In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3′ untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of—or breeding for—resistance to MCMV. 7GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5′ UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3′ CITEs.Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3′ untranslated region (UTR). The MCMV 3′ CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2′-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3′ CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an mIMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3′ untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of—or breeding for—resistance to MCMV. Maize lethal necrosis disease , first identified in the Americas in the 1970s –9. It ha–Potyviridae family (––MLND is caused by a mixed infection of maize chlorotic mottle virus (MCMV) and any potyvirid that infects maize, usually sugarcane mosaic virus (SCMV) , 15, 16.e family , or in te family , 18. Effe family –21, but  family –, 23 with family ––26, littMachlomovirus in the family Tombusviridae . The MTE is structurally similar to the panicum mosaic virus-like translation element (PTE) class of CITE, but with some key differences. We identify a key translation initiation factor with which the MTE interacts (eIF4E) and show how the MTE base pairs to the 5′ UTR to facilitate cap-independent translation, and that the functional MTE and the long-distance interaction are required for infection of maize. The results contribute to our understanding of structure-function relationships of cap-independent translation elements, and provide valuable information on the first step of gene expression for an important pathogen.Because all other tested tombusvirids harbor a 3′ CITE, we predicted that MCMV RNA harbors a 3′ CITE to facilitate its translation. 7GpppA cap analog and T7 RNA polymerase . Plasmid psgRNA1 was the template for transcription of full-length sgRNA1, the mRNA for the 25-kDa CP and the movement proteins . Linearproteins . Transcring ORFs . Interes as well –46. Unlias well –. It is nin vitro or in vivo when either the 3′-terminal 104 nt (nt 4334 to 4437) or the first 169 nt (nt 4095 to 4263) of the 3′ UTR were deleted .To rapidly map the 3′ CITE location at high resolution, a luciferase reporter (MlucM) was constructed such that the coding region of the virus was replaced by the firefly luciferase (Fluc) coding sequence . Deletio deleted . Additio deleted . Howeveral bases –49. Dele4215, A4216, and G4219 were highly modified by the SHAPE reagent benzoyl cyanide, while bases AGA4221–4223 became less modified (4235–4241) houses a pentamer, UGCCA4236–4240  experimentally by subjecting it to selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing . This remodified . The MTEoxes, 2+ .4221–4222:CU4249–4250 in the MTE. The SHAPE probing (4221–4222 decreased in the presence of Mg2+ (which favors pseudoknot formation), and they are thus probably base-paired. Yet, the already-modest SHAPE sensitivity of bases CU4249–4250 in the bridging domain does not decrease in the presence of Mg2+, as would be expected if the proposed pseudoknot forms. The bridging domains of other PTEs in which this pseudoknot is likely, however, also show little change in modification in the presence of Mg2+  to three (AGA4221–4223:UCU4248–4250) base pairs, translated only 55% as efficiently as wild type in WGE , estimated as the eIF4E concentration at which half of the MTE is shifted, is about 80 nM.Previously, PTEs have been shown to bind and require eukaryotic translation initiation factor 4E , despite the absence of methylation (cap structure) on the PTE RNA , 57. Becof eIF4E . Also asof eIF4E , 59. Likof eIF4E . Based oWe next tested the ability of mutant MTEs to bind eIF4E, in order to determine if eIF4E binding correlates with the translation enhancement function, as was observed previously for the TPAV PTE. Differences in binding affinity can be seen most clearly by the amount of shifted eIF4E at the 100 nM concentration in the gels in in silico analysis of the 5′-end structure of MCMV revealed two sites (GGCA12–15 or UGGCA103–107) that could potentially base pair with the MTE sequence UGCCA4236–4240 in loop 1. An RNA transcript containing the 117-nt 5′ UTR followed by the first 23 nt of the coding region, including the P32 (AUG118–120) and P50 (AUG137–139) ORF start codons, was subjected to SHAPE probing to determine which region was most likely to be available (single stranded) to interact with the MTE (12–15) is buried in a stem helix, while the UGGCA103–107 is in a favorable loop  of the above candidate sequences base pairs to the MTE. Mutations were introduced in the XGCCA regions in the 5′ UTR (nt 11 to 15 or 103 to 107) and the MTE UGCCA0 region . Mutatio systems . In cont% to 90% . These mmutation . While t2979–2983 in the short 25-nt 5′ UTR of sgRNA1 which matches the UGGCA103–107 that base pairs to the MTE. This sequence is predicted to be in the terminal loop of the stem-loop that occupies the 5′ UTR of sgRNA1  were observed. In agreement with the luciferase reporter constructs, mutants MCM41C4238G and MCM41G105C, which disrupt the long-distance base pairing between MTE and 5′ UTR, yielded less viral protein than wild type , was not expected to be translated much from genomic RNA as seen in U4218G/GA4247-4248CC, showed a substantial decrease in translation, in contrast to the same mutation in the luciferase reporter, in which translation was increased by 50% . For comparison, wild-type MTE folded the same as in C4238G and MCM41G105C, which had disrupted long distance base-pairing, yielded about 35 to 40% as much viral RNA replication product as wild type, while the double mutant MCM41G105C/C4238G, yielded about twice as much viral RNA as the single disruptive mutants  cell culture were transfected with full-length mutant MCM41 transcripts. Unfortunately, in BMS protoplast preparations, high levels of RNase obscured detection of distinct viral RNAs in Northern blot hybridization, so the level of viral RNA was quantified by simple dot blot hybridization. Single mutants MCM41 mutants . On the G4219U had a similar infectivity to wild type, but in about half of the infected plants the virus reverted to wild type . Maize B73 plants inoculated with wild-type MCM41 transcript began to exhibit chlorotic mottling symptoms between 8 and 10 days postinfection (dpi), while sweet corn (Golden Bantam) plants exhibited symptoms around 6 to 9 dpi. Viral RNA was detected via reverse transcriptase PCR (RT-PCR) in inoculated leaves of all plants at 8 dpi, including those that never showed chlorotic mottling. At 14 dpi, samples from the newest systemic leaves were subjected to Northern blot hybridization with a probe complementary to the 3′ end of the MCMV genome . Followiild type . Moreoveless RNA , suggestectivity  and RNA ectivity  was reduG105C/C4238G double mutant acquired additional mutations. These spontaneous mutations consisted of deletion of G94 in the 5′ UTR and a U492A point mutation in the overlapping P32 and P50 ORFs. This mutation introduced a stop codon in the P32 frame, shortening the protein from 289 aa to 125 aa, and changed amino acid 119 from valine to glutamic acid in the P50 and P111 (RdRp) proteins. This spontaneous mutant did not induce symptoms until 12 dpi, but after 14 dpi, symptoms were more extreme than wild type, giving nearly translucent leaves , but some MTE mutants that did not confer efficient translation still bound eIF4E with high affinity, while others did not. Importantly, all functional mutants bound eIF4E with high affinity (approximate Kd ≤100 nM), consistent with a requirement for this factor. eIF4E binding by nonfunctional mutants may be due to different folding of the RNA in the short, isolated sequence context used in EMSA, or these mutants may have lost the ability to bind other translation components required in addition to eIF4E. eIF4E facilitates translation while bound to its partner eIF4G, forming the heterodimer eIF4F. It is possible that the eIF4G component may affect binding affinity to the MTE either by altering eIF4E conformation or by binding directly to the MTE as well. While the PTEs bind directly to eIF4E, barley yellow dwarf virus-like 3' CITEs (BTEs) bind directly to eIF4G, and these and other types of 3′ CITE all bind with higher affinity to eIF4F than to eIF4E or eIF4G alone , as well as the entire 3′ UTR, stimulated translation more than the 3′ UTR alone . It is d103–107 in the 5′ UTR, could participate in the long-distance base pairing. For example, Simon’s group reported that a stem-loop in the 5′ end of the 5′-proximal ORF base pairs to a region adjacent to the PTE in PEMV2 RNA  UGGC179–182, and (iii) UGGC287–290. However, none of these would form as stable a base pairing to UGGCA as the UGGCA103–107 sequence we identified as required for base pairing to the MTE. Further, SHAPE probing indicates that the three middle bases of the UGGCG138–142 motif are base-paired and thus unlikely to be accessible for the long-distance interaction. These sequences may also be too far from the 5′ terminus to allow efficient delivery of translation factors for entry of the ribosomal 40S subunit at the 5′ end. In contrast, the PEMV2 sequence in the coding region is just 71 to 76 nt from the 5′ end, and in a clearly identifiable loop of a strong stem-loop , in addition to the UGGCAEMV2 RNA . For MCMThe translation of CP (P25) from full-length MCM41 RNA observed in C105G/G4238C transcript, which translates more efficiently than wild type in WGE, replicates indistinguishably from wild-type MCM41 in protoplasts, but accumulates to much lower levels in whole plants. This is to be expected because the G4238C mutation in the MTE would prevent base pairing to the 5′ end of sgRNA1, which remains a wild -type sequence. Thus, we predict translation of the CP and movement proteins is reduced in