C. elegans. While each single mutation rescued the motility of CAPS/unc-31 and synaptotagmin/snt-1 mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed in C. elegans. We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends to C. elegans’ characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.SNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds in  C. elegans movement requires coordination between the excitation and inhibition of the neuromuscular junction , synaptobrevin/VAMP and SNAP-25 come together to form a four-helix bundle called the SNARE complex that brings the membranes together, which is key for membrane fusion (C. elegans) (C. elegans) (2+ sensor synaptotagmin-1 (SNT-1 in C. elegans) (2+-dependent manner (SNARE (soluble NSF-attachment receptor) and Sec/Munc18 (SM) proteins are the essential factors that govern synaptic vesicle exocytosis. These proteins are conserved and play critical roles regardless of the types of synapses or types of exocytosis. The SNARE proteins syntaxin (UNC-64 in e fusion . In thise fusion . The SNAelegans) , Munc13selegans)  and the elegans) . Togethet manner .C. elegans, mice, and humans. On the other hand, gain-of-function mutations lead to enhanced exocytosis and can rescue the phenotypes of other exocytosis defective mutants. The investigation of such gain-of-function mutants provides mechanistic insights into how these proteins and their interactions with other exocytotic proteins contribute to exocytosis. Thus, an L166A/E167A mutation in syntaxin/UNC-64 (abbreviated as “LE”) and a P334A mutation in UNC-18  are well-known gain-of-function mutations that have been extensively studied in various model systems of exocytosis, including in vitro liposome fusion , respectively. Strikingly, the presence of both gain-of-function mutations within the same worm did not provide benefits to worm size, growth speed, or number of offspring given. Therefore, we further analyzed these single and double mutants by electrophysiology in detail. Our results show that these mutations have differential effects on exocytosis depending on the type of synapse and the type of exocytosis.In the present study, we investigated how the open syntaxin and P334A UNC-18 mutations interact with each other using unc-64(LE) KI mutant can rescue the defects in motility and acetylcholine release of various exocytosis mutants, including snt-1, unc-31, unc-13 and unc-10 (unc-18(PA) KI mutant rescues unc-31 and unc-13 mutants (unc-18(PA) has the ability to rescue a wide-range of exocytosis mutants remains unknown.We previously showed that an open syntaxin d unc-10 . Althoug mutants , whethersnt-1 gene in C. elegans, functions as a Ca2+ sensor for synchronous neurotransmitter release (Snt-1(md290) null worms show resistance to aldicarb and have smaller body sizes relative to wild-type worms (unc-18(PA) can rescue snt-1 null worms, like open syntaxin. We also tested whether absence of the inhibitory protein tomosyn  can rescue snt-1 mutants. The TOM-1 protein, encoded by the tom-1 gene, inhibits spontaneous and evoked release via the formation of an inhibitory tomosyn-SNARE complex (tom-1(ok285) null mutation is known to enhance acetylcholine release and partially rescues unc-13 mutants  or unc-18(PA) mutations were generated as previously reported (tom-1(ok285) null worms, were subsequently crossed with synaptotagmin null worms, snt-1(md290). Worms were assayed for their motility and acetylcholine release ability through thrashing and aldicarb sensitivity assays, respectively. We found that both snt-1; unc-64(LE) and snt-1; unc-18(PA) double mutants similarly and significantly increased thrashing compared to the snt-1 single mutant  null worms exhibit minimal paralysis after 4 hours  double mutants showed increased aldicarb sensitivity compared with snt-1, suggesting an increase in acetylcholine release  double mutant  worms have smaller brood sizes, body sizes and exhibit slow population growth speeds (unc-64(LE), unc-18(PA), or tom-1(ok285) mutations can rescue the reduced brood size, population growth speed, and body sizes observed in snt-1 null worms. For brood size, a single L4 worm was placed on an agar plate and after 48 hours of growth, the original worm was transferred to a fresh agar plate. Afterwards, the original worm was transferred to a fresh new plate every 24 hours until the worm died, or egg laying ceased. Offspring  were counted after the original worm was removed from the plate. We found that unc-64(LE) and unc-18(PA) worms both had comparable brood sizes to that of N2, while tom-1 had a significantly reduced brood size  double mutant, the double mutation of snt-1; tom-1 seemed to worsen the small brood size of snt-1 null worms  and snt-1; unc-18(PA) worm populations grew faster than snt-1 null worms. The rescue observed by open syntaxin was stronger than that of the P334A UNC-18 mutant; snt-1; unc-64(LE) worms reached their peak a day earlier than snt-1; unc-18(PA) worms. However, snt-1; tom-1 worms displayed decreased growth speeds compared to the snt-1 null mutant.For the population growth assay, three worms of each strain were placed on agar plates and allowed to grow over the course of 9 days, and L4 and above adult worms were counted daily. N2 worms showed a sharp increase in total worm number between days 3 and 4, reaching a peak at day 5 . Snt-1 nsnt-1; unc-64(LE) or snt-1; unc-18(PA), we did observe an increase in populational growth of these two strains when compared to snt-1 null worms  worms giving birth to a larger portion of their offspring earlier on. Indeed, when we plotted the brood counts by day  worms and snt-1;unc-18(PA) worms had a similar cumulative brood counts, snt-1; unc-64(LE) worms had a left-shifted curve and laid more eggs in the beginning. These worms that were laid earlier (F1 progeny) would then mature and give rise to the F2 progeny that may be reflected in the rapid peak observed in the population growth assay  weakly increased the size of snt-1 null worms, unc-18(PA) did not. We found that tom-1 null single mutants also exhibited a smaller body size, which is consistent with previous literature  and unc-18(PA) double mutation may have additive or synergistic effects when rescuing the snt-1 null mutant. On the other hand, if they use common or similar mechanisms, we may see saturating effects between the two. Therefore, we next asked if the double mutation would exhibit an additional ability to rescue motility and acetylcholine release in the snt-1 null background. As such, we generated the snt-1; unc-64(LE); unc-18(PA) triple mutant. Despite both open syntaxin and P334A UNC-18 mutants being able to rescue thrashing activity individually ; unc-18(PA) triple mutant worms showed a similar thrashing count to the snt-1 null mutant  and snt-1; unc-18(PA) double mutants were also abolished in the triple mutant – snt-1; unc-64(LE); unc-18(PA) worms, which showed a trend for reduced brood size  null worms are slow and sluggish, exhibiting a low thrashing rate and impaired aldicarb sensitivity (unc-31(e928) was significantly rescued by open syntaxin and P334A UNC-18  and unc-18(PA) in the unc-31(e928) background, namely unc-31(e928); unc-64(LE); unc-18(PA) triple mutant, strikingly abrogated the motility of the unc-31 worm (unc-31(e928) worms exhibited a comparable body size to that of N2 worms and both unc-31; unc-64(LE) and unc-31(e928); unc-18(PA) double mutants slightly increased the body size of the worm (unc-31(e928); unc-64(LE); unc-18(PA) triple mutant exhibited a significantly smaller body size when compared to the single and either of the double mutant worms. We once again find that the benefits gained by the unc-64(LE) and unc-18(PA) mutants are lost or even worsened when both mutations are simultaneously present.We next tested the two knock-in mutations in a different background to see if the loss of beneficial effects of the combined mutations is specific to the  docking . In addi docking . Unc-31(sitivity . We prevA UNC-18 . We orig-31 worm . Moreovethe worm . Howeverunc-31(e928) worms was partially rescued by the unc-64(LE) or the unc-18(PA) mutants (unc-31(e928); unc-64(LE); unc-18(PA) triple mutant. This trend is similar to that observed for the snt-1; unc-64(LE); unc-18(PA) triple mutant.The loss of aldicarb sensitivity observed in  mutants . The tri mutants . Thus, wunc-64(LE) and unc-18(PA) mutations in snt-1 null and unc-31 null backgrounds, we hypothesized that the double mutation may induce motility defects in a wild-type background. Thus, we crossed unc-64(LE) worms with unc-18(PA) worms to look at the resulting phenotype of the double mutants in a wild-type background. We observed that both single unc-64(LE) and unc-18(PA) mutants thrashed at a degree comparable to N2. However, the unc-64(LE); unc-18(PA) double mutants exhibited a dramatically decreased thrashing, with only 65 thrashes/min, which is about half of the wild-type thrashing (unc-64(LE) or unc-18(PA) single mutants display increased aldicarb sensitivity compared to N2 (unc-64 (LE); unc-18(PA) compared to the unc-18(PA) single mutant (unc-64(LE) and unc-18(PA) have beneficial effects on the motility, exocytosis, brood, and growth speed of exocytosis-defective mutants, such as snt-1 or unc-31 null mutants. However, when these mutants are present simultaneously, their beneficial effects are lost or the mutations become detrimental regardless of the presence or absence of exocytosis-defective mutations. Importantly however, the double mutant increased aldicarb sensitivity irrespective of the background.Since we found detrimental effects in motility when simultaneously expressing hrashing . As preved to N2 . At the e mutant . Howevere mutant . From the mutant , 2, 3, wcpx-1 null mutants, which exhibit reduced motility but enhanced aldicarb sensitivity  and