also indicates that this mechanism of initiation of viral genomic RNA synthesis by the RdRp is common among many genogroups, especially the GI, GII, GIV, and GVII genogroups, which infect humans and canines, respectively.The nt 74 to 100 region of GI, GII, GIV, and GVII noroviruses antisense genomic RNA is highly conserved among genogroups, although the GIII, GV, and GVI genogroups are different from this “conserved sequence” B. This rB). This implies that nt 74 to 100 of the antisense subgenomic RNA also forms the stem-loop structure the same as the antisense genomic RNA; also, the mechanism of the synthesis of the subgenomic RNA by the RdRp is probably the same.As we reported here, the 31 nt RNA located at the 3′-end region of the antisense HuNV genomic RNA, which contains the predicted stem-loop structure, is required to bind to the RdRp and to initiate the RNA synthesis by the RdRp. In GI, GII, GIV, and GVII noroviruses, the nt 74 to 100 regions corresponding to the 3′-end regions of the antisense subgenomic RNA are also highly conserved and homologous to those of the antisense genomic RNA B. This iThe RdRp also interacts with other stem-loop RNA regions (promoter for synthesizing the subgenomic RNA), the complementary sequences of which are located upstream of the antisense genomic RNA, and initiates the RNA synthesis , 13, 14.These results imply that the norovirus RdRp binds to at least three regions of stem-loop structures in the antisense genomic RNA ; as we r50 was 57 ± 20 μM. This inhibitory efficiency on HuNV was similar to that reported by Jin et al. (50 is 33.6 ± 5.8 μM). Similarly, several antiviral drugs have been developed so far, and most of them are direct-acting antivirals (DAAs) that selectively target viral components  without affecting cellular function, thus minimizing side effects , was initially an HCV inhibitor . It work effects , 19. Ourin vitro.Conclusively, we identified the regions in the antigenomic HuNV RNA that are required for the interaction and initiation of the genomic RNA synthesis by its RdRp The plasmid for expressing the RdRp protein coding for the cDNA (nt 3581–5113) of the human norovirus GII. P3_GII.3 U201 strain (accession no. AB039782) . ThereafAll plasmids for the template RNAs were prepared by PCR using nt 1 to 100 cDNA corresponding to the 5′-terminal of the genomic RNA of the human norovirus GII.P3_GII.3 U201 strain (corresponding to the positive strand) or its complementary sequence (corresponding to the antisense strand), specific primers containing the XbaI restriction site-T7 promoter sequences-three Gs (GGG) at the 5′-end, or three Cs (CCC) at the BsaI-EcoRI sites at the 3′-end   carrying the pET28b-RdRp were cultured in 1 l of Luria Broth Base medium (Invitrogen) at 30 °C until the turbidity at the optical density (OD)590 was approximately 0.6 (for up to 3 h) and then placed in ice-cold water for 20 min. Next, isopropyl β-D-1-thiogalactopyranoside  was added, and the cells were cultured for an additional 24 h at 13 °C. To purify RdRp (His-RdRp) containing a His tag at the N-terminus, the culture was then centrifuged at 4400g on a JLA-10.500 rotor (Beckman Coulter) for 5 min. Afterward, 20 ml of buffer (50-mM Tris-HCl (pH 7.5), 150-mM NaCl, and 5-mM β-mercaptoethanol) was added to the bacterial pellets, and the pellets were suspended in the buffer and sonicated by five rounds of a 30 s on ¾-in. chip at an intensity level of 5 with subsequent cooling in iced water . The sample was then centrifuged at 22,700g on a JA-20 rotor (Beckman Coulter) for 15 min, after which the supernatants were loaded onto a 2.5-ml buffer-equilibrated Ni-NTA column (Qiagen). After washing the column first with 200-ml buffer, and then with 15-ml His-A buffer (50-mM Tris-HCl (pH 7.5), 150-mM NaCl, 5-mM β-mercaptoethanol, 10-mM imidazole), His-RdRp was eluted with 10-ml elution buffer (50-mM Tris-HCl (pH 7.5), 150-mM NaCl, 5-mM β-mercaptoethanol，200-mM imidazole). The His-RdRp was subsequently purified further with a buffer-equilibrated gel filtration column .Here, 31.7 nmol (lane 1) or 2.3 pmol (lanes 2 and 3) of the purified RdRp was analyzed by SDS-PAGE. After SDS-PAGE, lane 1, the gel stained with Coomassie dye, GelCode Blue Stein Reagent (Thermo Fisher Scientific) was used to visualize the purified RdRp. Similarly, in lanes 2 and 3, proteins on the gel were transferred to the polyvinylidene fluoride (PVDF) membrane with iBlot Gel Transfer Stacks PVDF, regular . The two PVDF membranes were then blocked with PVDF-blocking reagent, CanGet Signal (TOYOBO) for 1 h at room temperature (RT). One PVDF membrane (lane 2) was incubated with 1/1000 diluted anti-RdRp antiserum . The other PVDF membrane (lane 3) was incubated with 1/1000 diluted anti-6X His-tag antibody  in a Can Get Signal Immunoreaction Enhancer Solution 1  at 37 °C for 1 h, respectively. The two PVDF membranes were washed with TBS Tween20  for 15 min three times, after which the PVDF membrane incubated with anti-RdRp serum or the other PVDF membrane incubated with anti-His tag antibody was then incubated with antibodies: 1/30,000 diluted Peroxidase-AffiniPure Donkey Anti-Guinea Pig IgG (H + L) , or 1/30,000 diluted Goat anti-Rabbit IgG (H + L) HRP-conjugated  in a Can Get Signal Immunoreaction Enhancer Solution 2  at RT for 1 h, respectively. After this step, the two PVDF membranes were washed with TBS Tween20 for 15 min three times again and then detected with a Super Signal WestFemto Maximum Sensitivity Substrate . The RdRp was detected using the LAS-3000 imaging analyzer (Fujifilm). The purity of RdRp was subsequently calculated using