this construct. CP and movement proteins are not necessary for replication of other tombusvirids in protoplasts but would, of course, be necessary for the virus to accumulate in plants, thus explaining the difference in accumulation in protoplasts versus plants  compared to that of genomic RNA 5′ UTR . We expected the sgRNA1 5′ UTR to confer superior translation efficiency because it should provide less resistance to ribosome scanning. Perhaps in infected cells, in the presence of the abundant sgRNA2, which contains the MTE, the sgRNA1 5′ UTR could outcompete the genomic RNA 5′ UTR for limiting eIF4E, as discussed below.Comparison of translation efficiencies of genomic (MlucM) and subgenomic RNA (Msg1lucM) reporter constructs revealed no striking difference in translation efficiency conferred by the 5′ UTR . This iscis, the MTE may regulate translation initiation in trans. Like certain other tombusvirids, such as red clover necrotic mosaic virus (RCNMV) (Tombusviridae) binds the eIF4G subunit of eIF4F and, as a result, inhibits translation of BYDV genomic RNA (gRNA) while favoring translation of BYDV sgRNA1, which, like MCMV sgRNA1, codes for movement and coat proteins. This would free genomic RNA of ribosomes, making it available for replication, encapsidation, and cell-to-cell movement. The selective inhibition of BYDV gRNA is due to its highly structured 5′ UTR, which likely increases dependence on eIF4F relative to the unstructured 5′ UTR of sgRNA1  , 80, tob (RCNMV) , and the (RCNMV) , MCMV ge (RCNMV) . These R (RCNMV) . Because (RCNMV) . sgRNA2 f sgRNA1 . The samproteins , 85–87, teins 85–.7G cap to eIF4E via the cap-binding pocket  such that a firefly luciferase  reporter gene was flanked by the 5′ and 3′ UTRs of MCMV (The full-length infectious clone of the MCMV genome (pMCM41) and pMCM721 were described previously . For psg of MCMV . A Q5 si of MCMV  and muta of MCMV  and 11 oin vitro transcription with T7 polymerase using MEGAscript (for uncapped RNAs) or mMESSAGE mMACHINE (for capped RNAs) kits (Ambion). RNAs used as probes for uncapped EMSAs were generated using MEGAshortscript kit (Ambion). RNA transcripts were purified according to the manufacturer’s instructions and the RNA clean and concentrator kit (ZymoResearch) was used for nonradioactive RNA preparation. RNase-free Bio-Spin columns P-30 (Bio-Rad) were used for radiolabeled RNA. RNA integrity was verified by 0.8% agarose gel electrophoresis. RNA concentration was determined by spectrophotometry for nonradiolabeled RNA. Radiolabeled RNA concentration was calculated by measuring the amount of incorporated radioisotope using a scintillation counter. At Oklahoma State University  as described , 93 mM potassium acetate, and 2.1 mM MgCl2 based on the manufacturer’s instructions. Translation reaction mixtures were incubated at room temperature (25°C) for 30 min. Translation products were separated by electrophoresis on a NuPAGE 4–12% Bis-Tris gel (Invitrogen), detected with Pharox FX plus Molecular Imager and quantified by Quantity One one-dimensional analysis software (Bio-Rad). For luciferase reactions, 10 μl of the translation reaction product was mixed into 50 μl of Luciferase assay reagent (Promega) and measured immediately on a GloMaxTM20/20 luminometer (Promega). Statistical and data analysis were performed using GraphPad Prism software.escribed . Nonsatu6 oat (Avena sativa cv. Stout) protoplasts along with capped mRNA encoding Renilla luciferase (1 pmol) (Renilla luciferase. Background firefly relative light units (RLUs), measured in the absence of added luciferase mRNA, were subtracted from the values obtained with MlucM and its deletions and/or mutant derivatives. Statistical and data analysis were performed using GraphPad Prism software.Uncapped MlucM (10 pmol) or its derived mutants were coelectroporated into ∼2 × 10(1 pmol)  as an in(1 pmol) . After 42) for 30 min at 30°C. RNA was modified by mixing 1/10 (vol/vol) ratio of renatured RNA with 60 mM benzoyl cyanide (Sigma) in anhydrous dimethyl sulfoxide (DMSO) (Sigma). After 2 min at room temperature, the RNA was mixed with 4-fold excess tRNA and precipitated in 3 volumes of 99% ethanol and 1/10 volume 3 M sodium acetate. Control RNA was treated with the same amount of DMSO without benzoyl cyanide. The primer  was 5′-end-labeled with [γ-32P]ATP and used for primer extension. Gel electrophoresis conditions and phosphorimager visualizations were performed as described previously  was applied to selected UTR fragments of MCMV following the procedure previously described  for the eviously , 97.E. coli (BL21) cells and eIF4E expression was induced at an optical density at 600 nm (OD600) of 0.7, with 0.5 mM IPTG . From here on, the purification procedure of the protein was similar to previous published work (E. coli cells was induced by incubating in IPTG for 2 h at 37°C with shaking (160 rpm). Cells were harvested by centrifugation at 6,000 × g for 15 min at 4°C, and cell pellets were quick frozen before purification. E. coli cells were disrupted by sonication in buffer B-50  containing complete protease inhibitor cocktail tablets (Roche). Cell debris was separated from the supernatant by 3 to 5 rounds of centrifugation , where for each centrifugation the supernatant was transferred to a clean round-bottom centrifugation tube. Wheat eIF4E protein was purified using m7GTP agarose beads as described previously , 0.1 μg/μl yeast tRNA, 1 μg/μl bovine serum albumen, 1 unit/μl RNaseOUT recombinant RNase inhibitor (Invitrogen), and 20 mM Tris-HCl/10% glycerol for 25 min in ice. Then, 3 μl of 50% glycerol was added to each reaction. Immediately after, 7 μl of RNA-protein mixture was loaded into a 5% polyacrylamide (acrylamide:bis-acrylamide 19:1), Tris-borate/EDTA (TBE) gel, which was run at ∼4°C at 110V for 45 min in 0.5× cold TBE buffer. Gels were dried on Whatman 3MM paper and exposed to a phosphorimager screen overnight. Phosphor screens were scanned in a Bio-Rad PhosphorImager, and radioactivity counts were analyzed using Quantity One software (Bio-Rad). Statistical analysis was performed using GraphPad Prism software.Calculated specific activities of probes were used to determine the molarity of RNA in each purified stock. RNA probes were subjected to 6 M urea-TBE gel electrophoresis to verify the quality of the RNA. RNA was diluted to 10 fmol/10 μl for electrophoretic mobility shift assays (EMSAs). As previously described ,32P-RNA-6) were transfected with 5 pmol of pMCM41 and pMCM41 mutant transcript RNA using PEG-1540 . Protoplasts were diluted slowly in 5 ml of solution M , followed by incubation at 4°C for 20 min. Protoplasts then were centrifuged at 100 × g for 5 min, followed by two washes with solution M. PGM buffer  was then added to each protoplast sample, followed by incubation in the dark at room temperature for 24 h. RNA was isolated using Plant RNAeasy kit (Qiagen) following the manufacturer’s instructions. Purified RNA was analyzed on a 0.8% native agarose gel and quantified using a Nanodrop spectrophotometer.Protoplasts were isolated from Black Mexican Sweet (BMS) suspension cultures as described previously , 29. ForMaize (B73 and sweet corn varieties) was grown in growth chambers on a 16:8 photoperiod with a temperature setting of 25°C/22°C (day/night). Maize seedlings were inoculated at the three-leaf stage. Plants were dusted with carborundum and inoculated on the third leaf with 10 μg of purified MCMV transcripts in 10 mM sodium acetate, 5 mM calcium chloride, and 0.5% bentonite by stroking three times with a freshly gloved finger. Inoculated leaves were rinsed with water 10 min postinoculation, and rinse water was collected and autoclaved prior to disposal. Total plant RNA was extracted using TRIzol (Invitrogen) from 100 mg of the newest systemic leaves for Northern blot hybridization (14 dpi) and cDNA synthesis. Quality of RNA was evaluated in a 0.8% native agarose gel, and RNA was quantified using a Nanodrop spectrophotometer. RNA extracted from infected plants was subjected to cDNA synthesis and reverse transcriptase PCR (RT-PCR) and sent to the Iowa State University DNA Facility for sequencing to evaluate the presence of introduced mutations.32P-labeled probe complementary to MCMV nt 3811 to 4356, which had been transcribed using SP6 RNA polymerase. Washed blots were wrapped in plastic and exposed to phosphor screens. Phosphor screens were scanned in a Bio-Rad PhosphorImager and radioactivity counts were analyzed using Quantity One software (Bio-Rad). Statistical analysis was performed using GraphPad Prism software.RNA isolated from BMS protoplasts had a higher ratio of degraded RNA genomic/subgenomic RNA in Northern blot hybridizations, such that lower molecular weight fragments overpowered the genomic and subgenomic RNAs. However, a trend on the amount of MCMV RNA detected suggested that replication was still occurring in protoplasts; thus, dot blots were used instead. Unfractionated RNA from protoplasts was denatured prior to immobilization on a nylon membrane using a vacuum manifold apparatus as described in Brown et al. 2004 . RNA froThe presence of MCMV virus RNA was evaluated by RT-PCR. Total RNA isolated from inoculated maize plants was extracted from washed 3rd leaves using TRIzol. The concentration and purity of extracted RNA was confirmed using a NanoDrop ND-2000 spectrophotometer and the quality of RNA was evaluated via 0.8% native agarose gel electrophoresis. One microgram of RNA was subjected to cDNA synthesis following the manufacturer’s instructions for Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific). The cDNA was amplified using MCMV-CP primers  for 25 cycles, and the products were separated on 1% agarose gels, visualized by SYBR safe DNA stain (Thermo Fisher Scientific) and photographed. Similar to MCMV-CP, maize ubiquitin cDNA expression  was analyzed in the same sample set to serve as an endogenous positive control.