unc-18(PA), and the double unc-64(LE); unc-18(PA) mutant. To measure evoked release from either excitatory or inhibitory synapses, we generated the respective mutant lines in the zxIs6 or zxIs3 background (zxIs6 and zxIs3 strains express channelrhodopsin-2 in excitatory cholinergic neurons (zxIs6) or inhibitory GABAergic neurons (zxIs3), enabling optogenetic stimulation for accessing transmitter release.Why do the double mutants exhibit reduced motility while it increases or rescues aldicarb sensitivity  - 3? We sitivity . Therefockground . zxIs6 azxIs6 background. Both unc-64(LE) and unc-18(PA) single mutants increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) (unc-64(LE); unc-18(PA) double mutant exhibited a trend for increase in mEPSC frequency (unc-64(LE) and unc-18(PA) mutations do not have additive effects on mEPSC frequency. The amplitude of the spontaneous mEPSCs was not significantly changed across all strains (unc-64(LE) mutants showed an increase in charge transfer of excitatory postsynaptic currents (EPSCs) without affecting the EPSC amplitude when compared to wild-type worms (unc-18(PA) single mutants exhibited a similar EPSC phenotype to unc-64(LE) and increased charge transfer without largely affecting EPSC amplitude (unc-64(LE) and unc-18(PA) were present together, the two gain-of-function mutations again seemed to cancel each other. The unc-64(LE); unc-18(PA) double mutant only showed a trend for increase in EPSC charge transfer, again yielding values that were lower than those of the single mutants and did not reach statistical significance compared to the WT values  , C, congs , C, co(mEPSCs) . While trequency , the ave strains . Next, w strains , unc-64 was slightly increased in unc-64(LE) and unc-18(PA) worms (unc-64(LE); unc-18(PA) double mutant did not show an increase (unc-18(PA), facilitated IPSC charge transfer and amplitude, but this facilitation was lost in the unc-64(LE); unc-18(PA) double mutant (We then investigated inhibitory synaptic exocytosis using the single and double mutant strains in a A) worms , 5C. Howincrease . Again, increase . We thene mutant –H. Not oe mutant . Thus, te mutant , our resunc-64(LE) and unc-18(PA) can rescue the motility and aldicarb sensitivity defects of unc-31(e928) null mutants (unc-64(LE); unc-18(PA) worsened the mobility of unc-31 mutants while restoring aldicarb to wild-type level (zxIs6 or zxIs3 background using electrophysiology. unc-31(e928) null mutants did not alter mEPSC frequency compared with wild type worms (unc-64(LE); unc-18(PA) mutant were crossed into the unc-31(e928) background, only unc-31; unc64(LE) significantly increased mEPSCs frequency  and unc-31; unc-64(LE); unc-18(PA) worms, they did not reach statistical significance. Again, mEPSC amplitudes were unchanged (unc-31(e928) exhibited decreased evoked EPSC amplitude and charge transfer compared with wild-type worms (unc-31(e928); unc-64(LE) and unc-31(e928); unc-64(LE); unc-18 (PA) showed slightly increased evoked EPSC amplitudes with respect to unc-31(e928), but without reaching statistical significance  null worms as both frequency and amplitude were comparable to wild type levels  nor unc-31; unc-18(PA) double mutants largely changed these properties. However, there was a significant decrease in mIPSC frequency and amplitude in unc-31; unc-64(LE); unc-18(PA) triple mutants  and unc-31; unc-18(PA) worms showed trend of rescue in amplitude and charge transfer. By contrast, the unc-31; unc-64(LE); unc-18(PA) triple mutant exhibited a further decrease in amplitude when compared to unc-31 null worms  and unc-31; unc-18(PA) worms were also lost in the triple mutant ; unc-18(PA) triple mutant suggest that there is an imbalance between excitatory and inhibitory transmitter release in both spontaneous and evoked release modes.Next, we investigated the inhibitory release potential of these worms by again crossing the various mutants into the e levels –C. Neith mutants , table 1controls , 7E, 7G.ll worms , table 1e mutant , table 1e mutant , the evoC. elegans has complex effects on exocytosis that depend on the type of (excitatory vs. inhibitory) synapse and on the mode of exocytosis (spontaneous vs. evoked) (2+-independent fusion between liposomes containing syntaxin-1 (S-liposomes) and liposomes containing synaptobrevin (V-liposomes), which is barely observable when using WT proteins, whereas the high efficiency of fusion observed upon addition of Ca2+ does not allow the observation of stimulatory effects.We found that the double mutation of open syntaxin and P334A UNC-18 in  evoked) . In shor evoked) . This as evoked)  as well  evoked) . These s2+, whereas using P335A Munc18-1 led to a strong increase in Ca2+-independent fusion .Overall, these observations show that the cancelation of the gains-of-function caused by the open syntaxin and P334A UNC-18 mutations in snt-1(md290) mutant, which involves the major Ca2+ sensor protein for exocytosis  synapse and on the type (spontaneous vs. evoked) of exocytosis . We firsocytosis , similarocytosis . We also mutants , P334A Ull worms . Tomosynormation .C. elegans that we tested, despite exhibiting an increased sensitivity to the acetylcholinesterase inhibitor, aldicarb  are present for each protein, and the compensatory and/or redundant effects of these isoforms makes it very difficult to evaluate the function of each protein : NaCl 150, KCl 5, CaCl2 5, MgCl2 1, glucose 10, sucrose 5, HEPES 15, pH 7.3 with NaOH, ~330 mOsm. All chemicals and blockers were from Sigma. Experiments were performed at room temperatures (20–22°C).Light stimulation of zxIs6 or zxIs3 was performed with a LED light source at a wavelength of 460 ± 5 nm (3.75 mW/mm2), triggered by the PULSE software with a duration of 10 ms. When recording mPSCs or eEPSCs, the muscle cells were held at −60 mV. To record mIPSCs or eIPSCs, 0.5 mM D-tubocurarine (d-TBC) were added to the bath solution to block all acetylcholine receptors with a holding potential of −10 mV. The recording solutions were described previously (Gao and Zhen). Specifically, the pipette solution contains (in mM): K-gluconate 115, KCl 25, CaCl1, C2B, MUN and C2C domains  were described previously  mutant (open syntaxin-1), a cysteine-free variant of full-length rat SNAP-25a, full-length rat synaptobrevin-2, full-length rat Munc18-1, full-length Chinese hamster NSF, full-length Bos Taurus αSNAP, and a fragments spanning the C-terminal region of rat Munc13-1 that includes the Ceviously . We noteeviously .2, 2 mM ATP, 0.1 mM EGTA, 5 μM streptavidin, 0.4 μM NSF, 2 μM α-SNAP, 1 μM Munc18-1 , 1 μM SNAP-25 and 0.5 μM Munc13-1C. Before mixing, S-liposomes were incubated with MgCl2, ATP, EGTA, streptavidin, NSF, αSNAP, and Munc18-1 at 37°C for 25 min. 0.6 mM of CaCl2 was added at 300 s to each reaction mixture. A PTI spectrofluorometer was used to measure lipid mixing from de-quenching of the fluorescence of Marina Blue–labeled lipids  and content mixing from the development of FRET between PhycoE-Biotin trapped in the S-liposomes and Cy5-streptavidin trapped in the V-liposomes . All experiments were performed at 30°C. Lipid and content mixing were normalized as the percentage values of the maximum signals obtained by addition of 1% b-OG at the end to each reaction mixture (for lipid mixing) or to controls without streptavidin to measure maximal Cy5 fluorescence (for content mixing).Assays that simultaneously measure lipid and content mixing between synaptobrevin-containing liposomes (V-liposomes) and syntaxin-1-containing liposomes (S-liposomes) were performed as previously described in detail . BrieflyAll statistical analyses were done in OriginPro2016 and Prism 9.5.1. Independent t-tests were used for two-group experiments with a p-value < 0.05 as the threshold for statistical significance. One-way analysis of variance (ANOVA) was used for comparisons of multiple groups, followed by Tukey’s range test, with a significance level of 0.05. For all box and whisker plots, mean line is represented, range indicate 25 and 75 percentiles, whiskers are the outliers.Supplement 1


Bacteria adjust gene expression at the post-transcriptional level through an intricate network of small regulatory RNAs and RNA-binding proteins, including ribonucleases (RNases). RNases play an essential role in RNA metabolism, regulating RNA stability, decay, and activation. These enzymes exhibit species-specific effects on gene expression, bacterial physiology, and different strategies of target recognition. Recent advances in high-throughput RNA sequencing (RNA-seq) approaches have provided a better understanding of the roles and modes of action of bacterial RNases. Global studies aiming to identify direct targets of RNases have highlighted the diversity of RNase activity and RNA-based mechanisms of gene expression regulation. Here, we review recent RNA-seq approaches used to study bacterial RNases, with a focus on the methods for identifying direct RNase targets. This review presents and explores the different RNA sequencing strategies to investigate the activity of bacterial ribonucleases genome-wide. Ribonucleases (RNases) are responsible for catalyzing the scission of the phosphodiester bond in RNA molecules. These enzymes are present in all living organisms, where they play a central role in RNA metabolism by cleaving RNA molecules internally (endoRNases) or from the extremities (exoRNases). Not only are they required for RNA turnover and subsequent nucleotide recycling, but they also participate in mechanisms of gene expression regulation.i.e. RNase E and RNase Y) or a 5′-to-3′ exoRNase  and some transfer RNAs (tRNAs) are co-transcribed, and multiple endoribonucleolytic processing followed by trimming events are necessary for the production of the individual rRNA molecules   systems and act either as toxins by cleaving