Escherichia coli multienzyme RNA degradosome, has a global role in RNA processing and degradation. RNase E localizes to the inner cytoplasmic membrane in small, short-lived clusters (puncta). Rifampin, which arrests transcription, inhibits RNase E clustering and increases its rate of diffusion. Here, we show that inhibition of clustering is due to the arrest of transcription using a rifampin-resistant control strain. Two components of the RNA degradosome, the 3′ exoribonuclease polynucleotide phosphorylase (PNPase) and the DEAD box RNA helicase RhlB, colocalize with RNase E in puncta. Clustering of PNPase and RhlB is inhibited by rifampin, and their diffusion rates increase, as evidenced by in vivo photobleaching measurements. Results with rifampin treatment reported here show that RNA degradosome diffusion is constrained by interaction with RNA substrate. Kasugamycin, which arrests translation initiation, inhibits formation of puncta and increases RNA degradosome diffusion rates. Since kasugamycin treatment results in continued synthesis and turnover of ribosome-free mRNA but inhibits polyribosome formation, RNA degradosome clustering is therefore polyribosome dependent. Chloramphenicol, which arrests translation elongation, results in formation of large clusters (foci) of RNA degradosomes that are distinct from puncta. Since chloramphenicol-treated ribosomes are stable, the formation of RNA degradosome foci could be part of a stress response that protects inactive polyribosomes from degradation. Our results strongly suggest that puncta are sites where translationally active polyribosomes are captured by membrane-associated RNA degradosomes. These sites could be part of a scanning process that is an initial step in mRNA degradation.The essential endoribonuclease RNase E, which is a component of the  RNase E is an essential endoribonuclease with major roles in maturation of stable RNA, degradation of mRNA, and posttranscriptional regulation of gene expression –6. Insta–9–Escherichia coli in which the RNA degradosome has been disrupted by deletion of part or all of the RNase E noncatalytic region are viable  has shown that RNase E-yellow fluorescent protein (YFP) forms short-lived clusters on the inner cytoplasmic membrane . Althougescentus . Rifampiescentus , 42, disescentus . Ectopiced cells . PhotoblE. coli. Photobleaching measurements showed that rifampin treatment relaxes constraints on the rate of diffusion of RhlB and PNPase, as was previously shown for RNase E  of RNase E-mCherry that are distinct from the puncta observed in untreated cells. The formation of foci could stockpile RNA degradosomes as part of a mechanism that protects inactive polyribosomes from degradation. We discuss the implication of our results for the cell biology of mRNA degradation in Escherichia coli and related Gram-negative bacteria.Here, we made strains with single-copy chromosomal constructs of RNase E, RhlB, and PNPase tagged with mCherry or monomeric superfolder green fluorescent protein (msfGFP), and we introduced a mutation that results in resistance to the drug rifampin. We show that RNase E, RhlB, and PNPase colocalize in puncta on the inner membrane of  RNase E . We showE. coli K-12 strain (SLM001) that is rifampin resistant  strains Kti162 and SLM001 are E. coli K-12 derivatives that encode RNase E-mCherry at the rne locus under the control of endogenous expression signals  before and 30 min after rifampin addition. The puncta, which are conspicuously present (+rpoB)  . Figure  present , top, di present , 45. Upo (rpoB+) . Since ro mature . The 21-The decrease in the normalized variance of average pixel intensity is a quantitative measure of the dispersion of RNase E-mCherry in a population of cells treated with rifampin . InhibitRifampin had no effect on the formation of RNase E-mCherry puncta in the SLM001 strain . Since tTo examine the localization and distribution of PNPase and RhlB, we constructed two strains, SLM018 and SLM024, expressing PNPase-msfGFP and RhlB-msfGFP, respectively. msfGFP is a monomeric derivative of superfolder GFP that minimizes aggregation and mislocalization . Both strpoB(D516) strains encoding PNPase-msfGFP and RhlB-msfGFP, respectively  background.We next analyzed SLM027 and SLM029, which are rifampin-resistant ectively . In contB-msfGFP . There wB-msfGFP  and D. TWe constructed a fusion protein in which the RNase E sequence from position 568 to 592, corresponding to the MTS, was fused to the N terminus of msfGFP. 