In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish  Autophagy can be induced by various stressors, such as nutrient deprivation and UV damage, but also infection. Detection of microbial invaders by the innate immune system activates the autophagy machinery to capture intracellular pathogens in double-membrane autophagosomes and target them to lysosomal degradation3. Autophagy proteins can also be recruited to single-membrane compartments when pathogens are engulfed by phagocytic cells3. These autophagic defense mechanisms form promising targets for the development of novel host-directed therapies for infectious diseases, many of which are currently complicated by the increasing occurrence of antibiotic resistance5. This is especially true for tuberculosis (TB), the most lethal infectious disease worldwide. The causative agents of human TB or TB-like disease in poikilothermic animals, Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm), are widely studied to increase understanding of the role of autophagy in host defense10.Autophagy is an intracellular degradation mechanism that functions to maintain homeostasis and intersects with the initiation of cell death programs when homeostasis is perturbed16. Among the five DRAM family members, human DRAM1 was first identified as a p53-induced protein that localizes predominantly to lysosomes and promotes autophagic flux as well as UV-damage-induced apoptosis11. In response to mycobacterial infection, DRAM1 transcription is induced by nuclear factor kappa B (NFκB), a central hub in the signaling network regulating the immune system6. DRAM1 colocalizes with Mtb in infected human macrophages and is required for host resistance of zebrafish larvae against Mm infection6. Mtb and Mm share the RD1 virulence locus, required for ESX1-dependent escape from phagosomes into the cytosol17. Selective autophagy, dependent on ubiquitin receptors, such as p62, may counteract this pathogenic mechanism by delaying the escape process or sequestering cytosolic bacteria8. In addition, selective autophagy has been shown to deliver anti-microbial ubiquitinated peptides to bacteria-containing compartments18. We have recently shown that selective autophagy receptors are required to control Mm infection in zebrafish and that the host-protective function of Dram1 requires p62 in this infection model19.DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 and other members of the DRAM family have been linked to cancer, myocardial infarction, HIV infection, and TB, but their molecular and cellular functions remain poorly understood8. In TB, the death of an infected macrophage triggers its phagocytosis by other macrophages that subsequently also undergo cell death, resulting in a cascade of cell death events and the formation of inflammatory infection foci, called granulomas20. The fate of individual infected macrophages is therefore a major determinant of whether granulomas can contain the infection or facilitate dissemination of the infection. It has been previously shown that mycobacteria-infected macrophages can undergo several types of regulated cell death, like apoptosis, necroptosis, and pyroptosis, resulting in different infection outcomes21. Apoptosis of infected cells is generally regarded as a host-protective defense mechanism against mycobacterial infection, and virulent Mtb therefore actively inhibit apoptosis23. In contrast, necroptosis and pyroptosis are lytic forms of cell death that create an inflammatory environment that may facilitate extracellular growth and disease progression25.Cytosolic escape of mycobacteria may accelerate the initiation of host cell death programsdram1 mutant zebrafish line to address this question. Analysis of the mutant fish showed that Dram1 is required for maturation of Mm-containing vesicles and for macrophages to restrict Mm infection. Without functional Dram1, Mm-infected macrophages prematurely die via a mechanism dependent on inflammatory Caspase a (Caspa) and Gasdermin eb (Gsmdeb) activities, indicative of pyroptosis. Collectively, our data support that Dram1 protects against mycobacterial infection by modulating autophagic targeting and maturation of Mm-containing vesicles. In the absence of Dram1, infected macrophages rapidly become overburdened by the bacteria and initiate pyroptotic cell death, resulting in increased dissemination of the infection.While our previous work demonstrated a role for Dram1 in autophagic defense against mycobacterial infection, its potential implication in the regulation of cell death during TB pathogenesis has not been explored. In this study, we generated a dram1∆19n/∆19n allele (designated dram1ibl53) contains a 21 nucleotides deletion combined with a two nucleotides insertion in the first coding exon 26 or a macrophage marker Tg(mpeg1:mCherryF)27, hereafter referred to as GFP-Lc3 and mpeg1:mCherry. The offspring from incrossed heterozygous fish (dram1+/∆19n) strictly followed Mendelian inheritance  signaling, phagosome-related processes, cytokine signaling, and apoptosis, were commonly affected in all Mm-infected larvae, other immune-related pathways, like Toll-like receptor (TLR) and RIG-I-like receptors signaling, were not affected in dram1∆19n/∆19n larvae infected with 150 CFU Mm  were not differentially expressed in dram1∆19n/∆19n mutants  compared with dram1+/+ when we applied a cellular stress factor, Bafilomycin A1 (BafA1), which inhibits vacuolar H+ATPase (V-ATPase) to prevent autophagolysosomal maturation , before differences in bacterial burden were detectable. Approximately 60% of Mm clusters were LysoTracker-positive in dram1+/+, while this was reduced to 20% in dram1∆19n/∆19n  is the Caspase-family member that induces pyroptosis of Mm-infected macrophages in zebrafish via Gasdermin Eb (Gsdmeb)40. Thus, we analyzed Caspa activity at 2dpf, the time point where we observed increased cell death in dram1∆19n/∆19n. We detected a minor but significant increase of whole larvae Caspa levels in dram1∆19n/∆19n infected with Mm compared with the non-infected mutant, but such infection-dependent increase was not observed in dram1+/+  in dram1 mutants could also contribute to this phenotype. Finally, the upregulated expression of genes in lytic cell death pathways may contribute to pathological inflammation and is consistent with the increased bacterial burden in dram1 mutants.The 11. We therefore tested whether zebrafish dram1 mutants display defects in autophagic processes. Autophagy is a host response to diverse stress factors, including starvation. Zebrafish larvae until 5 dpf can rely on their yolk proteins for nutrients43, and we therefore assumed that their autophagic processes are not activated above a level normal for their developmental stage, unless autophagy is triggered by a stressor such as infection. In agreement, we did not detect any differences when comparing the basal levels of autophagy activity in uninfected dram1 mutant larvae of 4 dpf to those of their wild type siblings. This finding is consistent with an in vitro study of the function of mouse DRAM144, which showed that basal autophagy was not altered in the absence of DRAM1 in primary mouse embryonic fibroblasts (MEFs). DRAM1 deficiency did not change autophagy induction in response to starvation or while blocking mTOR45. However, the lack of DRAM1 affected the activation of autophagy in human cells (HeLa and A549) following the induction of cellular stress by treatment with the mitochondria inhibitor 3-nitropropionic acid (3-NP)46. Besides infection, DNA-damage, and interference with energy metabolism14, it remains to be further investigated which stress factors can activate DRAM1/Dram1 in vitro and in vivo.The function of DRAM1 as a modulator of autophagy has been studied well in vitrodram1 mutant larvae with BafA1 revealed an increase of GFP-Lc3 puncta and Lc3-II protein levels. The dram1 mutants also accumulated higher Lc3-II protein levels than controls under conditions of Mm infection. The prolonged stress conditions imposed to zebrafish larvae during BafA1 treatment or infection may induce a compensatory response in dram1 mutants to produce more autophagosomes. Importantly, despite the increased Lc3-II levels in infected dram1 mutants, imaging in GFP-Lc3 transgenic fish revealed that mycobacteria are targeted by autophagic vesicles nearly three-fold less frequently in dram1 mutants compared to wild type zebrafish larvae. This reduced autophagic targeting of Mm was not due to a different phagocytic ability of zebrafish macrophages. We did, however, find that Dram1 deficiency reduced acidification of Mm-containing vesicles, which was associated with the premature death of infected macrophages. The resulting higher mycobacterial burden of infected zebrafish is in line with increased expansion of extracellularly growing Mm47. While we found that Mm-infected macrophages die more rapidly in the absence of Dram1, human DRAM1 has been shown to trigger lysosomal membrane permeabilization and cell death in HIV-infected CD4+ T cells, thereby lowering viral replication (14). Together, these studies indicate that DRAM1/Dram1 expression levels can have a major impact on cell death processes during infection, with different outcomes depending on the cell type and infectious agent.Blocking autophagy flux in 12, but an effect of Dram1 deficiency on apoptosis was not observed in our infection study. Strikingly, we found that Dram1 deficiency leads to increased inflammatory caspase activity and gasdermin-dependent pyroptotic cell death during infection. Previous studies revealed that pyroptosis can be induced by diverse pathogens and forms a critical mechanism to restrict microbial infection49. In line with this, there is also evidence that mycobacteria inhibit pyroptosis of infected macrophages via diverse mechanisms50. However, recent studies found that lytic cell death (pyroptosis and necroptosis) helps mycobacteria to evade host immunity and disseminate the infection51. Indeed, in the present study we found that pyroptotic cell death promotes the expansion of mycobacteria in Dram1-deficient zebrafish hosts. Moreover, genetic inhibition of Caspase a and Gasdermin Eb could rescue the exacerbated bacterial growth in dram1 mutants, confirming that pyroptosis