a wide range of RNAs under stress conditions and phage infection (Type II and III TAs), or as antitoxins by cleaving the mRNA encoding the toxin (Type V TA) , and are limited by the number of target RNAs tested. In addition, IVT RNA substrates may form different secondary structures and lack RNA modifications when compared to the in vivo conditions. Likewise, recombinant RNase variants may lack post-translational modifications, which could affect the RNase processing. Finally, the absence of RNase interacting proteins and/or sRNAs, which are key factors in the control of RNase activity, in these experiments does not allow a complete recapitulation of the mechanism of RNase cleavage and may impair the mapping of RNase processing sites.In the past, RNases have been studied largely Comparing RNA abundance between a wild-type (WT) strain and a deletion mutant strain of the RNase of interest  is highly informative, but time-consuming and not high-throughput.Transcriptomics approaches have become the method of choice for studying the roles of bacterial RNase, as they enable the simultaneous identification of multiple RNase targets and an understanding of the overall importance of the RNase under study. Transcriptomics studies, aimed at exploring the role and functions of bacterial RNases on a global scale, were pioneered using microarrays or tiling arrays. These techniques have made it possible to identify genes directly or indirectly regulated by an RNase and to monitor stability of mRNA transcripts in bacteria. In Here, we review the current RNA-seq methods for identifying RNase targets, discuss in detail the approaches for mapping cleavage positions and highlight the major advances in the field. Furthermore, we discuss the limitations of the available strategies and provide a perspective on how the study of bacterial RNases can benefit from the use of omics approaches.In RNA-seq, a specific group of RNA molecules is usually converted into a library, composed of fragmented cDNA molecules harboring adapter sequences at both or one of the extremities. The cDNAs are amplified and subsequently sequenced at high throughput. Although multiple protocols for library preparation are available, some critical steps are common to all existing methods.Regardless of the RNA species enriched, the majority of the protocols include steps for genomic DNA (gDNA) and rRNA removal. Exclusion of the gDNA by DNase treatment, ensures the reduction of background noise, while depletion of the rRNAs, which represent the most abundant RNA class present in the cell, allows more cDNAs nonderiving from rRNAs to be sequenced and therefore increases the information content of the sample.Due to the limitation in cDNA length that can be sequenced by the majority of available RNA-seq platforms, the RNAs sometimes need to be fragmented. RNA fragmentation can be accomplished mechanically (e.g. acoustic shearing)  and the accurate mapping of bacterial transcripts . The pretreatment with enzymes that either modify the RNA ends or exclusively degrade RNAs harboring a specific chemical group at the RNA termini allows distinct RNA subgroups to be distinguished. For instance, treatment of total RNA with terminator exonuclease (TEX), which degrades only processed transcripts (harboring a 5′ P), allows the cDNA library to be enriched for primary transcripts (harboring a 5′ PPP). After TEX treatment, Tobacco Acid Pyrophosphatase (TAP) is used to convert the 5′ PPP to 5′ P, and thus generate RNA 5′ ends suitable for adapter ligation. To enrich the cDNA library with processed transcripts (5′ P RNAs), it can be generated from the untreated RNAs. Comparison of these cDNA libraries by RNA-seq—approach named differential RNA-seq (dRNA-seq)—has been successfully used to map the primary transcriptome of Due to the detection limit of most sequencing platforms, cDNA libraries often need to be amplified before sequencing. A limited number of PCR cycles are performed, and several methods are now available to detect and eliminate PCR duplicates during data analysis to avoid bias in transcript abundance determination. For instance, the insertion of unique sequence tags consisting of either defined sequences or random nucleotides . A longer read length (more than 10 Kb)—although more error prone—enables transcripts to be mapped more precisely and makes it easier to identify isoforms  or both extremities (pair-end sequencing). The read length Prior to mapping the reads (i.e. sequenced cDNAs) to the genome of reference, quality control and normalization steps are required to ensure the accuracy of the results and the comparability of the samples, respectively. The low-quality reads are discarded, as are adapter sequences and duplicated reads due to PCR amplification. The coverage, i.e. the number of reads that are mapped to a defined region of the reference genome, is then calculated and visualized using a genome browser (e.g. IGV and Artemis). Comparison of the expression levels among different samples requires a normalization step, to eliminate biases due to the different gene lengths and sequencing depth. For this purpose, different gene expression measurements such as RPKM (reads per kilobase of transcript per million), FPKM (fragments per kilobase of transcript per million), or TPM (transcript per million reads) are used to account for factors that could affect the comparison of samples. In addition, the number of reads starting (5′ end coverage) or stopping (3′ end coverage) can be calculated at each single nucleotide. This analysis allows the precise mapping of transcript termini, which is highly informative for deciphering the origin of different transcript isoforms, for example derived from RNase activity.RNA-seq is often used to determine gene expression levels by counting the mapped reads and comparing them among different samples. This allows the identification of genes that are differentially expressed in response to changes in environmental conditions or variations in the expression of a gene under study. RPKM and FPKM are suitable for within-sample comparisons, but not for differential gene expression analysis. Therefore, other normalizing approaches are used for gene count comparisons between samples, such as the approaches included in the DESeq2 , indicating that these RNA fragments originated from RNase III processing of overlapping transcripts  followed by RNA-seq was used to determine whether variations in transcript abundance were due to changes in RNA stability  of rnc mRNA (coding for RNase III), a 5′ end of the product detected by sequencing after co-IP coincided with the RNase III processing site validated in vitro. Notably, RNase III has the ability to function in a noncatalytic mode and can bind some target RNAs without cleaving them  by forming reversible covalent bonds  . For instance, in the in vivo UV protein-RNA cross-linking prior to co-purification. Compared with formaldehyde, UV light is considered a more specific cross-linker, first because it favors formation of a covalent bound only when there is no distance between the interacting molecules, and secondly because it does not promote cross-linking between interacting proteins . Cross-linking and analysis of cDNAs (CRAC) S. aureus (MRSA), providing insight into the role of RNA-mediated regulation in the development of antibiotic resistance and during infection  has recently been used to discover RNA-protein complexes by fractionating bacterial lysates on linear glycerol gradient and analyzing each fraction by RNA-seq and mass spectrometry   were identified and in particular YhaM , which functions as a 3′-to-5′ exoRNase, was the most enriched protein. The YhaM-csRNA interaction was confirmed by CLIP-seq using an YhaM flag tagged version  Fig. . Differe20) Fig. . The csRion Fig. . YhaM waWhereas the above-described methods require the selection of a protein, which is then Co-IPed with the interacting RNAs, Lybecker et al. have used an alternative approach to identify RNase III targets based on the isolation of a specific class of RNAs.E. coli  , consisting of duplexes formed by the paring of sense and antisense RNAs or structured RNAs (e.g. stem-loops). This characteristic was exploited to selectively identify the dsRNAs that are targeted by RNase III in oli Fig. . The metoli Fig.  Fig. 5)E. coli  has been developed to identify novel riboswitches and attenuators in pathogenic and commensal bacteria  has been used to simultaneously map transcription start and end sites in E. coli and B. subtilis. Intermediate decay fragments, although more abundant than their parental transcripts, do not contribute to protein production, as they are less associated with ribosomes  opens the possibility of specifically detecting RNase processing sites and several tailored RNA-seq approaches have been developed for genome-wide identification of the cleavage sites of an RNase Table . Most ofRNase III is a dsRNA-specific endoribonuclease that cleaves the phosphodiester bond between two nucleotides, leaving 3′ OH and 5′ P termini and a 3′ overhang of 2 nt. Therefore, ideally, RNase III processing results in four RNA ends comprising two newly generated 5′ P.et al., sought to identify the putative RNase III processing positions in E. coli by sequencing RNAs harvested from a WT strain and a rnc (encoding RNase III) deletion mutant strain (∆rnc), before and after the addition of rifampicin  and a role for RNase III in the initiation of decay transcripts derived from premature termination. Although this method allows approximate localization of the RNase III cleavage sites, it does not provide information on the exact position of the cleavage.Gordon B. subtilis   defines the length of the nucleotide overhang, which typically consists of 2 nt protruding from the 3′ end. By analyzing the region surrounding