ImageJ v.2.1.0.et al.  to exclude the nonviral sequences at the 3′-end. RNA was then synthesized from a template of the linearized DNA using T7 polymerase and purified with a gel filtration column   as described by Mckenna et al.  and Shim  was used to dephosphorylate the 5′-end of the purified template RNAs. Subsequently, the 32Phosphate was bound to the 5′-end of the dephosphorylated template RNA by the T4 polynucleotide kinase (Takara), followed by phenol/chloroform, and precipitated by ethanol. The unreacted γ-32P-ATP in the RNA was then removed using Sephadex G-50 fine resin . The 32P-labeling efficiencies of RNAs were found to be 49% to 51%.The 5′-end 2, 0.8-U/μl ribonuclease inhibitor (Toyobo)) and incubated at 30 °C. The mixture was divided into two portions (20 μl each) 30 min after incubation. To one portion, 4 μl of 6× gel-loading buffer  was added, and nondenaturing polyacrylamide gel electrophoresis (native PAGE) with the running buffer  was performed at a constant voltage of 300 V for 25 min . To the other portion, 7 μl of ten times diluted S1 nuclease  and 3 μl of S1 ribonuclease buffer were added and incubated for 30 min at 30 °C to degrade the ssRNA and detect double-stranded RNA (dsRNA) comprising the template RNA and the newly synthesized complementary strands. After this step, the 6× gel-loading buffer was then added, and the native PAGE was performed as described above. After native PAGE, the gel was then stained with SYBR Green II (LONZA) and detected using a LAS-3000 imaging analyzer (Fuji Film).A 5-pmol template RNA  and 0.8-pmol HuNV RdRp  were mixed with the reaction buffer , 2-mM MnClThe nucleotide secondary structure of the 100AS RNA  was predicted using the Genetyx-Mac software v.19.0.3 (Genetyx Corporation) by “RNA secondary structure prediction”  using de32P-labeled 5′-end-labeled RNA (1 pmol) and the RdRp  were incubated in the reaction buffer  at 30 °C for 10 min . A 4 μl of 6× gel-loading buffer was then added to the reaction mixture. The RNA and RdRp complex was separated with native PAGE at a constant voltage of 60 V for 90 min at 4 °C in the 1/2 × running buffer  after the gel was prerun at a constant voltage of 60 V for 30 min at 4 °C and analyzed by Typhoon FLA 7000 image analyzer . In the case of the competition assay, the procedures are the same as above except that the nonlabeled RNA  was incubated with 32P-labeled 100AS RNA (0.2 pmol) and the RdRp (600 pmol), respectively.The 50) values of the RdRp activity.2′-C-methylcytidine  and 2′-C-methylcytidine triphosphate triethylammonium salts  were added  to the reaction mixture that is the same content described in the All data are contained within the manuscriptThis article contains The authors declare that they have no conflicts of interest with the contents of this article.


Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized.Tuberculosis (TB) is an infectious disease caused by  Mycobacterium tuberculosis (Mtb) remains one of the leading infectious causes of human mortality, supplanted only in 2020 by the COVID-19 pandemic triggered by the SARS-CoV-2 virus. Mtb evolved from an ancestral smooth tubercule bacillus , acquiring virulence elements to attain its preferred pathogenicity towards humans . La. LaM. smegulated  10]..M. smegmrv3282, fadA3, and lipB translation by binding a 7–11 nucleotide region upstream of the start codon. Supplementing fatty acids in the mycobacterial cultures overrides this regulatory process, revealing a feedback loop to control metabolic functions in Mtb. The regulatory role for sRNAs in Mtb metabolism is also revealed with ncRv11846 (106 nts). An ortholog of the E. coli sRNA RhyB, ncRv11846 is termed mycobacterial regulatory sRNA in iron (MrsI) , later named Mcr11, upregulates several genes required for Mtb fatty acid production  30]. Th. Th30]. n (MrsI)  12]. nc. ncrv328esxA and espI. Over-expressing sncRNA-1 alters the Mtb transcriptome, with multiple genes required for fatty acid biogenesis increased in expression. Screening putative targets of sncRNA-1 by seed-sequence complementarity searches reveals two targets of this sRNA, rv0242c, and rv1094. These encode two proteins involved in the oleic acid biogenesis pathway. Both genes have putative sncRNA-1 binding sites within their 5′ UTRs. Substituting selected nucleotides involved in Watson‐Crick base pairing, either within the 5′ UTR or in the sncRNA seed sequence, eliminated the positive regulation. One novel approach for studying microRNA functions is the use of locked nucleic acid power inhibitors (LNA-PIs). These have modified RNA sequences that prevent their cleavage by RNA processing enzymes. They also have chemical modifications to enable uptake into cells without any transfection or liposome-based carrier needs . Th. Th13]. er needs . They hyer needs . IncubatM. smegmatis and has homology to the 6S sRNA . Al. Al58]. E. coli and B. subtilis, used as model bacteria for RNA processing, Mtb encodes for RNase P, which is involved in the initial processing of the 5′ end of tRNA molecules [rv2681), and a divergent functional and structural ortholog of RNase T (rv2179c) [E. coli, RNase PH is implicated in degradation of structured RNAs, which accumulate in the mutant lacking this RNase [Transfer RNAs (tRNAs) share some common features with small RNAs, being relatively short and highly structured non-coding RNA molecules. tRNA maturation involves several steps, with both 3′ and 5′ ends being extensively processed in an orchestrated, sequential order. Besides the core RNA degradosome components, tRNA processing enzymes likely play roles in maturation