rather than necroptosis is responsible for the hypersusceptibility phenotype. Taken together, the death of infected macrophages is intricately related to TB pathogenesis and can result either in increased dissemination or restriction of the infection in the zebrafish host. The contradicting evidence discussed above concerning the beneficial or detrimental effects of the different modes of cell death suggests that the balance between different cell death modalities plays a crucial role in determining the outcome of the infection and a careful characterization of the specific type of cell death being studied is critical for a better understanding of TB pathogenesis.DRAM1 was previously shown to mediate apoptosis by blocking the degradation of the pro-apoptotic protein BaxIn conclusion, restriction of mycobacteria in infected macrophages during the early stages of infection requires functional Dram1. In this work, we have shown that Dram1 is involved in several processes important to defense against intracellular pathogens, potentially providing an intersection between modulation of autophagy, lysosomal function, and regulated cell death. Future studies are required to precisely elucidate the role of the lysosomal protein Dram1/DRAM1 in this network. Using an in vivo model for the early stages of TB disease we have demonstrated the importance of Dram1 for the elimination of intracellular mycobacteria and the cell fate of infected macrophages.dram1 mutant was approved by the Animal Experimentation Committee of Leiden University (UDEC) under protocol 14198. All experiments were done on embryos or larvae up to 5 days post-fertilization, which have not yet reached the free-feeding stage. Embryos/larvae were kept in egg water  containing 0.003% 1-phenyl-2-thiourea  at 28.5 °C and treated with 0.02% ethyl 3-aminobenzoate methanesulfonate  for anesthesia before bacterial injections, imaging and fixation.Zebrafish lines in this study Table  were handram1 (ENSDARG00000045561) were designed using the chop-chop website52 and generated by PCR complementation and amplification of full-length ssDNA oligonucleotides  and purified by RNeasy Mini Elute Clean up kit . The Cas9 mRNA was transcribed using mMESSAGE mMACHINE® SP6 Transcription Kit  from a Cas9 plasmid  and purified with RNeasy Mini Elute Clean up kit . A mixture of sgRNA and Cas9 mRNA was injected into one-cell stage AB/TL embryos (sgRNA 150 pg/embryo and Cas9 mRNA 300 pg/embryo). The effect of CRISPR injection was confirmed by PCR and Sanger sequencing. Genotyping was performed by PCR-amplification of the genomic region of interest using the following primers: Forward: 5′-AGTGAACGTCCGTGTCTTTCTT-3′, Reverse: 5′-ACATCTTGTCGATACAAAGCGA-3′; followed by Sanger sequencing to identify mutations 19.Short guide RNAs (sgRNAs) targeting the first coding exon of zebrafish D. rerio GRCz10.80 reference genome and read counting were performed by ZF-screens . Analysis of the count libraries was performed in RStudio 1.1.38353 running R 3.4.354 using in-house scripts (available at github.com/gabrifc). An initial quality check of the samples was performed using the tools provided in the edgeR package v3.20.755. Differential gene expression was assessed via pairwise comparisons using DESeq2 v1.18.156. Genes with a FDR-adjusted p-value  <0.05 were considered statistically significant. Venn Diagrams were created using the R package VennDiagram v1.6.1857. Gene lists were ranked using the published function “-log10*log2(fold-change)”, compared to the C2 “Curated Gene Sets” collection from the Molecular Signatures Database (MSigDB) using GSEA v3.058. Gene ontology enrichment was analyzed with goseq v1.3.059. Updated gene length and Gene Ontology data from the Zv10 assembly was retrieved from Ensembl with the packages ensembldb v2.2.160 and biomaRt v2.34.261, respectively. When necessary, mapping between different database gene identifiers was also performed using biomaRt v2.34.2. KEGG pathway analysis was performed with the kegga function provided in limma v3.34.562. Gene regulation data of significant pathways was visualized with pathview v1.18.063. Raw data are deposited into Gene Expression Omnibus under accession number GSE129035.Total RNA was extracted from 5 dpf infected and uninfected snap-frozen larvae (20 larvae/sample) from three independent crosses using Qiazol reagent  according to the manufacturer’s instructions and extracted with RNeasy Mini kit . RNAs were quantified using a 2100 Bioanalyzer . At least 10 million reads per sample were sequenced using Illumina Single read 50 nt runs in a Hiseq2500. Sequencing, mapping the reads against the 19. Antibodies used were as follows: polyclonal rabbit anti-DRAM1  , polyclonal rabbit anti-Optineurin  , polyclonal rabbit anti-p62  , polyclonal rabbit anti-Lc3 , monoclonal Caspase 3 antibody , anti-mono-and polyubiquitinated conjugates mouse monoclonal antibody , polyclonal actin antibody , anti-rabbit IgG, HRP-Linked Antibody , anti-mouse IgG, HRP-linked antibody .Western blot analysis was performed as previously described65, were microinjected into the blood island of embryos at 28 hpf as previously described66. The injection dose was 200 CFU for all experiments, except for the phagocytosis assay (500 CFU), and the RNA sequencing of infected wild types (300 CFU) and dram1 mutants (150 and 300 CFU). Embryos were manually dechorionated by tweezers before the injection. Infected embryos were imaged using a Leica MZ16FA stereo fluorescence microscope equipped with a DFC420C color camera, and the number of bacterial pixels per infected fish were obtained from the individual embryo stereo fluorescence images using previously described software67.Mm strain M or Mm strain 20 fluorescently labeled with mWasabi or mCherry, respectively68. For quantification of the autophagic response targeted to Mm clusters  and imaged using a Leica TCS SPE confocal microscope. For quantification of numbers of GFP-Lc3-positive vesicles, the fixed 4 dpf larvae were imaged by confocal microscopy with a ×63 water immersion objective (NA 1.2) in the pre-defined tail fin region to detect the number of GFP-LC3-positive vesicles Fig. . The numers Fig. , the fixers Fig. . To invedram1 or dram1∆19N (negative control) RNA was isolated from wild type or dram1∆19n/∆19n embryos using QIAzol lysis reagent  and purified with the RNeasy MinElute Cleanup kit . cDNA synthesis was performed using the iScript cDNA synthesis kit . Full-length dram1 cDNA and dram1∆19N cDNA was obtained by PCR amplification using Phusion High-Fidelity DNA Polymerase . The following primers were used: Forward: 5′-CTGCGGCGAGATGTTTTGGTT-3′; Reverse: 5′-CAAAAACAGTGGGACATACAGTGAA-3′. dram1 or dram1∆19N PCR products were ligated into a ZERO BLUNT TOPO vector  and the insert was confirmed by Sanger sequencing . dram1 and dram1∆19N mRNA was generated using the SP6 mMessage mMachine kit  and Poly(A) Tailing Kit ; purified using the RNeasy Mini Elute Cleanup kit  and 50 pg mRNA was microinjected into one-cell stage embryos.Cell death was examined by Terminal deoxynucleotidyl TUNEL staining with the In Situ Cell Death Detection Kit, TMR red  in 2dpi fixed embryos. The assay was performed as follows: embryos were fixed in 4% PFA in PBS solution O/N at 4 °C, de-hydrated and re-hydrated in serial dilutions of methanol (MeOH)  and washed in PBS containing 0.1% Triton-X100. Then, embryos were permeabilized in 10 μg/ml Proteinase K for 40 min at 37 °C followed by a quick rinse in PBS containing 0.1% Triton X100. TUNEL staining was performed according to kit instructions O/N at 37 °C in the dark. Samples were washed and stored in PBST until imaging by confocal microscopy as described above.69.Infected embryos were immersed in egg water with 10 μM LysoTracker Red DND-99  for 1 h. Embryos were washed three times with egg water before imaging. Mpx activity assay was performed with the Leukocyte detection Kit  for detection of neutrophils as previously describedEmbryos were bath treated with Bafilomycin A1 (BafA1)  diluted into egg water at the working concentration of 100 nM for 12 h.70. 35 embryos/group were sonicated and incubated in hypotonic cell lysis buffer (25 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 5 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 5 mM dithiothreitol, pH 7.5) on ice for 15 min. For each reaction, 10 μg protein was incubated for 90 min at 28 °C with 50 μM YVAD-AFC in 50 μl of reaction buffer (0.2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.2 M 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 20% sucrose, 29 mM dithiothreitol, pH 7.5). After the incubation, fluorescence was measured in a Tecan M1000 microplate reader at an excitation wavelength of 400 and emission wavelength of 505 nm.Inflammatory caspase activity was assayed with the fluorometric substrate Z-YVAD 7-Amido-4-trifluoromethylcoumarin  as described previouslycaspa and gsdmeb morpholinos (MO)71 were purchased from Gene Tools . MO oligonucleotide sequence: caspa 5′-GCCATGTTTAGCTCAGGGCGCTGAC-3′, gsdmeb MO 5′-TCATGCTCATGCTAGTCAGGGAGG-3′71. MOs were diluted in Milli-Q water with 0.05% phenol red and 1 nL of 0.6 mM (caspa MO) or 0.7 mM (gsdmeb) MO was microinjected into the yolk of one-cell stage embryos as previously described (39).Previously validated U tests for comparisons between two groups and Kruskal–Wallis one-way analysis of variance with Dunn’s multiple comparison methods as a posthoc test for comparisons between more than two groups. . For segregation of F1 or F3 heterozygous offspring, data were analyzed with a Chi-square test . Continuous measurements are represented as bar graphs. In non-continuous measurements, the data sets from each group are shown in a scatter plot (left) and a boxplot (right). In the scatter plots each data point represents an individual embryo/larva, with the mean indicated by a horizontal line. Boxplots include 50% of the data points, with a vertical line indicating the 95% confidence interval and a horizontal line indicating the median.Power calculations where performed to ensure that sufficient animals were included in the experiments to be able to detect a effect size of at least 20%. For infection experiments, improperly injected animals were excluded from the experiment. No animals were excluded from the analysis after the initial selection. Wild type and mutant animals were randomly distributed over the different experimental conditions and blinding was applied during data acquisition and analysis when possible. Statistical analyses were performed using GraphPad Prism software . All experimental data (mean ± SEM) was analyzed using unpaired, two-tailed Mann–Whitney Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Figure Legends