the mapped RNase III processing sites, the authors unraveled new features of RNase III's mode of action rnase strain  and ending (3′ reads) at each nucleotide position was calculated not only in the WT and ∆rnc strains, but also in additional strains used as further controls. RNase III cleavage sites were identified on the basis of the criterion that the number of read ends resulting from an RNase III processing events is decreased in the Δrnc strain compared with the reference strains. To identify RNase III cleavage sites, RNA ends that were more abundant in the WT strain than in the ∆rnc strain were filtered using strict parameters. In total, we identified 92 RNase III cleavage sites, 44 of which had never been identified before with the majority of the positions located in UTRs.In the methods described above, identification of the processing sites relies on mapping the 5′ ends of RNAs. However, if the cleavage product carrying the newly generated 5′ end is degraded, the RNase processing site will not be identified. To overcome this problem, we have developed a method, named ISCP, that in addition to the RNA 5′ ends, also maps the RNA 3′ ends , revealing novel targets and possibly new functions for this enzyme  has been widely used to identify RNase E cleavage sites in various proteobacteria . While Gram-positive bacteria with high guanosine and cytosine content (high-GC) display one orthologues (RNase J1), bacteria with low-GC content encode two RNase J orthologues (RNase J1 and J2).RNase J1 is a 5′-to-3′ exoRNase found mainly in Gram-positive bacteria, with the exception of S. aureus , the strain expressing this RNase Y variant showed impaired growth compared with the WT, ∆B. subtilis by mapping the 5′ ends produced by RNase Y  to study the involvement of RNase Y in operon maturation in S. pyogenes by mapping both 5′ and 3′ RNA ends using an optimized and improved ISCP protocol  has been applied to shed light on the specificity of the MazF toxin endoRNases  in E. coli. The MazF-mt3 is expressed in E. coli from a plasmid using a constitutive promoter and, as a control, the same plasmid, without the ORF encoding the toxin, was used. After toxin induction, total RNAs (including rRNAs) were collected and used to prepare two distinct cDNAs libraries, containing either 5′ P or 5′ OH RNAs, respectively.Mapping by overexpression of an RNase in M. tuberculosis. MazF-mt3 did not only target mRNAs but also rRNAs, and crucial cleavage sites were identified and validated in the helix/loop 70 of the 23S rRNA and in the anti-Shine-Dalgarno sequence of the 16S rRNA. Based on the MORE RNA-seq results, the authors formulated a model in which MazF would generate specialized ribosomes for the translation of leaderless mRNAs, as has been proposed in E. coli  of H. pylori compared with the WT strain, but also adapted the dRNA-seq protocol  containing only 5′ PPP RNAs; (ii) containing only 5′ P RNAs; and (iii) containing both 5′ P and 5′ PPP RNAs. These distinct libraries were generated using a combination of enzymatic treatments with TAP and TEX, as described previously (see ‘introduction on RNA-seq’) in the WT, ΔrppH, and complemented strains. 63 transcripts (53 mRNAs and 10 sRNAs) RppH direct targets were identified by selecting transcripts that—in the absence of RppH—were more abundant and harbored a 5′ PPP group. Based on the limited number of RppH targets in H. pylori, the authors hypothesized the presence of a second, nonredundant pyrophosphohydrolase, as has been proposed in other bacterial species such as B. subtilis  and the more abundant 3′ ends in the WT strain were defined as the start and stop positions of exoRNase trimming, respectively. The distance between the start and stop positions defines the length of the exoRNase trimming from 3′ to 5′. Knowledge of the position at which an exoRNase starts and eventually stops the degradation of an RNA molecule provided insight into the activity of 3′-to-5′ exoRNase in vivo.Sequencing and mapping of the RNA 3′ ends enabled the identification of 3′-to-5′ exoRNase targets, whose processing positions have never been determined genome-wide at nucleotide resolution. The ISCP method was therefore used to identify the direct targets of the 3′-to-5′ exoRNases PNPase, RNase R and YhaM in nes Fig.   enabled more precise mapping of the RNase Y targetome  in B. subtilis  and 3′ ends in a strain lacking all four 3′-to-5′ exoRNases   and an adenosine downstream and upstream of the processing site, respectively, within a region characterized by low GC content. In addition, a preference for a structured RNA was detected downstream of the processing site. Bioinformatic analysis of the RNA ends detected in all the strains tested revealed the presence in B. subtilis of a yet unidentified RNase capable of trimming both 5′ P and 5′ OH RNAs . This would would uncover processing events that would not otherwise be possible to map, thereby refining the endoRNase targetome  could functionally replace the missing RNase. This characteristic makes the screening of RNase cleavage sites more challenging, as some of the targets would not be detected as such. In this scenario, the inclusion in the study of strains in which other RNases are deleted in addition the one under study could facilitate the discovery of a greater number of processing events. Noteworthy, deletion of one RNase can have effects on the expression of other RNases  sequencing], enable longer reads to be generated and thus full-length transcripts to be sequenced  in combination with ribosome profiling and standard RNA-seq revealed the structural signature of RNase E in E. coli (Del Campo 2015 Plos Genetics). 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP) coupled with RNA-seq was used to identify the E. coli structurome at the transcriptome scale . RNA-seq-based approaches have been developed to map RNA modifications such as N6-Methyladenosine (m6A) (Dominissini et al. 6A-CA’; of note, this characteristic has been harnessed for the precise mapping of m6A sites (Imanishi et al. While our understanding of RNA modifications derives mainly from studies in eukaryotes, a growing body of research in bacteria has revealed that modified ribonucleotides are not limited to tRNAs and rRNAs, but are also present in regulatory RNAs and mRNAs (Marbaniang and Vogel Ribosomes trafficking along mRNAs has a major impact on RNase activity and therefore RNA stability. This year, an atlas of the mRNA degradome across the bacterial tree of life, based on sequencing of 5′ P degradation products, revealed that cotranslational RNA degradation is widespread and conserved in both Gram-negative and Gram-positive bacteria (Huch et al. Overall, RNA-seq methodologies have revolutionized the field of RNA biology in bacteria, and the development of novel strategies will continue to reveal new insights into the functions of RNases as key players in the post-transcriptional mechanism of gene expression regulation.fuad049_Supplemental_FileClick here for additional data file.


This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and  RNA viruses, such as influenza A viruses (IAV) or coronaviruses (CoV), are important pathogens that cause mild to severe respiratory disease in humans. RNA virus spread among humans occurs in seasonal epidemics, outbreaks of emerging viruses, or occasional pandemics . SeasonaAnalysis of IAV strains has shown that human-adaptive mutations occur in nearly all viral proteins, but in particular in the IAV surface glycoproteins hemagglutinin (HA) and neuraminidase (NA); the proteins involved in viral replication polymerase basic 1 and 2 (PB1 and PB2), polymerase acidic (PA), and the nucleoprotein (NP); and the protein involved in modulating host immune responses, non-structural 1 (NS1) . During Secondary RNA structures in RNA virus genomes play critical roles in IAV and CoV infection cycles and perform functions in packaging, translation, and viral replication. In addition, RNA structures can be the target of therapeutics . SecondaG) for RNA structures upstream, downstream, and around the RNA polymerase along an RNA sequence and confirmed our predictions with biochemical assays , a transient RNA secondary structure that folds effectively ‘around’ the viral RNA polymerase  and affel assays . For fulG of t-loops in the IAV H3N2 genome linearly increased, and thus the overall t-loop stability in the genome reduced. Reductions in t-loop stability are also observed in H1N1 IAV following the 1918 and 2009 pandemics. No destabilization of t-loops is observed in the IBV genome, which has been circulating in humans for decades. However, an analysis of SARS-CoV-2 isolates does reveal destabilization of t-loops. Overall, we propose that the ΔG of RNA structures in the RNA genome of emerging respiratory RNA viruses may play a role in the adaption of these viruses to humans.To test the above hypotheses, we performed cell culture assays as well as analyses of IAV, influenza B virus (IBV), and SARS-CoV-2 sequence data obtained through surveillance. We observe that the sensitivity of H3N2 IAV RNA polymerases to t-loops increased between 1968 and 2017. At the same time, the ΔRenilla luciferase control plasmid. In addition, we provided a cellular RNA polymerase I-driven plasmid expressing a 71-nt long template without a t-loop in the first half of the template (NP71.1) and a 71-nt long template containing a t-loop in the first half of the template (NP71.11). These templates were extensively tested previously and used to show that the IFN-β promoter activity is indicative of poor RNA polymerase activity  . In our activity . Westernactivity . Measureactivity , suggestThe above result suggested that the RNA polymerase of a recent IAV H3N2 isolate is more sensitive to t-loops than an IAV H3N2 isolate from 1968. We hypothesized that any t-loops in