10.1128/mBio.01932-21.1FIG S1lac promoter. Cells were grown in LB with 40 μg/ml spectinomycin and 100 μM IPTG  to mid-logarithmic phase; 1 to 2 μl of each culture was mounted on glass microscope slides with 1.2% (wt/vol) agarose pads. (A) Epifluorescence and TIRF images of the MTS-msfGFP fusion protein. The icon on the bottom right corner of each image shows the plane of focus. (B) Average pixel intensity and normalized variance in pixel intensity before and 30 min after the addition of rifampicin (150 μg/ml) were measured by quantification of line scans as for P < 0.0001; ns, not significant). Download FIG S1, TIF file, 1.2 MB.Characterization of the MTS-msfGFP fusion protein. MTS-msfGFP is a protein in which the RNase E sequence from position 568 to 592, corresponding to the MTS, was fused to the N terminus of msfGFP. MTS-msfGFP is expressed from the pLP120 plasmid, which was transformed into NCM3416 (SLP122 strain). pLP120 is a low-copy-number plasmid conferring spectinomycin resistance. Expression is under the control of a Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the B. subtilis, 65% of more than 200 proteins are localized in patchy patterns (E. coli over a period of seconds give the impression of movement (fK) and a low rate (sK). fK and sK are not independent. However, fK can be thought of as the initial rate of photobleaching of molecules in the illuminated field, whereas sK is related to the rate of diffusion. Under carefully controlled conditions, sK can be used to measure relative rates of diffusion  strain. We observed a similar effect of rifampin on RNase E-GFP photobleaching (sK), the goodness of fit (R2), the standard error of the mean (SEM), and the number of measurements (n). These results show that the diffusion rate of RNase E-mCherry and RNase E-GFP expressed from the native rne locus under the control of endogenous regulatory elements increases with rifampin treatment. The rpoB(D516Y) mutation is validated as a control, since photobleaching is not affected by rifampin.We measured photobleaching of RNase E-mCherry in Kti162 and SLM001 before and after treatment with rifampin. Snapshots  and B suleaching . Table 210.1128/mBio.01932-21.2FIG S2FIG S2, TIF file, 1.1 MB.The diffusion rate of RNase E-GFP increases after rifampin treatment. Strain Kti164, which expresses RNase E-GFP, was grown to mid-log phase in LB at 37°C. (A) Images showing the photobleaching of RNase E-GFP were extracted from a 20-s TIRFm time lapse video  with and without 30 min of rifampin treatment (150 μg/ml). (B) Averaged, background-subtracted, and normalized fluorescence values plotted versus time. Dotted lines show standard errors of curve fitting (SEM). Values from two-phase curve fitting are listed in Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rpoB(D516Y) background, there was no change . Cells were grown and mounted on agarose pads as for Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli cell, affect colocalization measurements. We therefore applied a pixel-by-pixel analysis of the RNase E-mCherry/PNPase-msfGFP and RNase E-mCherry/RhlB-msfGFP pairs. Rr values, which are a measure of the degree of correlation, are shown in the insets of the graphs (Rr = 1.000) indicates perfect correlation , as described for FIG S4, TIF file, 2.1 MB.Fluorescence intensity correlation graphs. Scatter plots of mCherry and msfGFP pixel intensities of the cells in Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P values, expressed as percentages, are shown in the merged images  and AtpB-msfGFP (middle), and merged images (right). Cells were imaged in epifluorescence (top) and TIRF (bottom) illumination modes. Bar, 2 μm. (B) Scatter plots and Pearson’s correlation coefficients (Rr) as described for FIG S5, TIF file, 2.8 MB.AtpB localization. (A) Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Our epifluorescence images  and 5 suin vitro photobleaching of purified RNA degradosomes containing PNPase-msfGFP or RhlB-msfGFP  geometry of the RNA degradosome on the inner cytoplasmic membrane, in which PNPase-msfGFP is further from the glass slide than RhlB-msfGFP, resulting in lower excitation levels, and (ii) an exchange between degradosome-associated PNPase-msfGFP and cytoplasmic PNPase-msfGFP, which would serve as an additional pool of photobleachable molecules. This phenomenon was not investigated further.PNPase-msfGFP photobleaching is 5- to 10-fold slower than RhlB-msfGFP photobleaching . We therB-msfGFP  and S7. 