CHASERR (AC013394.2 or LINC01578) that could regulate target genes co-transcriptionally via interaction with a nascent transcript by directing CHD2 helicase. The obtained results suggested that this nuclear lncRNA may be able to activate expression of the target genes in trans by base-pairing with the nascent transcripts and directing the CHD2 helicase to the regulated promoters leading to open the chromatin and active transcription. Our study highlights the possible importance of base-pairing between nuclear lncRNAs and nascent transcripts for the regulation of gene expression.Although thousands of mammalian long non-coding RNAs (lncRNAs) have been reported in the last decade, their functional annotation remains limited. A wet-lab approach to detect functions of a novel lncRNA usually includes its knockdown followed by RNA sequencing and identification of the deferentially expressed genes. However, identification of the molecular mechanism(s) used by the lncRNA to regulate its targets frequently becomes a challenge. Previously, we developed the ASSA algorithm that detects statistically significant inter-molecular RNA-RNA interactions. Here we designed a workflow that uses ASSA predictions to estimate the ability of an lncRNA to function via direct base pairing with the target transcripts . The workflow was applied to 300+ lncRNA knockdown experiments from the FANTOM6 pilot project producing statistically significant predictions for 71 unique lncRNAs (104 knockdowns). Surprisingly, the majority of these lncRNAs were likely to function co-transcriptionally, i.e., hybridize with the nascent transcripts of the target genes. Moreover, a number of the obtained predictions were supported by independent iMARGI experimental data on co-localization of lncRNA and chromatin. We detected an evolutionarily conserved lncRNA  Long non-coding RNAs (lncRNAs)—transcripts of at least 200 nucleotides long lacking protein-coding potential—are the most populated class of transcripts in the mammalian genomes. They are expressed from more than 50,000 loci in the human genome , which iUp to date, several genomic screening approaches to functional annotation of lncRNA have been developed. Based on lncRNA knockdown (knockout) followed by cellular or molecular phenotyping, these approaches characterize a fair share of lncRNAs in one series of experiments ,13,14,15Complementary approaches to lncRNA annotation aim to search for their targets via detecting interactions between lncRNAs and chromatin or other RNA molecules. LncRNAs use different molecular mechanisms to bind to the chromatin, including RNA-DNA binding mediated by protein complexes, direct RNA-DNA hybridization via triplexes, RNA binding to single-stranded DNA regions (known as R-loops) and co-transcriptional RNA-RNA interactions via duplexes with nascent RNA transcripts (reviewed in ). ExperiIn this work, we focus on RNA:RNA interactions. We investigated two possible scenarios— lncRNA bound co-transcriptionally to the nascent RNAs via duplexes and lncRNA bound to mature transcripts—by applying the ASSA RNA-RNA interaction prediction tool  to the dhttps://github.com/vanya-antonov/assa) tool was used in this study to predict inter-molecular RNA:RNA interactions [The AntiSense Search Approach  with the first 1kb of the target gene . We included the introns assuming that lncRNA-RNA binding may precede splicing.By contrast, post-transcriptional lncRNA:RNA hybridizations occur with mature spliced target transcripts in the cytoplasm or the nucleus . We hypothesized that this type of interaction results in post-transcriptional regulation of gene expression. It should be noted that CAGE-seq technology which has been used in the FANTOM6 project measures expression changes at the promoter level. Thus, to predict post-transcriptional interactions we considered the longest isoform that can be expressed from the corresponding promoter only.In the present work ASSA was run with default settings in both the co- and post-transcriptional modes.p-values To estimate the correspondence between the iMARGI peaks and ASSA predictions for each lncRNA we only considered differentially expressed genes (DEGs) that had predicted antisense interactions with the corresponding lncRNA  were cophyper R function). We used 45480 as the total number of the human genes that was computed as the sum of the protein-coding (19954), long (17957) and small (7569) non-coding RNA genes as annotated in the GENCODE version 35. The human orthologs of the DEGs Chaserr-/- mEFs [getLDS function of the biomaRt R package. The expression of the CHD2 gene was obtained from the FANTOM5 SSTAR online tool (https://fantom.gsc.riken.jp/5/sstar/EntrezGene:1106) for the “Fibroblast-skin normal, donor2 (nuclear fraction).CNhs12582.14302-155B9” sample.The statistical significance of the gene overlaps was estimated using the hypergeometric distribution  taking into account the lengths and the GC contents of both transcripts.We used the AntiSense Search Approach (ASSA) tool  to identWe analyzed all the lncRNAs knockdown experiments available from the recent pilot FANTOM6 project . In brieCHASERR and the promoters that were differentially expressed upon its knockdown and detected strong ASSA hits in the co-transcriptional mode [p-value < 0.01) was observed for 21 of those ambiguous 41 ASO knockdowns . This indicated the CHASERR functional significance and reproducibility of the results.Two knockdown experiments for the AC013394.2 lncRNA  demonstrate coordinated and strong enrichment of the ASSA predicted RNA:RNA duplexes in DEG D. The prCHASERR is mainly localized in the nucleus where it is bound to the chromodomain helicase DNA binding protein 2 (Chd2) [CHASERR orthologs are highly similar to each other (Chaserr-/- mouse embryonic fibroblases (mEFs) (see Methods) and compared them to the lists of the DEGs from the two FANTOM6 knockdowns. We observed a statistically significant overlap between the three sets of genes  supporting conservation of the CHASERR target genes (It has recently been shown that the mouse ortholog of 2 (Chd2) . The chr2 (Chd2) . Thus, C2 (Chd2) . Importach other . To checet genes A.CHASERR orthologs as well as the fact that the CHD2 helicase is also expressed in the human fibroblasts (FANTOM5 expression = 113.25), we assumed that CHASERR lncRNA and CHD2 helicase interact in the nucleus of human fibroblasts as well. Our computational analysis suggested that CHASERR may directly interact with the beginning . It should also be noted that ASSA predicted statistically significant interactions between CHASERR and transcripts of these two genes . A more detailed analysis of the


Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a  Heterochromatic domains of DNA are a conserved feature of eukaryotic chromosomes and play central roles in regulation of transcription, inactivation of transposons, and maintenance of genome integrity . HeterocSchizosaccharomyces pombe heterochromatin is assembled at pericentromeric DNA regions, telomeres, the mating type locus, and the ribosomal DNA repeats , growth on tetracycline-containing medium results in a variegated phenotype, due to the stable propagation of the heterochromatic domain in roughly 60% of cells  mutants  in cells carrying this reporter and screened for  mutants . We plat mutants . We scre mutants . About hemd mutants lost silencing on tetracycline-containing medium, they differed in their ability to establish efficient silencing on medium lacking tetracycline , one had no coding variant despite good genome coverage, and the remaining 9 had insufficient sequencing coverage to be analyzed. The identified mutations mapped to 19 genes, most of which encode subunits of one of five complexes , a subunit of the major Ccr4-Not deadenylase complex (ccr4), a universal stress family protein (dbs2), and a vacuolar sorting protein (bro1). Finally, we identified four mutations in three genes – crb3, grc3 and mdn1 – coding for subunits of an essential ribosome biogenesis complex that we refer to as the rixosome  or rich medium containing 5-fluoroacetic acid (5FOA). Uracil supplementation is required for cell growth in the absence of +ura4 expression, while 5FOA is toxic to cells expressing +.ura4 Thus, growth on -Ura and 5FOA medium can be used as a readout of +ura4 expression. In the presence of ongoing establishment from the siRNA generating otr1R and cenH repeats, silencing at these regions was robust in mutant cells  . The rixreporter . We thenreporter  and analreporter  and its reporter . When wee mutant , which we mutant . Howevere mutant , were aleratures . By contractions . Furthertability . These rtability . The subtability , with mecrb3-D198N and grc3-V70M cells, followed by sequencing or qPCR. As shown in IR-L and IR-R, of the mat locus  of Crb3-TAP from wild-type, at locus , which cat locus . In the  reduced . This red levels . The grcith Swi6 . Further0M cells , demonst H3K9me2 . In addiM mutant , includint cells , the mutnt cells . These rS. cerevisiae, the endonuclease and polynucleotide kinase activities of the rixosome cleave the rRNA precursor and prepare the resulting RNA ends for downstream processing  mutant cells, which are maintenance defective. The catalytically dead mutant carried substitutions of two conserved catalytic site residues , which have been shown to be crucial for the kinase activity of Grc3 in S. cerevisiae  and RNA Pol II ChIP. Under establishment condition, we did not observe an increase in gfp reporter RNA levels in the grc3-V70M mutant relative to wild-type cells. We reasoned that other RNA degradation pathways may act redundantly to mask the effects of the rixosome on RNA levels under strong establishment conditions at the reporter locus. We deleted a component of the nuclear TRAMP complex (cid14Δ) and a component of the Ccr4-Not