the 1968 IAV H3N2 genome would be absent in the 2017 IAV H3N2 genome if they negatively affected viral replication. To explore this, we focussed on an initial set of genome sequences of IAV H3N2 isolates from 1968, 1972, 1982, 1993, 2003, 2008, 2014, and 2017. These isolates capture the full IAV H3N2 tree  and did Next, we analysed the putative RNA structure content in the IAV H3N2 genome. To this end, we modified the t-loop analysis that we described previously  to accouin virio , we included both the positive and negative ΔG values in this analysis. Interestingly, we observed that of the three RNA polymerase encodings segments, in particular, the PB1 vRNA segment showed a trend corresponding with t-loop destabilization  for the PB2 segment, whereas the PB1 and PA segments demonstrated a loss of MFE stability (in silico variants (G was significantly different (P < 0.001). The steepest increase in t-loop ΔG was observed for the PB1 segment between 1968 and 2008. An apparent plateau in PB2 and PB1 t-loop stability followed 2008, but additional (future) years would need to be analysed to confirm the start of a new trend. For simplicity, we fit the trends with a linear regression (P < 0.0001). In general, both the vRNA and cRNA sense showed similar directions in their increase or decrease in MFE or t-loop ΔG. However, the direction in MFE and t-loop ΔG change was not consistent, as was particularly evident in the PB2 segment  may contribute or drive the changes in t-loop stability observed in  changes  for the  changes . Quantifegment 2 . For segon level . Taking The above data showed that the putative t-loops in the H3N2 PB1 segment were destabilized more quickly than the t-loops in the H3N2 PA and PB2 segments. We wondered if the different origin of the PB1 segment, which had been introduced into IAV H3N2 through a reassortment event with an IAV from the avian reservoir prior to 1968, was driving this increased rate in transient RNA structure destabilization . To explIn recent memory, H1N1 IAV has been associated with two pandemics in which one or more genome segments were of avian IAV origin . In 1918G in the H1N1 genome segments showed that introduction of a new segment also caused a shift in MFE ΔG. In contrast to the t-loop ΔG, no trend in MFE ΔG change was observed following the reassortment event. In line with our analysis of the H3N2 genome, this suggests that t-loop adaptation occurs independently of changes in other RNA structures in the genome. Overall, the above analysis suggests that IAV reassortment is followed by changes in t-loop stability, and that these changes can occur in both avian-adapted as well as human-adapted IAV genome segments following reassortment.Analysis of the t-loop stability in the three longest H1N1 IAV genome segments revealed a gradual loss of overall t-loop stability in all three segments between 1918 and 1957. Between 1977 and 2009, the t-loop stability remained relatively unchanged for the PB1 segment, whereas the PA segment continued to show some destabilization. The t-loop stability in the PB2 segment was more diverse, and particularly widespread from 1998 to 2009. In 2009, a shift in t-loop stability occurred in the PA segment, in line with our expectations for the introduction of an avian-adapted gene segment through reassortment. At the same time, the H3N2-derived PB1 segment displayed a rapid reduction in t-loop stability, whereas the avian-adapted PB2 segment showed only a modest reduction in t-loop stability. Analysis of the overall MFE ΔG over time. Analysis of the t-loop stability indeed showed no trend that was indicative of stabilization or destabilization. The lack of a trend was both evident for both the vRNA and the cRNA sense of the three segments investigated . Over the course of the pandemic, point mutations and deletions occurred and accumulated in the SARS-CoV-2 genome. Some of these mutations have been linked to amino acid changes that improved host receptor binding, while others caused deletions of known secondary structures. However, many synonymous mutations and less obvious deletions have not yet been linked to improved viral functions, although it has been observed that some variants display increased replication in tissue culture, suggesting that these mutations and deletions may contribute to viral replication efficiency . Based oG and the t-loop ΔG. We therefore set the footprint to the lower of these values for the data presented below, in order to collect more t-loop datapoints per genome.To investigate the above question, we first estimated the footprint of the SARS-CoV-2 replication and transcription complex (RTC). To this end, we aligned motif C of the cryo-EM structure of the SARS-CoV-2 RTC (PDB 7CYQ) with motif C of the cryo-EM structure of the IAV RNA polymerase (PDB 6T0V) in Pymol and estimated the route that template RNA would take through the polymerase. We concluded that 20–22 nt of the template would be covered by the RTC during RNA synthesis . Our iniG and t-loop ΔG trended towards a less negative ΔG, and thus less stable RNA structure content, over the course of the COVID-19 pandemic (P ≤ 0.0001).We next analysed the original SARS-CoV-2 isolate and several SARS-CoV-2 variants of concern using our computational model. For each variant, we analysed the first 50, full-length, high-quality sequences published. We found that both in the positive and negative sense of the full-length genome, the MFE Δpandemic . The tre−3 and 3.95 × 10−4 per nucleotide per year as the upper and lower bounds of the whole genome mutation rate  matched. We observed a similar trend in the evolution of SARS-CoV-2, which recently emerged as a zoonotic virus from an unknown animal reservoir. Such a trend was not evident in the IBV genome, which has circulated in humans without the introduction of non-mammalian adapted sequences for decades. Possible drivers for the observed trends in IAV and SARS-CoV-2 include differences in cellular environment between hosts, including differences in the temperature at the site of replication .A bias in mutations towards bases not involved in base pair formation has been previously observed . It is pin silico models that were derived from experiments performed on short IAV templates lucter] and the transfection control plasmids constitutively expressing Renilla luciferase (pTK-Renilla) were described previously (Human embryonic kidney 293T (HEK293T) cells were originally sourced from ITCC and routinely checked for mycoplasma infection. HEK293T cells were maintained in DMEM containing 10 per cent FBS, glutamate, pyruvate, and high glucose (Gibco). WSN pcDNA3-based protein expression plasmids and pPol I-based viral template RNA expression plasmids were described previously . The opeeviously .Renilla luciferase. Transfections were performed with lipofectamine 2000 (Invitrogen) to the manufacturer’s specifications. Twenty-four hours later, the medium was aspirated, cells were washed with 1 ml PBS and split into two fractions. Cells were pelleted at 2,500 rpm using a benchtop centrifuge. The pellets from one fraction were resuspended in 30 μl of 1x Laemmli buffer for western blotting and the other fraction was resuspended in 50 μl PBS for the IFN-β promoter activity assay. The assay was done in duplicate using 25 μl of cell suspension in PBS per well in a white 96-well plate format. Next, 25 μl of DualGlo reagent (Promega) was added per well, samples were incubated at RT for 10 min (in dark), and the firefly luciferase readings were taken using a Synergy LX Multimode Microplate Reader (Biotek). Twenty-five μl of Stop-Glo reagent/well was added next, plate was incubated for 10 min at RT (in dark), and the Renilla luciferase readings were taken. Firefly luciferase values were normalized by the Renilla luciferase values. Data analysis was performed in Prism 9.5.0 (Graphpad).Transfections to perform RNP reconstitutions and IFN-β reporter assays in HEK293T cells were essentially performed as described previously . RNP recIAV proteins were detected using rabbit polyclonal antibodies anti-PB1 , and anti-NP  diluted 1:2000 in blocking buffer , 0.1 per cent Tween 20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin  diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit  and IRDye 680 goat anti-rat  were used to detect Western signals with a LI-COR Odyssey scanner.Initially, our analysis was performed on a limited, representative set of H3N2 sequences. To expand this set, 50 full-length IAV H3N2 sequences per year were acquired from EpiFlu (gisaid.org) for 1968–2014, and 2017 was downloaded from NCBI see . Fifty fSequences were acquired from EpiCoV (gisaid.org) are listed in After selection, sequences were aligned using EMBL-EBLI Clustal Omega . We note−3 and 3.95 × 10−4 per nucleotide per year (−3 and 2.5 × 10−4 to represent upper and lower bounds of estimated mutation rates (The SARS-CoV-2 reference genome (NC_045512.2) was mutated at frequencies of 1.8 × 10per year . These fon rates . Mutatioon rates .in silico analysis starts with the RNA polymerase binding to the 3′-end of a viral genome (segment), which places residue 16 within the 20-nt footprint in the active site . Next, the total ΔG that the RNA polymerase encounters while copying the template was calculated.The above sliding window was started at the 3′-end of the viral genome and moved in 1-nt intervals along the length of the template RNA. For each window, the RNA sequence upstream of the RNA polymerase footprint, downstream of the RNA polymerase footprint, and between the upstream and downstream sequences was analysed and the stability Δthon 3.9 . LocatioH3N2 consensus sequences for PB2, PB1, and PA were generated using EMBOSS Cons and GC content was calculated using Bio.SeqUtils package from BioPython .vead056_SuppClick here for additional data file.