and turnover of certain sRNA species. In eukaryotes, many RNP complexes involved in tRNA biology participate in the generation and subcellular trafficking of other small structured RNAs such as snRNA, snoRNA, 5S RNA, and others . In manyolecules . The suiolecules , RNase Drv2179c) . As a lais RNase . In the Streptomyces coelicolor, another actinomycetes related to Mycobacteria [Gln32-CUG and tRNALeu3-CAG [tRNA cleavage is seen in all kingdoms of life as a regulatory mechanism, adding another layer to the complexity of gene regulation mechanisms. This has been observed in bacteria . tRNA clbacteria . TubercuLeu3-CAG . Mtb encLeu3-CAG . Direct Leu3-CAG , but theM. smegmatis growth when overexpressed, a screen for RNA chaperons that mediate the interaction between 6C, and its targets was developed [M. smegmatis clones overexpressing 6C were exposed to saturation mutagenesis, there was some growth. However, the few colonies that recovered were those that had mutations in the overexpression cassette, indicating the lethality of 6C. No other genetic mutations were observed in the colonies from the saturation mutagenesis library. Presuming a chaperon was targeted, the growth inhibition by B11/6C was not overcome, suggesting a chaperone protein was not involved [In the majority of bacteria species, trans-encoded sRNAs require RNA chaperones, either Hfq or ProQ, to ensure appropriate sRNA/mRNA base pairing . Taking eveloped . In expeinvolved . It remaBacillus subtilis [CsrA, a conserved small RNA binding protein, was recently shown to assist in a complex between the sRNA and its mRNA targets in subtilis . The RNAsubtilis . Howeversubtilis . While tE. coli and S. aureus have revealed that cold shock domain containing proteins (CSPs), involved in binding and melting RNA species [E. coli has reported the requirement of the CsdA DEAD-box helicase for low temperature riboregulation of rpoS mRNA via sRNA-mediated mechanism, where the activity of Hfq was not sufficient for translational activation of rpoS expression [Recent studies from  species ,77, also species . Hence,  species . Future pression .M. smegmatis, M. dioxanotrophicus, and M. goodie encode a eukaryotic-like protein with a full length TROVE domain (KEGG database search www.genome.jp (accessed on 27 May 2021)), sharing over 35% identity with the human 60 kDa SS-A/Ro ribonucleoprotein ortholog ). It remains unknown whether the TROVE protein has acquired some functions related to sRNA metabolism in fast growing mycobacterial species or whether it is simply a useless remnant of a previous bacteriophage infection.Intriguingly,  results ). The 60 results . It is t results . In mycoMtb-encoded small RNAs are emerging as new regulators of mycobacterial growth, survival, and pathogenesis. To date, the functions for only a handful of these sRNAs have been described. Given their distinct sizes, their functional contributions likely differ when comparing those <50 nt and those between 50 and 350 nt. The recently developed CRISPR interference-based assays hold great potential for studying the function of sRNAs and sncRNAs ,26,61. I


Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in controlling the temperature regulation of plant-virus interactions are poorly characterised. To elucidate these further, we analysed the responses of potato plants cv Chicago to infection by potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of which is known to significantly increase plant susceptibility to PVY. Using RNAseq analysis, we showed that single and combined PVY and heat-stress treatments caused dramatic changes in gene expression, affecting the transcription of both protein-coding and non-coding RNAs. Among the newly identified genes responsive to PVY infection, we found genes encoding enzymes involved in the catalysis of polyamine formation and poly ADP-ribosylation. We also identified a range of novel non-coding RNAs which were differentially produced in response to single or combined PVY and heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites. Finally, to gain more insights into the potential role of alternative splicing and epitranscriptomic RNA methylation during combined stress conditions, direct RNA nanopore sequencing was performed. Our findings offer insights for future studies of functional links between virus infections and transcriptome reprogramming, RNA methylation and alternative splicing. Solanum tuberosum L.), one of the most important non-grain crops in the world, similar to all other crop plants, is constantly exposed to a plethora of pathogens. Among these pathogens, viruses account for up to 50% of all novel and emerging plant diseases )In nature, plants, including potato crops, not only face an onslaught of biotic stress invoked by pathogen exposure, but they are often simultaneously exposed to various environmental stresses, such as elevated temperatures. Increased temperatures are particularly detrimental to potatoes, which is a cool-weather crop and has optimal growth at temperatures between 14 and 22 °C; above these temperatures, its yield is significantly reduced, which is likely to become an increasing problem given climate change expectations .To cope with higher temperatures, plants have evolved mitigation mechanisms for the acquisition of thermotolerance. Heat stress may invoke the reprogramming of various signalling pathways, such as the production of reactive oxygen species (ROS), induction of mitogen-activated calcium-dependent protein kinase signal transduction cascades, increased expression of a variety of heat shock transcription factors (HSFs), heat