complex (caf1Δ)  , the oth (caf1Δ) . TRAMP c (caf1Δ) . Deletio4Δ alone . By cont1Δ alone . The inc1Δ alone . We concdg repeats in wild-type, grc3-V70M, and dhp1-1 cells by northern blotting and, consistent with the above hypothesis, found that both mutations caused a marked increase in siRNAs accumulation :ade6 transgene (cenH) region into the mat2p gene and the adjacent IR-L boundary region :ade6 silencing. As expected, the +L(BglII):ade6 transgene became fully derepressed in clr4-W31G cells, indicating that Clr4 read-write was essential for the spread of silencing away from the cenH nucleation region :ade6 transgene silencing was completely rixosome- and Dhp1-dependent  did not significantly affect RNA Pol II occupancy at the cenH region and the +ade6 transgene (+L(BglII):ade6 transgene silencing in grc3-V70M and dhp1-1 cells was accompanied by impaired spreading of H3K9me2 and H3K9me3 from the cenH region into the +ade6 transgene and leftward into the IR-L boundary, while spreading into the mat2P gene occurred with similar efficiency in mutant and wild-type cells  complex at euchromatic mRNAs or the integrator complex which has been shown to release paused RNA pol II complexes . However4 end, which becomes a substrate for trimming by the 5’−3’ exonuclease Dhp1/XRN2 leading to formation of mature 25S rRNA  tagged strains were expressed under the control of their endogenous promoters and terminators. Cells were cultured on rich YES (yeast extract with supplements) medium, unless otherwise indicated.The genotypes of all strains used in this study are presented in +10xtetO-ade6 reporter  or YES medium supplemented with 5-fluoroorotic acid (0.1% and 0.2% 5FOA). For +ade6 reporter gene silencing assays, serial dilutions were plated on YE and YE+TET media. Phenotypes were scored after 3 days at 32°C.Yeast cells were grown in YES medium with shaking at 32°C to stationary phase (OD600 = 12 and above). The cultures were adjusted to a density of OD600 = 4 and serially diluted tenfold. Three microliters of each dilution were spotted on the appropriate medium. For assessing For TET treatment, yeast cells were grown for 24 hr in YES medium supplemented with TET to exponential phase (OD600 = 1–2). For all other ChIP experiments, yeast cultures were grown in rich (YES) medium to OD600 densities of 1–2. For Crb3-TAP ChIP, 50 OD600 units of cells per ChIP were fixed with 1.5% EGS for 45 min and 1% formaldehyde for 15 min, quenched with glycine and collected. For all other ChIPs, 50 OD600 units of cells were crosslinked with 1% formaldehyde for 15 min. Cell pellets were resuspended in lysis buffer  and cells were lysed by the glass bead method, using MagNA Lyser (Roche) . The lysates were sonicated by 3 pulses of 20 s (amplitude 40%) in a Branson microtip sonifier with intermittent cooling on ice, to a fragment distribution of 200–1000 bp. Cell debris was removed by centrifugation (16000xg) at 4*C for 15 min and the cleared lysates were incubated for 2 hr at 4*C with magnetic beads coupled to antibodies against the targets of interest. For H3K9me2 and RNA Pol II ChIPs, 30 µl Protein A Dynabeads (Invitrogen) were coupled with 2 µg anti-H3K9me2 antibody  or 4 µg anti-Pol II , respectively. For H3K9me3 ChIPs, 30 µl Streptavidin Dynabeads M280 (Invitrogen) were coupled to 1 µg of anti-H3K9me3 antibody (Diagenode C15500003) and blocked with 5 µM biotin. For Crb3-TAP ChIPs, 4.5 µg Dynabeads M-270 Epoxy beads (Invitrogen) were coupled to 1.5 µg rabbit IgG  and incubated with the cleared lysate. The bead-protein complexes were washed three times with lysis buffer, once with chilled TE buffer  and eluted using 120 µl 50 mM Tris/HCl pH 8.0, 1 mM EDTA, 1% SDS with heating (65°C) for 20 min. Crosslinks were reversed at 65°C overnight and the eluates were treated with RNase A and Proteinase K (Roche), prior to an extraction with phenol:chloroform:isoamyl alcohol. qPCR was performed on Applied Biosystems qPCR instrument with the primer sequences listed in For ChIP-seq, reverse crosslinked DNA treated with RNase A and proteinase K was purified using the Qiagen PCR purification kit. The DNA was then sonicated in a QSonica water bath sonicator  to a fragment size of ~200 bp, concentrated and quantified using Qubit dsDNA high sensitivity kit. 1–10 ng of DNA were used to prepare libraries as described previously . Librarigrc3-V70M or crb3-D198N) backgrounds, as well as corresponding untagged cells, were grown in rich YES medium to exponential phase (OD600 = 1–1.5) and 600 OD600 units were collected per immunoprecipitation. The pellets were resuspended in lysis buffer . The cells were lysed by the glass bead method in a MagNA Lyser (Roche) with a succession of short pulses , followed by rapid cooling for 2 min in an ice water bath. Lysates were clarified at 16,000xg for 15 min and incubated with 300 µl of Pan Mouse IgG Dynabeads (Invitrogen) per IP. Four washes were performed with lysis buffer with full resuspension of the beads, followed by four short washes with wash buffer . Proteins were eluted with 0.5M NH4OH and dried down in a speed vac. Five to ten percent of each elution fraction was analyzed by silver staining using the Silver Stain SNAP Kit (Pierce) according to the manufacturer’s protocol. The remainder of each elution was analyzed by mass spectrometry.Purifications of Crb3-TAP tagged rixosome complexes were performed by a chromatin purification method described previously  with min18 chromatography via stage tips. Data were collected on an Orbitrap Lumos instrument with a multi-notch MS3 method. Prior to injection, peptides were separated by HPLC with an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) with a 100 μm inner diameter column and a 2.6 μm Accucore C18 matrix (Thermo Fisher Scientific) with a 4 hr acidic acetonitrile gradient. MS1 scans were recorded in the Orbitrap  and MS2 scans in the iontrap after collision-induced dissociation  and a maximum injection time of 150 ms. For TMT quantification, an SPS-MS3 method was used with HDC fragments scanned in the Orbitrap at a resolution of 50,000 at 200 Th with a maximum injection time of 200 ms. Peptides were searched with an in-house software based on SEQUEST  against a forward and reverse database of the S. pombe proteome database (Uniprot) with human contaminants added. Searches had a mass tolerance of 50 ppm for precursors and a MS2 fragment ion tolerance of 0.9 Da. For searches, 2 missed cleavages were allowed and methionine oxidation was searched for dynamically (+15.9949 Da). After linear discriminant analysis (LDA), peptide false discovery rate was 1%, as was the FDR for final collapsed proteins. MS1 data were post-search, calibrated, and the search performed again. For TMT quantification, peptides with an isolation specificity of >70% and a summed TMT signal-to-noise (s/n)>200 were filtered. Proteins with at least two peptide quantification events were considered for analysis. For all 1139 quantified proteins, an enrichment score was calculated as the summed TMT signal-to-noise (TMT sum s/n) ratio of the tagged wild-type sample over the untagged TMT sum s/n of the wild-type control. The top 10% most enriched proteins (118) contained all known components of the Rix1 core complex , many of the pre-60S ribosome particle components , Swi6 (required for the recruitment of the Rix1 complex to heterochromatin) and nuclear export factors known to be involved in the shuttling of the pre-60S complex to the cytosol. The wild-type/mutant fold change score for each protein was calculated as the ratio of the summed TMT s/n in the wild-type over the mutant IPs, after subtraction of the background from the untagged control and correction for the abundance of the bait protein. The log transformed fold-change scores were plotted against the log transformed enrichment scores to identify proteins which were over- or under-represented in the mutant sample relative to the wild-type. Z scores were calculated from the distribution of fold-change scores of the top 10% most enriched proteins and the Z-scores of outliers were displayed on the plot.All liquid reagents used for mass spectrometry sample preparation were HPLC grade. Proteins were digested with trypsin (Promega #V5111) in 200 mM HEPES buffer pH 8.5 with 2% acetonitrile (v/v) overnight at 37°C. For direct TMT peptide labeling of digests, TMT-10plex reagents from Thermo Fisher Scientific (#90406) were used. After quenching with hydroxylamine at 0.3% (v/v), reactions were acidified with formic acid and peptides purified by reversed phase CTo prepare whole cell protein extracts, yeast cells were grown in YES medium at 32°C with shaking to exponential phase (OD600 = 1) and 25 ml of culture (25 OD600 units) were collected. Cells were resuspended in lysis buffer  and lysed by the glass bead method in a MagNA Lyser (Roche) with a succession of short pulses , followed by rapid cooling in an ice water bath. Aliquots of 0.25 and 0.5 OD600 units were resolved by SDS-PAGE electrophoresis, immunoblotted with anti-TAP antibody  and anti-Swi6 antibody  and 50 OD600 units of cells were collected. Cells were lysed by the hot phenol method, RNA was purified on-column using RNeasy Mini Kit (Qiagen), treated with Turbo DNase (ThermoFischer) and cleaned up by a second RNeasy Mini kit purification. 500 ng of pure RNA was reverse transcribed  using the primers indicated in Yeast cultures were grown to exponential phase (OD600 = 1) and 50 OD600 units of cells were collected. Cells were lysed by the hot phenol method, RNA was precipitated with ethanol and resuspended in water. RNA was separated by denaturing gel electrophoresis in a MOPS-formaldehyde buffer system and visualized by staining with EtBr.7 cells were collected. sRNAs were purified with the mirVana miRNA isolation kit (Ambion) and 25 µg of sRNA were used for Northern blotting as described previously , a cell count was determined by a BrightLine hemacytometer and 5 × 10eviously . Sequenc In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This work identifies the rixosome, a RNA degradation complex, that regulates heterochromatin spreading and inheritance in fission yeast. The authors discover the intersection of this RNA degradation by the rixosome and canonical RNAi pathway to ensure heterochromatin spreading. Overall this work identifies important mechanisms for the interplay between RNA and heterochromatin.