Mitoviridae are the mitochondria-replicating “naked RNA viruses” with genomes encoding only the replicase RNA-dependent RNA polymerase (RdRp) and prevalent across fungi, plants, and invertebrates. Arbuscular mycorrhizal fungi in the subphylum Glomeromycotina are obligate plant symbionts that deliver water and nutrients to the host. We discovered distinct mitoviruses in glomeromycotinian fungi, namely “large duamitovirus,” encoding unusually large RdRp with a unique N-terminal motif that is endogenized in some host genomes. More than 400 viral sequences similar to the large duamitoviruses are present in metatranscriptome databases. They are globally distributed in soil ecosystems, consistent with the cosmopolitan distribution of glomeromycotinian fungi, and formed the most basal clade of the Mitoviridae in phylogenetic analysis. Given that glomeromycotinian fungi are the only confirmed hosts of these viruses, we propose the hypothesis that large duamitoviruses are the most ancestral lineage of the Mitoviridae that have been maintained exclusively in glomeromycotinian fungi.Mitoviruses in the family  Mitoviridae. Mitoviruses were likely to evolve from leviviruses in the family Leviviridae, RNA bacteriophages, by losing the capsid protein, which yielded mitochondria-replicating “naked RNA replicons” that encode only the replicase RNA-dependent RNA polymerase (RdRp)  . They werasitica . Decadesrasitica  and inverasitica . Althougrasitica  and planrasitica  has beenrasitica . Howeverrasitica  and evenrasitica  suggestsrasitica  by delivrasitica . These frasitica -12, inGigaspora rosea DAOM194757, Rhizophagus clarus CK001, and R. clarus OL1, respectively , are rare  glomeromycotinian mitoviruses were retrieved, and those that encode complete protein (putative RdRp) of 500 aa or longer were extracted  sequences that encode complete protein of 500 aa or longer in 2,888 sequencing libraries, including 8,361 sequences in the 184 libraries from which the 415 large duamitovirus-like sequences were identified. From the 8,361 sequences, those that showed significant similarity to the 105 reference species and (non-large) glomeromycotinian mitoviruses were extracted , and host taxa (phyla/kingdom) and virus genera were assigned to them in reference to the top hit reference species   + 415 (RVMT) large duamitovirus-like viruses were isolated. The large duamitoviruses and their related sequences were globally distributed, except for African and Australian continents of which virus-targeted metatranscriptome data are currently absent , and thi species . Althoug species . Not onl< 0.001) . These rTuber excavatum (Ascomycota) (Geopora sumneriana (Ascomycota) (Albatrellopsis flettii (Basidiomycota) (Ceratobasidium sp. (Basidiomycota)  , Geoporaomycota) , and Albomycota) . One mitomycota) . Recentlidium sp . Given tentified , but theitovirus . All thein vitro conventionally in association with root-organ cultures (R. clarus and R. irregularis (Rhizoctonia triamitoviruses that are closely related to some (non-large) glomeromycotinian mitoviruses employ no or few UGA codons for Trp  and Narnaviridae (“naked RNA viruses” replicate in the eukaryotic cytosol). Although the origin of this motif, as well as its biological function, deserves investigation, the two events of endogenization of the motif sequence suggest a long history of coevolution between glomeromycotinian fungi and large duamitoviruses. The possibility that a prototype of large duamitoviruses obtained the sequence from the fungal host can also be envisaged because glomeromycotinian fungi are, so far, the only organisms that possess the motif sequence in their genomes. Further surveys on fungal genomes/transcriptomes are necessary. Taken together, we provide new insights into the evolution and ecology of mitoviruses, showing that they are a highly diverse group of mycoviruses and shedding light on their unique evolution across the prokaryote-eukaryote domain boundary.We demonstrated that glomeromycotinian fungi harbor structurally distinct mitoviruses, namely large duamitoviruses, that form the most basal clade in the


Targeted RNA-based therapeutics with multiple specificities and high efficacies are now available. In this review, we discuss progress in RNA-based antitumor therapeutics, including mRNAs, siRNAs, miRNAs, ASOs, saRNAs, RNA aptamers, and CRISPR-based gene editing. We focus on the immunogenicity, stability, translation efficiency, and delivery of RNA drugs, and summarize their optimization and the development of delivery systems. In addition, we describe the mechanisms by which RNA-based therapeutics induce antitumor responses. Furthermore, we review the merits and limitations of RNA cargos and their therapeutic potential for cancers.RNA-based therapeutics  have considerable potential for tumor treatment. The development and optimization of RNA modifications and delivery systems enable the stable and efficient delivery of RNA cargos  RNA-based therapeutics, including messenger RNAs (mRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligomers (ASOs), small activating RNAs (saRNAs), RNA aptamers, and CRISPR-based gene editing—have considerable therapeutic potential for genetic diseases These advantages are particularly beneficial in tumor treatment. Such therapeutics can control the expression of target proteins, reshape the suppressive tumor microenvironment (TME) by regulating cytokine expression, and induce an innate or adaptive immune response RNA-based therapeutics remains a significant challenge due to the inherent susceptibility of natural RNA to degradation by nucleases. As a result, there is a need to modify RNA structures and optimize delivery vehicles to efficiently deliver nucleic acid cargos to the target tissues and/or organs Here we review recent progress in antitumor RNA-based therapeutics—mRNAs, siRNAs, miRNAs, ASOs, saRNAs, RNA aptamers, and CRISPR-based gene editing. We focus on the mechanisms by which RNA-based therapeutics induce antitumor immune responses. We also discuss RNA modification strategies and classify their carriers according to composition. Moreover, we review the relative merits and bottlenecks of various RNA-based therapeutics in tumor treatment Figure .RNA molecules are inherently unstable. Exogenous RNA molecules trigger immune responses, leading to limited level of protein expression. RNA molecule modifications can be used to address these issues, which is the subject of the next section.in vitro transcription (IVT). They consist of five main structures: the 5' cap, the 5' and 3' untranslated regions (UTRs), the open reading frames (ORFs) encoding the target proteins, and the 3' poly(A) tail. These structures affect the stability, translation efficiency, and immunogenicity of mRNA-based therapeutics mRNA drugs are synthetic versions of mature eukaryotic mRNAs and are typically produced by The 5' and 3' UTRs are non-coding regions whose secondary structures, elements, and lengths affect ribosome recruitment and mRNA translation The 5' cap structure contributes to mRNA stability and translation efficiency in vivo. Activation of Toll-like receptor (TLR) 3 can be inhibited by replacing the original nucleotide with 6-methyladenosine (m6A) or 2-thiouridine (s2U), whereas replacement with 5-methylcytidine (m5C), 5-methyluridine (m5U), s2U, m6A, or pseudouridine (Ψ) blocks the activation of TLR7 and TLR8, thus preventing an innate immune response 5C enhance RNA stability and translational capability while diminishing its immunogenicity 1-methyl Ψ-modified nucleotides are employed in IVT to improve the safety and stability of mRNA vaccines. Two mRNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were modified using N1-methyl Ψ and each showed > 90% protection against coronavirus disease 2019 (COVID-19) Nucleotide modification can also optimize mRNA stability, translation efficiency, and immunogenicity siRNA can be chemically modified to enhance the selectivity of their antisense strands for RNA-induced silencing complex (RISC) loading and to reduce off-target RNAi activity. However, unmodified siRNA may induce immune toxicity by activating TLR3. A variety of chemical modification strategies can be used to maximize the therapeutic efficacy and minimize the side effects of siRNAs.Chemical modification typically targets the sugar ring, base, and phosphate skeleton of nucleotides. The goals are to improve the binding affinity of siRNA and protect it from nuclease degradation The most common phosphate skeleton modification is phosphorothioate (PS), which prolongs the half-life in the circulation. Clinically approved siRNA therapeutics  have PS modifications. The phosphorodithioate (PS2) substitution in siRNAs involves the replacement of two non-bridged oxygen atoms. The potency and nuclease resistance of PS2-modified siRNAs are slightly higher than those of PS and unmodified siRNAs. Boron phosphate siRNA may be more effective than phosphorothioate 5C and m6A, tend to be used Base replacement is another modification strategy for siRNA-based therapeutics. 