shock proteins (HSPs) and accumulation of osmolytes that affect membrane fluidity and morphology . HSPs coR or N gene-mediated resistance. Most of these potato resistance genes, such as Ny-1 in potato cultivar Rywal and Ny found in S. sucrense and S. sparsipilum, confer resistance only at low temperatures (16–20 °C). At higher temperatures (24–28 °C) resistance does not develop, and PVY spreads systemically throughout the plant  are covalently attached to proteins by PAR polymerase enzymes . The cenPARG, NUDIX and ADPR-PPase  and ADP-PPase (Soltu.DM.08G000850.1) in response to PVY infection at both the normal and higher temperature A,B. The PR-PPase C. It is Viral RNA accumulation was verified in non-inoculated  leaves of PVY-infected plants grown at normal (22 °C) and elevated (28 °C) temperatures via a RT-qPCR analysis of total RNA at 8 dpi, a time point at which samples for RNAseq analysis were also collected. PVY-accumulation rates were essentially similar at both these temperatures at this early stage of infection F, howeveIt should be noted that when the polyadenylated RNA was analyzed, short-read sequencing revealed only 10,048 virus-specific reads, equating to 0.003% of the total reads  obtained at 28 °C (20.3 FPKM), whereas the number of viral reads at 22 °C was much lower . The reasons for this inconsistency remain unclear, but a possible contributor is that although PVY RNA is typically polyadenylated [SAMDC, SPDS) and PARylation process , and one non-regulated gene (PARP1) were further analyzed by RT-qPCR  their transcriptional level under at least one stress condition (n = 307), exonic or intronic (n = 79), and antisense transcripts (n = 35) (It has recently been shown that plant lncRNAs can play regulatory roles in plant responses to stress conditions . To provondition . These D(n = 35) A. The si(n = 35) B. We fou(n = 35) B. Some l(n = 35) B. These We identified 221 DE-lncRNAs from Clusters 3, 4 and 5 which were up-regulated under the elevated temperature regardless the presence of PVY, suggesting that these DE-lncRNAs could belong to temperature-responsive lncRNA transcripts in potato. Some of these transcripts (27 from 77 in cluster 3) were significantly up-regulated at an elevated temperature, but down-regulated during infection at the normal temperature B. In addIt has been previously reported that antisense lncRNAs are involved in gene-expression regulation under various stress conditions . TherefoIt has been previously shown that lncRNAs can act as decoys, limiting the availability of different regulatory factors, such as miRNAs . To gainAnother example of miRNA involved in regulating many processes, including heat-stress response, is miR390. Previously, MiR390 was shown to take part in the regulation of auxin response factors ARF3/4 . The lncAdditionally, A lncRNA MSTRG.27358.1, that contains binding sites for miR396, was up-regulated under the elevated temperature in the presence or absence of the virus E. The miA further fifteen lncRNAs with predicted binding sites for potato specific miRNA were down-regulated under at least one of the stress conditions. For example, a lncRNA MSTRG.7747.1 has a predicted binding site for miRNA102 and was up-regulated under the elevated temperature (in the presence or absence PVY), but down-regulated under PVY infection at 22 °C E. This lAlternative splicing (AS) is a mechanism responsible for the generation of numerous protein isoforms from a single gene. AS plays an important role in plant stress responses, however, the correct detection and quantification of isoforms is a challenging task. Using the advantages of nanopore sequencing to currently identify full-length transcripts, we analyzed differentially spliced potato genes in response to combined stress (PVY + heat) treatment. For this, the transcriptome assembled from nanopore reads was used. We compared the proportion of each isoform in the level of gene expression using the Fisher exact test. Overall, 8705 genes with two or more isoforms were analyzed. We identified 283 transcripts from 176 loci that showed significantly different proportions between mock-inoculated plants and plants infected with PVY under elevated temperature . Twenty-6A) is the most prevalent modification in both plants and animals [6A RNA methylation may be pivotal for posttranscriptional gene-regulatory events such as mRNA splicing, stability, and translation. The ability to control the fate of RNA molecules through nucleotide modifications is vital to plant health and survival under diverse environmental conditions. To provide more insights into the role of RNA methylation in the stress responses of potato we used direct RNA nanopore sequencing, which provides an opportunity to predict sites of RNA methylation. Here, we used the xPore tool for analysis of the differences in m6A RNA methylation between control mock-inoculated plants maintained at 22 °C and plants infected with PVY at the higher temperature. Overall, 57 transcripts might be differentially modified, some of which are involved in responses to stress conditions  that has a higher methylation rate for sites located in the 3′-UTR region  of the transcript . This geIntriguingly, in addition to mRNAs, we also predicted methylation events on PVY RNA in two of the three biological replicates from plants infected with PVY at 28 °C. Since PVY RNA was obviously absent in the mock-inoculated samples, we could not use the xPore tool for an analysis of differently methylated transcripts. Therefore, we used the m6Anet software for the prediction of methylation events. The M6Anet tool