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: John L Rinn (Reviewer #2).Thank you for submitting your article "A Conserved RNA Degradation Complex Required for Spreading and Epigenetic Inheritance of Heterochromatin" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Moazed and colleagues report the identification of rixosome, a RNA degradation complex previously implicated in rRNA biogenesis , that is important for heterochromatin spreading and inheritance in fission yeast. The authors conduct a forward genetic screen with an inducible heterochromatin system, and show that several hits comprise a RNA degradation complex they term the rixosome. Rixosome interacts with Clr4 H3K9 methylase and generates RNA fragments that are substrates for XRN2/Dhp1 exonuclease. Additional functional and genetic interaction studies show that the rixosome functions redundantly with RNAi pathway to ensure heterochromatin spreading. Altogether, this study expands on previous findings  and brings them into a new and exciting perspective. Overall this work identifies important mechanisms for the interplay between RNA and heterochromatin, and should be published after the following points are clarified.Essential revisions:grc3-V70M mutant . On the other hand, the authors argue that rixosome and RNAi work together in a redundant manner for heterochromatin spreading and inheritance. E.g. in Figure 2E and F ago1Δ seem to increase the effect of rixosome mutation suggesting a cooperative action in the maintenance of H3Kme at endogenous heterochromatic domain. Similarly Figure 5 and 6 argue for the redundancy model. The confusion arises from how rixosome has specificity for which RNA. If rixosome only cleaves euchromatin RNA, then RNAi and rixosome could work together. If rixosome cleaves heterochromatic RNA that is necessary for RNAi-dependent heterochromatin, then rixosome would counteract RNAi. Please address experimentally or comment on whether rixosome has specificity toward different classes of RNAs.1) Interplay between rixosome and RNAi needs to be clarified. On one hand, authors claim that these pathways compete and bring as evidence increase of sRNA in caf1grc3-V70M vs. caf1 at 10xtetO-ura-gfp; Figure 4F) or no increase of RNA Pol II (grc3-V70 vs. wild type at L(BglII):ade6; Figure 5F), which let the authors conclude that transcription is not affected. However, also RNA levels were only moderately increased in those mutants, and even clr4 mutants did not show changes for RNA Pol II for one of the reporters . Thus, rather than ruling out transcriptional changes, these RNA Pol II ChIP experiments appear to be not sufficiently sensitive. A better approach for measuring transcription and RNA turnover would be NET-seq and pulse-labeling with 4-thiouridine (4SU) , respectively. Otherwise, these statements should be toned down.2) Another approach the authors took for testing a post-transcriptional role of RIXC in silencing are ChIP experiments with RNA Pol II. These revealed only mild  should be included.3) By northern blots, the authors showed increased sRNA in  Essential revisions:1) Interplay between rixosome and RNAi needs to be clarified. On one hand, authors claim that these pathways compete and bring as evidence increase of sRNA in grc3-V70M mutant . On the other hand, the authors argue that rixosome and RNAi work together in a redundant manner for heterochromatin spreading and inheritance. E.g. in Figure 2E and F ago1Δ seem to increase the effect of rixosome mutation suggesting a cooperative action in the maintenance of H3Kme at endogenous heterochromatic domain. Similarly Figure 5 and 6 argue for the redundancy model. The confusion arises from how rixosome has specificity for which RNA. If rixosome only cleaves euchromatin RNA, then RNAi and rixosome could work together. If rixosome cleaves heterochromatic RNA that is necessary for RNAi-dependent heterochromatin, then rixosome would counteract RNAi. Please address experimentally or comment on whether rixosome has specificity toward different classes of RNAs.We apologize for any confusion regarding the relationship between the rixosome and the RNAi pathway. Thank you for explicitly describing the source of the confusion. Our findings clearly indicate that the 2 pathways can act on the same RNA substrates at the pericentromeric repeats . Thus, when rixosome function at heterochromatin is eliminated, more RNA substrate is available for processing by the RNAi machinery, resulting in higher siRNA levels in rixosome mutant cells . Conversely, in the absence of RNAi, the residual H3K9me at the pericentromeric repeats becomes entirely rixosome-dependent, indicating that the rixosome, and by implication its RNA degradation activity, is required for H3K9me maintenance in the absence of RNAi .not necessary for RNAi-dependent heterochromatin, as indicated by the observations that (1) RNAi-dependent H3K9me and silencing at pericentromeric DNA repeats is not affected by rixosome mutations [Figure 2 and Figure 2—figure supplement 2] and (2) mutations in the rixosome result in increased pericentromeric siRNA levels [Figure 2G]. We have strived to modify the text and figure legends to make these points clearer.The data presented in our manuscript clearly demonstrate that the rixosome is 2) Another approach the authors took for testing a post-transcriptional role of RIXC in silencing are ChIP experiments with RNA Pol II. These revealed only mild  or no increase of RNA Pol II (grc3-V70 vs. wild type at L(BglII):ade6; Figure 5F), which let the authors conclude that transcription is not affected. However, also RNA levels were only moderately increased in those mutants, and even clr4 mutants did not show changes for RNA Pol II for one of the reporters . Thus, rather than ruling out transcriptional changes, these RNA Pol II ChIP experiments appear to be not sufficiently sensitive. A better approach for measuring transcription and RNA turnover would be NET-seq and pulse-labeling with 4-thiouridine (4SU) , respectively. Otherwise, these statements should be toned down.grc3-V70M versus wild type for 2 different reporter genes at 2 different heterochromatic regions [10xtetO::ura4-gfp and +L(BglII):ade6 loci]. Regarding the possibility that “RNA Pol II ChIP experiments appear to be not sufficiently sensitive”, we believe that the data we had already presented rules out this possibility for the first locus. In Figure 5E, we included a crucial control in which heterochromatin is absent and the reporter gene is maximally derepressed . Comparison of tetR-clr4-I, clr4Δ mutant with wild type (clr4+) control in Figures 5E shows that our RNA Pol II ChIP signal at this locus displays a large dynamic range of ~120 fold. Despite little or no change in RNA Pol II occupancy in the caf1Δgrc3-V70M double mutant cells , we observe a 5-fold increase in RNA levels . It is important to note that these experiments were performed under establishment conditions in which we show that the rixosome is not required for silencing . The data show that even under conditions in which the reporter gene is strongly silenced, we could detect some increase in RNA levels in the absence of rixosome-mediated RNA degradation, which supports the model that the RNA processing/degradation activities of the rixosome are required for heterochromatin maintenance. In the revised manuscript, we provide further support for this idea by demonstrating that the catalytic polynucleotide kinase activity of the Grc3 subunit of the complex – which prepares RNA substrates for degradation by Dhp1/XRN2 – is required for heterochromatin maintenance .We thank the reviewer for this comment but believe that our presentation of the RNA Pol II ChIP and the qRT data may have led to a misunderstanding. As the reviewer states, we see only modest changes in RNA Pol II occupancy in mat2P gene . Our results are therefore consistent with previous findings and indicate that loss of rixosome localization to the mating type locus has little or no effect on the H3K9me-independent repression of mat2P.Regarding modest increases in RNA Pol II occupancy and RNA levels in rixosome and clr4 mutants at the mating type locus , we failed to note that some transcriptional repression at the mating locus occurs in a heterochromatin-independent manner. It has previously been shown that a heterochromatin-independent (Clr4- and H3K9me-independent) mechanism represses the expression of the +L(BglII)::ade6] the rixosome may function in silencing by promoting RNA degradation without an effect on RNA Pol II occupancy. We note that whether or not the rixosome affects RNA Pol II occupancy at target loci, our conclusion that its RNA degradation activities are critical for heterochromatin spreading and epigenetic inheritance remain valid.We were unable to more fully address the possible lack of sensitivity in detection of changes in RNA Pol II occupancy noted by the reviewer due to shutdown of laboratories to combat COVID19 spreading. We are eager to perform the nascent transcript experiments mentioned by the reviewers in the near future. We have removed the section in the Discussion where we proposed that the at some target genes  and (2) the grc3-V70M mutation disrupts the interaction of the rixosome with Swi6 and its localization to heterochromatin . We would therefore not expect the grc3-V70M mutant to have any effect on dg siRNA levels as each mutation (swi6Δ and grc3-V70M) abolishes the localization of the rixosome to heterochromatin, i.e., they act in the same pathway.We apologize for not describing these results more clearly. As noted by the reviewer, we observed increased sRNA levels in Finally, we would like to note that the lack of an increase in siRNA levels in cells lacking Swi6 is an expected result. We and others have previously shown that Swi6 stabilizes the RNAi machinery on chromatin and is required, to a variable degree depending on the locus, for accumulation of siRNAs. Therefore, although in swi6D cells, the rixosome is no longer recruited to heterochromatin, in these cells, siRNAs do not accumulate to the same level as swi6+grc3D or swi6+dhp1D cells. We have now included this explanation in the text .