5'-nitroindole modification of siRNAs reduces passenger strand-mediated off-target effects N-acetylgalactosamine -siRNA conjugates enable drug delivery to the liver. Tris-GalNAc binds to asialoglycoprotein receptor, which is highly expressed on the surface of hepatocytes, resulting in rapid endocytosis ASOs are susceptible to nuclease degradation An example of a modified ASO molecule is peptide nucleic acids (PNAs) In vivo administration of 5'-cholesterol-modified aptamers significantly prolongs plasma half-life and exposure The inherent susceptibility of RNA aptamers to nuclease degradation determines their stability 2, 2'-LNA Sp or Rp configuration), unlike the natural conformation, and may therefore have adverse effects on biological function.Modifications of the sugar ring can improve nuclease stability and prolong serum half-life. Similarly, the 2'-OH positions of RNA aptamers can be substituted with 2'-F m +1.6°C, higher than the 2'-OMe modification) in vivom by mediating hydrophobic stacking interactions Chemical modification strategies are under development for other RNA-based therapeutics. The nucleotide sugar ring, base, and phosphate skeleton are modified in anti-miRNA oligonucleotides (AMOs). Most widely used chemical modifications on AMOs are LNA, 2'-F-RNA, 2'-OMe, PNAs. Modified AMOs with higher binding affinity and superior stability have greater regulatory potency RNA therapeutics must be delivered to the correct tissues without triggering an immune reaction. However, the high molecular weight and negative charge of RNA hampers their delivery to target sites. RNA delivery systems can be divided to viral or nonviral. Below we summarize several nonviral RNA delivery systems being extensively studied Figure .in vivo transfection capability and reduce their toxicity Polymeric NPs are typically prepared from biocompatible and biodegradable polymers, in which the drug is dissolved, entrapped, encapsulated, or attached to a nanoparticle matrix. Polymers bind nucleic acids to form polymeric complexes at physiological pH, facilitating gene delivery. Polymeric NPs promote electrostatic binding to nucleic acid cargo by interacting with positively charged units. Nucleic acids and polymers can be covalently linked using degradable linkers. The addition of cationic groups such as chitosan to polylactic-co-glycolic acid (PLGA) enables its use for siRNA delivery Lipid-based NPs have high biocompatibility and biosafety, and their production is simple. They include liposomes, micelles, emulsions, and solid lipid NPs (SLNs). LNPs have been evaluated as mainstream delivery systems in multiple preclinical trials.Because the size, shape, surface charge, and materials of NPs affect their cellular uptake Inorganic NPs  have a narrow size distribution and a surface chemistry suitable for ligand conjugation in vivo for extending the plasma half-lives of AuNPs and enhance their targeted accumulation and controllable release AuNPs typically have diameters of 1 to 100 nm, a large surface-to-volume ratio, good optoelectronic properties, excellent biocompatibility, and low toxicity MSNs are biodegradable and chemically stable nanostructured materials composed of silica particles with pore channels Versatile multifunctional nanomaterials whose synthesis is simple and inexpensive, as well as hybrids that integrate the advantages of different materials, have been developed in vivo delivery of RNA-based therapeutics in clinical applications.Hydrogels consist of a three-dimensional network swollen with water in vitro production and purification of mRNAs prevents host protein and virus contamination in vivo degradation and promote the intracellular delivery and endosomal escape of mRNAs mRNA-based therapeutics are promising alternatives to DNA for cancer immunotherapy due to their lower mutagenicity and easier transient expression. Furthermore, the LNPs consist of phospholipids, cholesterol, PEGylated lipids, and cationic or ionizable lipids. Phospholipids and cholesterol mediate LNP endocytosis and accelerate mRNA release during endocytosis. PEGylated lipids prolong the half-life in the circulation Acute inflammatory responses, such as pain, swelling, and fever, can be caused by mRNA-LNP vaccines  et al.in vitro, enhancing CD11c-cell maturation and CD8 T-cell proliferation in aggressive E·G7-OVA lymphoma models Depending on their charge and composition, LNPs can be broadly classified as cationic LNPs, ionizable LNPs, and lipid calcium phosphate (LCP) NPs. Classical mRNA cancer vaccines target tumor-associated antigens (TAAs) preferentially expressed in malignant cells. For instance, Oberli et al.in vivoSeveral ionizable lipoplex-type mRNA carriers are available. Tateshita et al. et al. reported that a decreased cationic lipid-to-DOPE ratio of mRNA-loaded lipoplexes affected organ specificity. Based on this rationale, they developed a lipoplex-based system for cargo delivery to splenic DCs The clinical applications of the above delivery system are hampered by the lack of specific targeting, which make them useful only for local inoculation and liver-targeted therapy. Efficient lymphatic drainage and accumulation can be promoted by PEGylation and modifying NP size and surface charge Use of mRNAs to restore tumor-suppressor expression has therapeutic potential for cancer. An example is a polymeric NP platform for delivering mRNA encoding phosphatase and tensin homolog deleted on chromosome ten (PTEN), a cancer-inhibiting factor. Reactivating PTEN in PTEN-mutated melanoma cells and PTEN-null prostate cancer cells by mRNA delivery reversed the immunosuppressive TME by promoting CD8 T-cell infiltration and lifting the expression level of proinflammatory cytokines in vivo. The CXCR4-targeting NP system transported p53 mRNA to HCC cells, restoring p53 activity and decreasing HCC cell viability. This mRNA nanotherapy-based p53 restoration strategy in combination with anti-PD-1 therapy induced a potent antitumor effect in intrahepatic and ectopic models of HCC with p53 loss. Therefore, combining p53 mRNA therapeutics with ICB could reverse immunosuppression in HCC p53 is a tumor suppressor involved in cell cycle arrest, apoptosis, senescence, and other cellular pathways + TiDCs in vivo into stimulatory cells that efficiently process spontaneously engulfed TAAs, upregulate costimulatory molecules, and migrate to TDLNs to activate cytotoxic T lymphocytes (CTLs), ultimately delaying the growth of established tumors mRNAs encoding cytokines or chemokines can also induce APC maturation and activation, activate T-cell-mediated immunity, and adjust the dysfunctional immune TME. The mRNA-based adjuvant TriMix consists of mRNAs encoding the costimulatory molecule CD70, the activation stimulator CD40 ligand (CD40L), and constitutively active TLR4 (caTLR4). Upon codelivery of tumor-associated antigen (TAA), the TriMix mRNA reprograms CD8+ T-cell infiltration and IFN-γ expression. The systematic administration of such cytokines leads to high exposure, possibly resulting in toxicity. For this reason, MEDI1191, an optimized IL-12 mRNA, was developed for tumor-targeted local delivery. To minimize off-target liver toxicity, the miRNA-mediated binding site was incorporated into the 3'-UTR of the IL-12 mRNA to promote its elimination Moderna has collaborated with AstraZeneca to develop a local intratumoral mRNA therapy for IL-12 delivery γ mRNAs and is intended for the treatment of lymphoma (NCT03739931). OX40L enhances the expansion and survival of CD4 and CD8 T cells, and IL-23 and IL-36γ are pro-inflammatory cytokines of the IL-12 and IL-1 families, respectively, which activate and mature DCs and other immune cells. Compared to mono-cytokine mRNA therapy, addition of an mRNA encoding the T-cell costimulator OX40L increased the complete response rates of treated and untreated distal tumors. Mice treated with the mixture exhibited complete immune responses and effective protection Preclinical data suggest limited antitumor activity for IL-12 mRNA monotherapy. For this reason, other mRNA therapeutics encoding mixtures of cytokines and chemokines have been developed. Intratumoral injection of DAL4-LNP-IL-12 and IL-27 mRNAs showed synergistic suppression of tumor growth with robust infiltration of immune effector cells Engineered T cells that express chimeric antigen receptors (CARs) for adoptive cell therapy (ACT) have considerable benefits for the treatment of certain blood malignancies Clinical-scale manufacturing of engineered T cells requires their isolation, transfection, modification, amplification, and re-injection, which is difficult and costly. Although virus-mediated transfer can prolong transgene expression by T cells Nanocarrier-mediated targeted delivery of an mRNA encoding a rare-cleaving megaTAL nuclease disrupts T-cell receptor expression ex vivoIonizable lipid NPs have been used to deliver mRNAs to primary human T cells in vivo delivery efficiency.Most solid malignancies failed to effectively respond to CAR-T cell infusion because of tumor resistance, tumor-antigen-escape relapse, and the suppressive TME In one study, a liposomal antigen-encoding RNA was intravenously administered to stimulate tumor-associated T cells in patients with cancer CAR targeting macrophages rather than T cells has emerged as another meaningful strategy for the treatment of solid tumors. MT-101 is a new class of non-T cell CARs produced by transforming monocytes with mRNAs. Most monocytes in blood differentiate into macrophages after migrating to tissues. MT-101 targets tumor cells in peripheral tissues by expressing CARs targeting CD5 on the surface of monocytes. A phase 1/2 clinical study demonstrated the safety and tolerability of MT-101 in patients with refractory or relapsed peripheral T-cell lymphoma (PTCL) at day 28, with no dose-limiting toxicity, cytokine release syndrome (CRS), or immune effector cell-associated neurotoxicity syndrome (ICANS) (NCT05138458). In 2020, Moderna and Carisma Therapies established a cooperative relationship to combine Carisma's engineered macrophage technology with Moderna's mRNA and LNP technology and launched the first clinical study of their so-called CAR-M therapy (CT-0508). The combination of CT-0508 with an anti-PD-1 antibody (pembrolizumab) was used in the phase 1 clinical development stage to treat solid tumors with HER-2 overexpression.siRNAs are double-stranded RNA molecules of 21 to 23 nucleotides, typically with two free bases at the 3'-end, that silence target genes by RNA interference. The precursor is recognized by Dicer RNase and subsequently binds to the target mRNA via the RISC and cleaves it at bases 10 to 11 from the 5'-end, resulting in post-transcriptional gene silencing  et al.2O3 and HER2-siRNA shows an excellent antitumor effect in an orthotopic gastric tumor model. As2O3 induces apoptosis and suppresses tumor metastasis, and HER2-siRNA blocks the expression of the oncogene HER2, inhibiting tumor invasion and metastasis siRNAs can be used in combination with nanomaterials. Currently, various lipid-based delivery systems have been used for co-delivering siRNA and drugs Table . CationiSuitable modification of nanocarriers enables siRNA delivery to target tissues for cancer treatment. One study introduced a versatile codelivery platform for the treatment of triple-negative breast cancer  et al.Silencing key elements of tumor progression or downregulating immunosuppressive genes can induce an antitumor immune response  et al.