showed 2033 modified positions, 78 of which were located on the virus sequence . The hig6A machinery methylates the viral RNA genomes of several animal and plant viruses [Previous studies have shown that the m viruses ,78 incluO) was used [Nicotiana bentamiana), which was then extracted in a potassium phosphate (KP) buffer pH 7.5 (1:3 w/v). Four-week-old potato plants  were inoculated with 200 µL PVY extract; control plants were mock-inoculated with KP buffer. Plants were kept in a controlled environment chamber  with a photoperiod of 16/8 h day/night at a relative humidity of 50% and a temperature of 22 °C, with a light fluence of 250 µmol m−2·s−1. At 2 dpi (days post-inoculation), half of the plants were transferred to 28 °C for heat-stress simulation. Leaf samples from four virus-inoculated  or control mock-inoculated plants were collected at 8 dpi. From each plant, 3–4 leaves were collected and pooled together. The harvested material was processed and analysed as described below.To infect potato plants, an ordinary (O) strain of PVY (PVYwas used . The virTM technology platform. The Direct RNA sequencing kit from Oxford Nanopore (SQK-RNA002) including the optional reverse transcription step was used to prepare libraries from thepoly(A) RNA. Total library sequencing was performed on 200 ng of each poly(A) RNA library using the MinION platform in conjunction with FLO-MIN106 (ONT R9.4) flow cells and the standard MinKNOW software. All obtained reads were deposited in the NCBI database; BioProject accession PRJNA786109.Total RNA was isolated from the liquid-nitrogen-frozen and ground up leaf tissue using the Invitrogen™ TRIzol™ Reagent , following the manufacturer recommendations. The precise concentration of total RNA in each sample was measured using a Quant-iT™ RNA Assay Kit in conjunction with a Qubit 3.0 fluorometer , with a standard curve range of 20–1000 ng of RNA. A total of 100 μg aliquots of total RNA was diluted in 100 μL of nuclease-free water and poly(A) containing RNA was isolated using and following the recommendations of the Thermo Fisher Scientific Poly(A)Purist™ MAG Purification Kit Invitrogen. The resulting poly(A) RNA was eluted in nuclease-free water. Paired-end RNA-Seq libraries were generated from the poly(A) RNA isolated from mock-inoculated and PVY-infected plants; short read sequencing was performed using the DNBSeqS. tuberosum genome V6.1 [S. tuberosum plastid and mitochondrial sequences, and Saccharomyces cerevisiae enolase control RNA sequence. Minimap2.20 [We used Guppy 4.0.15 (Oxford Nanopore Technologies) for basecalling direct RNA sequencing data. MinIONQC was used for quality control . Reads wome V6.1  with addimap2.20  with parimap2.20  and Bedtimap2.20  were useimap2.20  and Striimap2.20  were useimap2.20 .The FeatureCounts  tool wasTo find the enrichment of GO terms g:Profiler  was usedOpen reading frames with lengths >30 a.a. were predicted in assembled transcriptome with Python package orfipy. These ORFs were used for domain prediction with InterProScan . Loci wip-values of xPore and isoform ratio differences were adjusted using Benjamini–Hochberg procedure. Sashimi plots were built in the Integrative Genomics Viewer (IGV) [Isoform ratios were counted based on long reads TPM values. Differences in the ratios between mock-inoculated (22 °C) and PVY-infected (28 °C) plants were counted using Fisher exact test. RNA modifications were predicted with m6Anet  and xPorer (IGV) .For the isolation of plant nuclei and nuclear protein extraction, the CelLytic PN kit (Sigma Aldrich) was used . The prohttps://phytozome.jgi.doe.gov/pz/portal.html (accessed on 26 January 2022)) and PRIMER EXPRESS software . The Ct values for PVY RNA and each mRNA of interest were normalized using two internal reference genes encoding StEF-1α [Total RNA was isolated as described above. DNase-treated RNA was reverse-transcribed into cDNA using the SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen), with either an oligo-dT primer (for host plant-specific mRNAs) or a PVY-specific primer see . The pri StEF-1α  and cyto StEF-1α . The aveWe have previously shown that the enhanced susceptibility of Chicago potato plants to PVY at high temperatures may be triggered by MTC perturbation and the resultant reduction in transmethylation activities ,22. The PARG, NUDIX and ADPR-PPase  stress treatments also cause dramatic changes in the expression of many lncRNAs, RNA methylation and alternative splicing. In our analyses, we were able to recover some known regulators and pathways in the responses to virus infections and abiotic stress as well as some putative novel regulators and pathways, which provide new insights for understanding the mechanisms of plant stress tolerance. Collectively, these data demonstrate that plant responses to biotic (virus) and abiotic (heat) stresses are highly complex and involve changes at many levels. Moreover, plants respond to combined (multiple) stresses differently from single stresses, completely reprogramming the gene-expression pattern corresponding to the particular environmental conditions. These interactions between biotic and abiotic stresses are concerted by hormone-signalling pathways and changes in gene expression, including coding and non-coding RNAs transcription, alternative splicing and epitranscriptomic and epigenetic events that act together in a complex integrated network. The identification of master regulators that integrate all these mechanisms is therefore crucial for the development of new approaches applicable for the improvement of broad-spectrum stress-resistance in crops.


Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin-inducible degron (AID) system to immobilize both adult and larval hermaphrodite and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes . Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode  The discovery of green fluorescent protein (GFP) and subsequent proliferation of both engineered and additional fluorescent proteins has revolutionized biological research by enabling direct visualization of the biological processes occurring within living organisms. This breakthrough changed how scientists viewed cellular processes from snapshots in time generated by fixed images to the complete dynamic picture occurring in real time within the organism . Live imCaenorhabditis elegans worms. This small, soil-dwelling nematode is completely transparent, thereby making it ideal for live imaging since the intact worm can be placed on a slide and imaged without requiring any tissue dissection or tissue clarification. However, immobilizing the worms on slides can be challenging since wild type worms are highly mobile and spend their life traveling in a sinusoidal pattern across agar plates eating lawns of bacteria. Further, worms display phototaxis when exposed to light stimulus, avoiding ultraviolet , blue (470 nm), and green (500 nm) wavelengths of light , which allows for manipulation and placement of the worm within the hydrogel  and then solidify at room temperature. This method allows worms to be embedded into the cold hydrogel liquid and become immobilized as the gel warms  and width (∼50 µm). Consequently, many methods that immobilize the hermaphrodites fail to immobilize males. Caenorhabditis elegans is an excellent model system for sexual dimorphism studies with many sex-specific differences having been identified related to germline processes, nervous system development, and animal aging  system, we designed a conditional immobilization system where worms are only paralyzed upon exposure to the plant hormone auxin. Here, we validate this conditional immobilization tool using the C. elegans germline and demonstrate that this system works efficiently in both sexes. Notably, we also found that male worms require a higher concentration of auxin to display the same amount of paralysis as hermaphrodites, thereby revealing a potential novel sexual dimorphism within the AID system.In comparison to hermaphrodites, °C under standard conditions on nematode growth media (NGM) with lawns of Escherichia coli. InVivo Biosystems tagged unc-104, unc-18, and unc-52 with the AID [AID*, asterisk denotes the 44 amino acid tag rather than the 68 amino acid miniAID (mAID) tag , PhytoTechnology Laboratories, cat no. N610)  L4 worms from SYP-2::GFP strains were placed at 25°C overnight to enhance GFP expression  and sealed using nail polish. All slides were stored at 4°C and imaged within 1–2 days.EdU staining was performed as described in Immunofluorescence slides of gonad stained with RAD-51 were imaged on a GE DeltaVision microscope with a 63X/N.A. 1.42 lens and 1.5X optivar at 1024 × 1024 pixel dimensions. Images were acquired using 0.2 µm Z-step size and deconvolved with softWoRx deconvolution software. EdU slides and all brightfield timelapses were imaged on GE IN Cell microscope. Edu slides were imaged with a 40X/N.A. 0.95 lens using a Z-step size of 0.72 µm, male brightfield timelapses were imaged with 20X/N.A. 0.75 lens using a Z-step size of 1.3 µm, and hermaphrodite brightfield timelapses were imaged 10X/N.A. 0.45 lens using a Z-step size of 3.91 µm. All IN Cell timelapses and images were deconvolved using the IN Cell 3D deconvolution software. SYP-2::GFP, L1 autofluorescence, and mCherry::H2B timelapses were imaged on a Nikon CSU SoRa Spinning Disk Microscope with a 60X water lens/N.A. 1.2 using a Z-step size of 0.3 µm.z-projected in FIJI. Then, the position of the EdU straining front was determined by the last nucleus within the germline labeled with EdU. Max intensity z-projection montages and movies were made in FIJI, and only GFP::SYP-2 movies were stabilized using the FIJI plugin “StackRegJ” . This stabilization was necessary to reduce the motion of the germline inside the worm and generate a stable movie for viewing. Additionally, photobleach correction was applied to the GFP::SYP-2 male movie using the photobleach correction application in FIJI. All images and movies have been slightly adjusted for brightness and contrast using FIJI.RAD-51 gonad images were stitched together using the FIJI (NIH) plugin Stitcher  rgef-1p::TIR1, which expresses only in neurons; and (2) eft-3p::TIR1, which has pan-somatic expression. For all experiments, we found that animals needed to be grown for a single generation on plates containing NGM with auxin to exhibit the strongest paralysis phenotype (see Methods).Using CRISPR/Cas9, each of these genes was tagged with the AID sequence and genetic crosses were performed to incorporate unc-104::AID* and unc-18: AID* strains using the neuron-specific expression of TIR1 (rgef-1p::TIR1) and AID*::unc-52 with pan-somatic expression of TIR1 (eft-3p::TIR1) . We noticed that unc-104::AID* did not display as strong of a mobility defect as unc-18::AID* when depleted using the neuron-specific TIR1 . Moreover, depleting UNC-104::AID* with the pan-somatic eft-3 driven TIR1 exhibited a similar degree of mobility defects to neuron-specific depletion of UNC-104::AID* with the rgef-1 driven TIR1 . Although the effects of each driver on UNC-104::AID* depletion were statistically indistinguishable, the median average speed was slightly lower with the pan-somatic eft-3 driver .We first assayed p::TIR1) . AID*::uunc-104::AID* and unc-18::AID* worms recovered some degree of normal motion . However, the overall median average speed in recovered unc-104::AID* animals was lower than wild type worms . unc-18::AID* worms were able to recover some motion off auxin compared to the unc-18::AID* in the presence of auxin; however, this recovered motion was significantly slower than wild