The circular (circ)RNAs are a newly recognized group of noncoding (nc)RNAs. Research to characterize the functional features of circRNAs has uncovered distinctive profiles of conservation, stability, specificity and complexity. However, a new line of evidence has indicated that although circRNAs can function as ncRNAs, such as in the role of miRNA sponges, they are also capable of coding proteins. This discovery is no accident. In the last century, scientist detected the ability of translate in some virus and artificial circRNAs. Artificial circRNA translation products are usually nonfunctional, whereas natural circRNA translation products are completely different. Those new proteins have various functions, which greatly broadens the new ideas and research direction for our research. These series findings also raise questions about whether circRNA is still classified as non-coding RNA. Here, we summarize the evidence concerning translation potential of circRNAs, including synthetic and endogenous circRNA translation ability, and discuss the mechanisms of circRNA translation. The classic “central dogma of molecular biology” suggests that the DNA constituent of our chromosomes is transcribed into RNA and subsequently translated into proteins. High-throughput sequencing technology has not only verified the dynamic complexity of gene expression but also revealed the existence of delicate regulatory processes at the RNA level . The RNAAlthough the newly discovered ncRNAs were at first largely dismissed as “transcriptional noise”, focused investigations began to reveal functional roles in cell biology and many disease types. Researchers’ attention has now turned towards defining the roles of ncRNAs in regulating and modulating host gene expression . The curIn 2012, CircRNAs are single-stranded covalently closed circular RNA molecules generated from a broad array of genomic regions, ranging from intergenic, intronic and coding sequences to 5′- or 3′-untranslational sequences . Two modTo date, four functions have been defined for the circRNAs. First, circRNAs harbor miRNA complementary sequences, facilitating their combination with and ability to adjust the biological function of a large number of miRNAs by functioning as molecular sponges. A specific example of this is the circMTO1, which acts as the sponge of miR-9 to suppress hepatocellular carcinoma progression . FurtherIt is commonly believed that mRNAs are the primary controller of cells, carrying out the necessary functions for life. Since the endogenous circRNAs appear to not be associated with polysomes, they presumably lack the potential for translation . AlthougIt is well known that there are two translation modes, cap-dependent translation and cap-independent translation. The traditional cap-dependent translation accounts for a basal level of protein synthesis under normal growth conditions. In contrast, cap-independent translation contributes to cell proliferation or cellular adaptation/survival when traditional protein synthesis is severely inhibited; this second mode is mediated by the IRES. Therefore, IRES-mediated translation serves as an urgent breakdown maintenance mechanism during cell stress, ensuring basic protein needs are met ; as suchhttp://reprod.njmu.edu.cn/circrnadb)  , the firrcrnadb) . However6A, ψ and m5C chemical modifications  serves three main functions, namely, changing the codon, enhancing the transcript stability and regulating the stress response. To date, only the m6A modification has been verified in circRNAs, wherein it plays a role in promoting translation  but were distinctive in their location patterns; the data also suggested that the m6A modification did not appear to promote degradation of circRNAs as it does for mRNAs. Ultimately, interpretation of these findings indicates that switching the state of m6A modifications may allow for functional control of circRNAs.Thereafter, patterns . The wriIn 2018, β-catenin encoded a novel protein (β-catenin-370aa) which shared homologous N-terminus sequence with wild type β-catenin, but it contained a new C-terminus with 9 specific amino acids. The β-catenin-370aa might function as a decoy for GSK3 β, leading to escape from GSK3 β-induced β-catenin degradation (In 2019, radation .Interestingly, In 2020, Detailed information for the published coding circRNAs is summarized in The field of RNA research has continually emphasized the structural and functional versatility of RNA molecules. This versatility has in turn inspired translational and clinical researchers to explore the utility of RNA-based therapeutic agents for a wide variety of medical applications. Several RNA therapeutics with diverse modes of action are currently being evaluated in large late-stage clinical trials, and many more are in the early clinical development stage, including strategies to modulate target gene expression, such as mRNA, siRNA and miRNA . For insIn terms of the discovery and exploration of endogenous circRNA translation proteins, we speculate that there will be a large number of circRNAs with translational function gradually discovered. But these need to be supplemented by a large number of experiments, especially the function of those unknown proteins translated by circRNAs. For example, two cases of circRNA translation of small proteins have been found to act as molecular inhibitors or agonist of their mother protein . TherefoThe collective evidence to date implies that the translation of endogenous circular RNA into proteins or peptides may be a widespread phenomenon, though the coding potential of circRNAs previously had been largely disregarded. Therefore, further studies on the translational capacity of circRNAs should be encouraged and should focus on the existing problems, such as the functions and detailed mechanisms of circRNA modifications, the 5′ cap-independent translation of circRNAs and circRNA-derived protein or peptides. The resulting insights will also be helpful towards furthering our understanding of ncRNA functions in general.


RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability. A wide variety of functional base modifications are present in cellular RNA in addition to the regular four ribonucleosides. Over 160 known chemical modifications that modulate the structure and function of RNA molecules have been described ,4,5,6,7.O-methylation of ribose moieties   A. Deposiis steps . Only a is steps ,15. On tis steps ,16.Further regulation of RNA modification is achieved through a specific association of the target RNA molecule and guide snoRNA in determined cellular locations, a molecular matchmaking that has been extensively described for pseudouridylation ,18,19. MSeveral reports connect defects in the rRNA modification machinery, which encompasses snoRNAs and protein components of the snoRNP complexes or stand-alone rRNA-modifying enzymes, with genetic diseases and cancers. However, it is still unclear whether pathogenic effects are driven by the lack of modification per se .Here, a selected set of known rRNA modifications and their functions are presented .Pseudouridine is considered the fifth ribonucleoside due to its abundance, and it is found in both the large and small subunits of the ribosome ,19,25,26Pseudouridine is enzymatically introduced by the isomerization of uridine. In humans, the main pseudouridylase is dyskerin (DKC1) . DKC1 exDKC1. Patients present lower levels of Ψ compared to the healthy controls that may ultimately impair internal ribosome entry site (IRES)-mediated translation of a subset of mRNAs, such as TP53 and CDKN1B . I. I130]. The 25S NRD substrates, which accumulate around the nuclear envelope in perinuclear foci, are eliminated after export to the cytoplasm in a process involving the exosome . The proS. cerevisiae, where in the absence of pre-rRNA dimethylation, for example, Dim1p blocks pre-rRNA processing steps required for the maturation of 18S rRNA [In the nucleus, when rRNAs have defects during the maturation step, they can be polyadenylated by the Trf4–Air2–Mtr4 polyadenylation (TRAMP) complex before degradation by the nuclear exosome. The TRAMP complex adds short poly(A) tails to aberrant transcripts, forming a favorable substrate for the exosome and, thereby facilitates degradation . In addi18S rRNA . The dis18S rRNA .tRNAs are long-lived RNA molecules. Defective tRNAs are degraded both in the nucleus and the cytoplasm via the combined action of the TRAMP complex, the nuclear exosome, and the rapid tRNA decay (RTD) pathway . These pRecent advances in high-throughput sequencing techniques have also improved our understanding of degradation of non-coding RNA. ncRNAs play key roles in cells, mostly through the formation of ribonucleoprotein complexes. The 3′-5′ exoribonuclease DIS3L2 is involved in the quality control of ncRNAs in the DIS3L2-mediated decay (DMD) pathway . DMD subWhile the processing of rRNA is well described, it is less clear which molecular event initiates the activation of the rRNA quality control machinery. In chemotherapy settings, it is well known that ribogenesis defects might be caused by the presence of modified or damaged bases in RNA. For example, in cells treated with 5-fluorouracil (5-FU), a synthetic analogue of uracil with a fluorine atom at the C5 position, 5-FU accumulates in RNA and RNA-mediated toxicity appears to be an important contributor to cytotoxicity . InteresN-glycosylic bond between the damaged base and the 2′-deoxyribose of the nucleotide. The damaged base is then removed to generate an apurinic/apyrimidinic (AP) site that is recognized and cleaved by an AP endonuclease, APE1 or AP lyase [DNA repair plays critical roles in the maintenance of genome integrity; around 150 proteins have so far been implicated in this process . BER is AP lyase . After tAP lyase ,164.E. coli and human uracil DNA N-glycosylases (UNG), can recognize and excise uracil in single- and double-stranded DNA regardless of the opposite base. In contrast, E. coli mismatch uracil-DNA glycosylase (MUG) or human thymine DNA glycosylase (TDG) only removes uracil in the U:G context [One of the most frequent lesions found in DNA is uracil (U), and it arises from the deamination of cytosine and misincorporation of dUMP instead of dTMP during replication. It can lead to G:C to A:T transition mutations ,166. The context . As a mo context ,169.N-glycosylic bond. The conserved N-terminal GMNPG motif together with the C-terminal HPSPR motif form the pocket that positions the substrates for cleavage  A,B. hSMUcleavage C, left [lic bond C, right No experimental structural data is available to verify whether a similar mechanism is utilized on RNA substrates, but there are reasons to suspect that RNA is processed in a similar manner: the C-terminal nucleic acid binding domain and the catalytic residue His239 are required for full activity on RNA substrates a well as DNA ,173. OurhTERC). We proposed that SMUG1 might regulate the presence of two modified bases (C323 and C445) positioned in the CR4/CR5 and H-box domains of hTERC [hTERC is a highly structured non-coding RNA [TERC, we computationally predicted the binding pose of the homology model of hSMUG1 to the available structure of medaka TERC (PDB ID: 4026 chain E). As shown in TERC, corresponding to C323 in the hTERC, lies at the active site of hSMUG1. The residues involved in the binding with hm5U of fish TERC are highlighted in Recently, our group demonstrated a new role for hSMUG1 in controlling telomere maintenance through processing of the human telomeric RNA component  and the dyskerin complex  [hTERC. As the interaction between hSMUG1 and hTERC might differ when hTERC is present in the telomerase holoenzyme and when hSMUG1 interacts with DKC1, we decided to superimpose the SMUG1–DKC1 complex, previously characterized in [hTERC (nd GAR1) ,175. Exprized in , on the rized in . Our SMUn [hTERC C,D.hTERC; others are transiently introduced in a specific subset during highly regulated processes . Some modifications are stable components of long-lived RNAs, e.g., pseudouridines in rRNA molecules and elopment ). In fact [hTERC . In the In summary, SMUG1 and other BER enzymes are emerging as regulators in RNA metabolism and RNA surveillance. The role of SMUG1 in recognizing subtle chemically modified bases could be a key feature of this newly discovered mechanism for distinguishing aberrant RNA from the normal RNA pool. However, the development of new sequencing techniques for hm5U detection and distribution within RNA molecules, is required for better characterization of the combinatorial presence of RNA modifications and their biological significance.