+ T-cell activation, enhancing the effectiveness of cancer immunotherapy and inhibiting HCC progression in situ-injectable chitosan hydrogel containing C-C chemokine ligand 5 (CCL5) siRNA-loaded NPs, together with mRNA encoding lipid-immune regulatory factor 5 (IRF5), reshaped the TME in a model of pancreatic cancer by promoting M1 macrophage polarization Research is now focusing on combination therapy or codelivery via nanomaterials with siRNA. Guo et al.in vitro and in vivo. Coadministration of nanocarriers loaded with Xkr8 siRNA and the FuOXP prodrug into tumors via intravenous injection significantly inhibited tumor growth in colon and pancreatic cancer models and enhanced antitumor immune responses. In addition, doxorubicin (Dox) induces ICD in tumors, a type of apoptosis that enhances the protective immune response. Stable nucleic acid-lipid particles (SNALPs) have been used for the simultaneous delivery of Dox and an siRNA that knocks down CD47 (siCD47) Combination therapy with siRNA and chemotherapeutic drugs has shown promise for some types of cancer. ChenASOs are synthetic single-strand nucleic acids of 12 to 30 nucleotides. Upon entering a cell, they specifically bind to the target mRNA, forming a DNA-RNA hybrid that triggers mRNA cleavage via recruitment of RNase H, reducing the mRNA level  et al. used a 2.5-generation cEt-ASO (AZD9150) to suppress STAT3 expression, resulting in antitumor activity in lymphoma and lung cancer models Because conventional methods cannot downregulate the protein level of STAT3, Hong et al.The limited cell membrane permeability and lack of nuclear targeting of ASOs hinder their clinical application. To address these issues, ChengmiRNAs are single-stranded, non-coding RNAs typically of 20 to 24 nucleotides. They complementarily bind the 3' UTR of the target mRNA, and then cleave the mRNA or inhibit its translation in vitro and in mouse models of cancer. In addition, mice treated with MRX34 exhibited significant tumor growth inhibition, and a significantly increased survival rate. However, liposomal delivery systems have issues with liver and kidney accumulation and acute hypersensitivity reactions.The first clinical miRNA-based therapeutic was the liposomal formulation of miR-34a, named MRX34 Inorganic NPs are superior to liposomes in terms of their adjustable size and superior pharmacokinetics. For example, a lipid-coated calcium phosphonate NP has been designed for macrophage-targeted miRNA delivery in tumor immunotherapy Vesicular exosomes could be used for miRNA delivery. Exosomes, the smallest extracellular vesicles (EVs) (40 to 160 nm), can transport miRNAs in a paracrine and endocrine manner Small activating RNAs (saRNAs) are small dsRNAs of approximately 21 nucleotides. After generating active Ago-RNA complexes, they trigger gene expression activation by targeting promoter regions. In addition, they have an effect in any genomic region with antisense transcripts Lipid-based delivery systems are commonly used for systemic or local saRNA delivery. Take target gene P21 as example, results illustrated that using targeting saRNA transcriptionally activated p21 expression and promoted cell-cycle arrest in the G1/G0 phase, inhibiting cell proliferation and enhancing chemosensitivity Surface modification, for example, with biodegradable polymers such as PEG, can overcome the toxicity and off-target effects of LNP delivery systems RNA aptamers are sequences 20-100 nucleotides in length with complex three-dimensional structures that bind to target molecules with high affinity and specificity via non-covalent pocket interactions Several clinical trials of aptamers for tumor therapy are ongoing. The guanosine-rich AS1411 is the first oligodeoxynucleotide aptamer to be evaluated in phase I and II clinical trials. It suppresses tumor-cell proliferation by interfering with the stability of Bcl-2 mRNA RNA aptamers are used in combination with other therapeutic agents  for targeted delivery. For example, the combination of a PSMA-specific aptamer for prostate cancer with therapeutic oligonucleotides inhibited oncogene activity in PSMA-expressing cells, thereby having an antitumor effect The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has unprecedented therapeutic potential for genetic diseases The two critical components of the CRISPR/Cas9 system are the Cas9 protein and gRNA. The principle of this system involves the insertion of a specific DNA sequence (spacer) from the invading virus and plasmid into the CRISPR locus. Upon re-infection, the CRISPR sites containing the spacer acquired previously are transcribed and the products are processed into mature gRNAs. The gRNA targets Cas9 to a particular genomic locus, where it induces double-strand breaks (DSBs). For binding to and cleavage of DNA by Cas9, the 3'-side of the target sequence must have an NGG protospacer adjacent motif (PAM)  et al.Cancer arises from the accumulation of genetic/epigenetic abnormalities. CRISPR-Cas9-mediated genome editing enables precise manipulation of a genomic sequence, enabling the identification of genes involved in carcinogenesis and correction of oncogenic mutations  et al.in vivo, promoting tumor-cell apoptosis. In addition, the technique allows antibody-targeted delivery for the treatment of diffuse tumors. Intraperitoneal injection of EGFR-targeted sgPLK1-cLNPs resulted in selective uptake into diffuse ovarian tumors, leading to up to ~80% gene editing in vivo, inhibiting tumor growth and improving survival  et al.However, the CRISPR/Cas9 system has limitations related to cell injury, limited packaging capacity, and immune activation. In addition, the large sizes of Cas9  and sgRNA  preclude the use of viral and nonviral delivery systems in vivo delivery system have been developed, RNA-based drugs can be rapidly designed and synthesized. However, several technical bottlenecks affect RNA-based antitumor therapies, including their targeting specificity, safety, and efficacy. To address these issues, a variety of optimization and modification strategies for RNA molecules and delivery systems have been explored.Although cell and antibody-based therapies currently dominate tumor immunotherapy, more work is needed to overcome the risk for side effects, induction of an inappropriate and potentially harmful immune response, the complexity of treatment, and the high cost of production. RNA-based therapeutics  can modulate the expression of target genes to varying degrees. In addition, they can stimulate an immune response or reshape the suppressive TME by producing antigens or restoring the levels of beneficial proteins. The ability to target multiple genetic components is an advantage of RNA therapeutics over other small-molecule or protein-based drugs. Furthermore, once the chemical structure of the RNA molecule and the Therapeutic effectiveness is influenced by off-target effects induced by nonspecific accumulation. To prolong accumulation at tumor sites, promote target uptake, and control drug release, surface modifications with targeting ligands have been developed The safety of RNA cargos is a key consideration for their clinical application. The innate immunostimulatory properties of RNA molecules and their function as immune adjuvants enhance the immune response to antigens during vaccine development. However, excessive immunogenicity can lead to severe adverse reactions. Clinical translation requires the striking of a balance between safety and efficacy. The development of more biocompatible delivery vehicles with better biodegradability would accelerate the clinical translation of nanomedicines in vivo targeting of immune cells. Such approaches could enhance the effectiveness of immunotherapy and so reduce the RNA-based drug dose required. Because drugs have different pharmacokinetic and pharmacological characteristics, as well as interindividual differences in drug distribution after systemic administration, multiple-drug treatment strategies must consider the optimal dosage ratio and sequential release to reduce toxicity and enhance efficacy. P-gp inhibitors should be released by delivery systems prior to any co-delivered drugs. Otherwise, the co-delivered drug(s) might be exported extracellularly by P-gp, thus reducing their therapeutic efficacy.Another crucial issue is therapeutic effectiveness. Tumorigenesis involves complex regulatory networks and immune-escape mechanisms. Treating tumors with a single modality is often unsatisfactory. Treatment of tumors requires multiple modalities, potentially including codelivery of cytokines and chemokines, alleviating immunosuppressive signaling in the TME, and In conclusion, we reviewed the modifications of, delivery systems for, and potential applications of RNA-based therapeutics for cancers. Although RNA delivery platforms have limitations, advances in RNA-based bioengineering mean considerable therapeutic potential for cancer treatment. Indeed, these modalities offer hope for types of cancers with few or no treatment options.


RNA plays a vital role as the central and necessary intermediary in the flow of genetic information between DNA and proteins. Throughout their lifecycle, RNA molecules are accompanied by a multitude of RNA-binding proteins (RBPs). RBPs mediate the transcription, processing, export, translation, and degradation of RNA molecules. To perform all these functions, living organisms possess a large number of RBPs, with more than 800 in plant species .targets of RBPs identified by editing, was developed in the animal field   and subsequent improvements . These aal field  and recehaliana) . This mehaliana) .Plant Physiology, Oryza sativa) DOUBLE-STRANDED RNA BINDING PROTEIN 1 (OsDRB1) to identify putative RNA ligands, opening the door to use this technique in other plant species outside the model plant Arabidopsis.In this issue of To characterize the RNA editing properties of ADARdd in plants, the authors made use of 2 well-established RBP–RNA ligand pairs from a virus and Arabidopsis . By coexOnce ADARdd was confirmed as functional in rice protoplasts and that it could target single-stranded RNA, the authors generated stable transgenic rice lines expressing ADARdd fused to OsDRB1 and conducted RNA-seq experiments with roots and leaves from adult plants. High-confidence editing sites were identified applying a stringent pipeline to the RNA-seq data. Interestingly, 25% to 50% of the identified editing sites were organ specific, suggesting important tissue specificity of OsDRB1–RNA interactions. These editing sites were located in almost 800 annotated rice transcripts. Most of these transcripts arose from repetitive elements and coding genes. In the case of coding genes, the editing sites were located predominantly in the noncoding parts . As most small RNAs (sRNAs) are generated from noncoding regions of transcripts and from repetitive elements , the autIn summary, O. sativa) OsDRB1. A fusion protein of OsDRB1 with the deaminase domain of ADARdd is expressed either stably in transgenic plants or transiently in protoplasts. ADARdd deaminates the adenosines of the RNA molecules bound to OsDRB1, transforming them into inosines that can be detected by sequencing, adapted from A schematic representation of the TRIBE approach was employed in this study to identify the RNA ligands of rice (