type worms  and the no auxin cohort . Taken together, these motion recovery experiments suggest that unc-18::AID* may require more time to completely recover wild type motion compared to the unc-104::AID* worms. Overall, both unc-104::AID* and unc-18::AID* are able to both partially recover movement after removal from the auxin treatment.With this worm immobilization technique, worms can be recovered postimaging and assayed for viability off auxin. We found that upon removal from auxin for 18–24 h both l motion . unc-104C. elegans germline. Since the effects of UNC-104 or UNC-18 loss on germline function are unknown, we examined multiple aspects of germline biology to determine if a loss of UNC-104 or UNC-18 causes germline-specific defects. We began by assaying the fertility of hermaphrodite worms containing unc-104::AID* and unc-18::AID* under both depletion (in the presence of auxin) and wild type conditions (Methods). Scoring fertility over multiple days allowed for observation of most of the hermaphrodite reproductive lifespan, which begins with a large abundance of living progeny and subsequently ends with unfertilized eggs once the hermaphrodite sperm is depleted . In contrast, on days four and five unc-104::AID* displayed larger brood sizes than wild type animals with these changes being only significantly different on Day 4 . This result suggests that depletion of UNC-104 might cause a slight delay in oocyte progression through the germline allowing more oocytes to be laid later during the hermaphrodite reproductive lifespan. Further experiments are necessary to determine if indeed this is a delay or if something else is contributing to the discrepancies in brood size compared to wild type animals.In the presence of auxin, ertility . The prounc-104::AID* worms also displayed a high number of worms with a bag of worms phenotype, where worms retained their progeny within their body cavity, and worms that went missing over the 5 days of scoring  remains indistinguishable from wild type (unc-18::AID* worms during the first 24 h (Day 1) on auxin exhibited no significant changes in brood size. In contrast, the progeny on auxin at Day 2 displayed significant reductions in brood size compared to wild type . Notably, this decrease in brood size on Day 2 did not correlate with an increase in the number of dead eggs, indicating that oocyte viability is not the likely cause of this brood size reduction  (C. elegans germline was divided up into four different regions based on nuclear DNA morphology: premeiotic to transition zone (PMT-TZ), early pachytene to mid pachytene (EP-MP), mid pachytene to late pachytene (MP-LP), and diakinesis to germline end (Diakinesis+)  to allow EdU incorporation into nuclei undergoing DNA replication, including those in the premeiotic tip. Then, worms were dissected at 0, 10, 24, and 48 h post-EdU soaking to “chase” the EdU staining in the germline (see inesis+)  and thosar rates . GermlinC. elegans, DSBs are formed during the transition zone through early pachytene, and then RAD-51 is loaded on to the resected single-stranded DNA ends of the DSB site (unc-18::AID* worms (Methods for details). Overall, unc-18::AID* worms are able to both induce and repair DBSs at similar frequencies to wild type. The transition zone did display a slight, but significant, decrease in RAD-51 foci in unc-18::AID* worms (average RAD-51 foci: 0.9 ± 1.7 SD) compared to wild type . However, RAD-51 foci numbers in early, mid, and late pachytene were indistinguishable from wild type in unc-18::AID* worms . Taken together, formation of DSBs and subsequent off-loading of RAD-51 is largely unaffected by depletion of unc-18::AID* by auxin.To determine if meiotic recombination is altered by depletion of UNC-18::AID*, we assayed for the initiation of recombination through the quantification of meiotic double-strand DNA breaks (DSBs) using the recombinase protein RAD-51. At the onset of meiosis, the topoisomerase-like enzyme SPO-11 induces DSBs from which a subset are repaired via homologous recombination into crossover events, which ensure accurate segregation of the homologs during meiosis I  , thereby indicating depletion of UNC-18::AID* has minimal effects on meiotic recombination. Taken together, subtle differences in both germline progression and brood size in UNC-18::AID* depletion do not have obvious negative consequences on meiosis.To determine if UNC-18::AID* depletion had any effect on crossover formation, we assayed for the presence of crossover events by quantifying the number of DAPI staining DNA bodies at diakinesis. Since ed DSBs) . Both wiunc-18::AID*; rgef-1p::TIR1 were selected from 1 mM auxin plates. These worms were placed into a 1–2 µL drop of imaging M9 media containing 25 mM serotonin and 1 mM auxin on a poly-lysine coated coverslip. Previous studies have shown that serotonin is important to maintain motion within the germline during live imaging  in the germline was equivalent between males and hermaphrodites on 4-mM auxin plates  did not cause detrimental changes to the C. elegans germline. To determine if depletion of UNC-18::AID* altered the progression of nuclei in the male germline, we used EdU labeling to track germ cell nuclei progression. Previous studies have demonstrated that the male germline progresses much faster than the hermaphrodite germline (24 h to complete spermatogenesis vs 48–72 h to complete oogenesis)  . In addiunc-18::AID* worms had on average about 2–3 more RAD-51 foci per nucleus than wild type . This slight increase of approximately 2–3 RAD-51 foci in unc-18::AID* worms persisted through early pachytene and mid pachytene, but by late pachytene, both unc-18::AID* worms and wild type displayed similar amounts of RAD-51 foci . Thus, the slight increase in DSBs in unc-18::AID* worms does not appear to affect the ability for these DNA breaks to be repaired by off-loading RAD-51 in late pachytene. Further, once removed from auxin, these unc-18::AID* males are able to mate and generate progeny . Taken together, the increase in DNA breaks following depletion of UNC-18::AID* does not appear to cause any large defects to spermatogenesis.In addition to normal germline progression, the initiation and repair of DSBs exhibited a similar pattern to wild type following depletion of UNC-18 in the male germline. However, analysis of RAD-51 foci using immunofluorescence revealed that knockdown of UNC-18::AID* caused a slight increase in RAD-51 foci during both the transition zone and the beginning of pachytene . In the unc-18::AID*; rgef-1::TIR1 conditional immobilization technique, we were able to observe both the directional motion of germline progression and the chromosome motion within each nucleus . All live imaging experiments were performed using the same imaging settings as the hermaphrodites except for the brightfield timelapses, which due to the smaller size of the male worms were captured at 20X magnification instead of 10X . NotablyC. elegans worm makes this model organism ideal for live imaging studies, however, effectively and reliably immobilizing the worm without injury has been a challenge for many C. elegans labs seeking to do live imaging experiments. We developed and validated a new tool that enables conditional immobilization of C. elegans for live imaging. This conditional immobilization tool uses the AID system, which we show works for immobilizing both larval and adult hermaphrodite and male worms. Notably, we found that depletion of the gene product responsible for this immobilization phenotype does not cause any substantial changes within the germline of either sex. Finally, with this tool, we were able to demonstrate that both male and hermaphrodite adult worms can be minimally restrained as whole animals with an agar pad and imaged live for at least 2 h (Supplementary Movies S5 and S8).The transparent nature of the C. elegans are known to respond differently than hermaphrodites to different chemicals and toxins including the widely used anesthetic levamisole (Methods), we were able to consistently and reliably mount male worms without any of the technical challenges associated with the agar pad handling or placement. Specifically with our combination of these techniques, we found that regardless of how the agar pad was placed over the males, all of the mounted male worms were suitable for imaging and remained immobile. Further, our auxin-based immobilization system could be combined with other standardized mounting procedures. Overall, multiple options for immobilization of male worms increase the possibility of live imaging experiments that can be performed in a multitude of laboratory settings with different resources.The conditional immobilization technique described here to immobilize worms enhances the existing toolkit for live imaging worms. While many modalities exist from microfluidic chips to pharmaceuticals for immobilization of the worm, here, we present an accessible genetic tool that can be used to easily immobilize worms and can be implemented in any lab without needing to purchase specialized equipment or use hazardous chemicals. Notably, the use of anesthetics has been widespread for live imaging studies; however, male C. elegans germline, an increasing number of studies are indicating hermaphrodites and males have multiple sexually dimorphic features that lead to differential regulation of genome integrity germline . Further, genetically linking the rgef-1p::TIR1 transgene to the unc-18:AID* locus will simplify moving this system into existing tagged lines through genetic crosses. Moreover, there are likely more genes within the C. elegans genome that could be tagged with AID and used to induce immobilization analogous to UNC-18 depletion. Identification of additional genes that cause paralysis on different chromosomes and in different tissues will permit further flexibility with this immobilization system. Currently, this immobilization system works well for visualization of the germline but may not be ideal for neuronal studies since UNC-18 is important for nervous system function. However, many genes within the worm have been identified to cause severe paralysis and some of these do not alter the nervous system, such as unc-54 which is part of the muscle myosin complex. Thus, future expansion of this conditional immobilization system by AID tagging other genes will provide even greater genetic flexibility for use of this system in a multitude of live imaging studies.Our immobilization system has both flexibility for use in a variety of experiments and the ability for future modifications and expansions. To enable live imaging of any existing tagged fluorescence reporter strains, the components of our live imaging system can easily be incorporated through either genetic crosses or CRISPR-Cas9 injections  are available at the CGC. All Supplemental materials are available at figshare: https://doi.org/10.25387/g3.14673441. Supplementary Figure S1 shows a diagram of the steps used to immobilize worms for live imaging. Supplementary Table S1 contains all the primers and sgRNAs used for CRIPSR generations of unc-18::AID*, unc-104::AID*, and AID*::unc-52. Supplementary Movie S1 is a brightfield timelapse of an immobilized hermaphrodite at 10X magnification. Supplementary Movie S2 is a timelapse of the autofluorescence from an immobilized L1 worm. Supplementary Movie S3 is a timelapse of mCherry::H2B in the germline of an L4 worm. Supplementary Movie S4 is a timelapse of SYP-2::GFP in an immobilized hermaphrodite at 60× magnification. Supplementary Movie S5 is an entire germline view of SYP-2::GFP at 60Xin an immobilized hermaphrodite. Supplementary Movie S6 is a brightfield timelapse of an immobilized male at 20X magnification. Supplementary Movie S7 is a timelapse of SYP-2::GFP in an immobilized male at 60X magnification. Supplementary Movie S8 is an entire germline view of SYP-2::GFP at 60X in an immobilized male.All strains are available upon request. Strains developed as part of this study (