10.1128/mBio.01932-21.6FIG S6FIG S6, TIF file, 1.3 MB.Nanoscale photobleaching of purified RNA degradosome. (A) Silver-stained gel of native RNA degradosome purified by affinity of RNase E containing a C-terminal FLAG tag . Size markers (M1) are indicated to the left. E1 and E2 are fractions eluted with FLAG peptide. A protein standard (M2) was used to estimate concentration of RNase E. The asterisks indicate LDH subunits that bind to the anti-FLAG beads due to autogenous FLAG epitopes. RhlB-msfGFP copurifies with a minor contaminant. (B) Photobleaching of 1-μl droplets of affinity-purified RNA degradosome (∼2 ng RNase E) was measured by time lapse videos using wide-field illumination. The Cy5-oligoRNA was used to center the droplet. Photobleaching is quantified by integrating the intensity of the entire field in the time-lapse video. The green-to-black transition in the time-lapse video is an illustration. It does not represent real data. Download Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01932-21.7FIG S7In vitro rates of photobleaching of purified RNA degradosome. Representative photobleaching curves of RNA degradosomes labelled with PNPase-msfGFP (A) or RhlB-msfGFP (B) purified as described for fK) and slow (sK) rate constants from two-phase curve fitting, standard deviation (sd), and number of measurements (n). Download FIG S7, TIF file, 1.1 MB.Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We asked if the formation of RNA degradosome puncta involves a direct interaction with mRNA by treating cells with kasugamycin, which results in inhibition of translation of canonical mRNAs with a 5′ leader containing a Shine-Dalgarno sequence, arrest of growth, and low-level translation of leaderless mRNA . ContinuWe also measured the effect of kasugamycin on the photobleaching of AtpB-msfGFP. 10.1128/mBio.01932-21.8FIG S8fK) and slow (Ks) rate constants from two phase curve fitting, standard error (SEM), and fit (R2). Download FIG S8, TIF file, 0.4 MB.AtpB-msfGFP photobleaching. The graph shows photobleaching in the absence or presence of kasugamycin (Kas). The table shows fast  The dispersion of puncta by rifampin treatment argues that RNA degradosome clustering involves an additional factor whose presence depends on transcription.Epifluorescence measurements show that rifampin treatment results in the dispersion of puncta containing RNase E, PNPase, and RhlB. Photobleaching measurements by TIRFm show that rifampin treatment relaxes constraints on the diffusion of RNase E, PNPase, and RhlB. Punctum formation is thus due to clustering of the RNA degradosomes. There are three arguments against the hypothesis that RNA degradosome puncta are an artifact of fluorescence protein aggregation. (i) Punctum formation has been seen with fusions of four different fluorescence proteins  to three different components of the RNA degradosome . (ii) With the exception of the MTS-msfGFP control, all fusion proteins in this work were expressed from single-copy chromosomal constructs at the Kasugamycin treatment results in dispersion of RNA degradosome puncta and relaxation of constraints on diffusion. Although this effect is the same as that observed with rifampin, the mechanism is necessarily different. E. coli triggers a stress response that results in the formation of foci containing RNA degradosomes and the RNA chaperone Hfq (The inhibition of translation by chloramphenicol results in large clusters (foci) of RNase E-mCherry that are distinct from the puncta observed in untreated cells. The loss of an RNase E-mCherry signal in TIRF mode after chloramphenicol treatment strongly suggests that RNA degradosomes in the foci are not membrane attached. Recent work has shown that nitrogen starvation in rone Hfq , 64. By rone Hfq . The forProteobacteria emphasizes their importance in evolutionary fitness  mutation was selected as a spontaneous mutation of the NCM3416 strain, as described elsewhere (Liquid medium (LB) and agar (LA) plates were prepared as described elsewhere . Strainslsewhere , 66. Brilsewhere .t tests for the statistical analysis were performed using GraphPad Prism v.8.0 with an alpha level of 5%.TIRFm photobleaching experiments were carried out as described in reference The affinity purification protocol was adapted from procedures described in references Additional experimental detail is given in 10.1128/mBio.01932-21.10TEXT S1in vitro. Download Text S1, DOCX file, 0.01 MB.Cell imaging growth conditions, wide-field epifluorescence, TIRFm imaging, affinity purification of native RNA degradosome, and nanoscale photobleaching Copyright © 2021 Hamouche et al.2021Hamouche et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Cervus canadensis) with neurological signs associated with an Elaphostrongylus cervi (E. cervi) infection. The original host of the nematode parasite is the Eurasian red deer (Cervus elaphus), although other cervids and small ruminants may also be affected. The two wapitis imported from Canada were kept in an enclosure with the Slovenian red deer herd. After developing debilitating neurological signs, the wapitis were euthanized and examined for possible causes. A histopathological examination of the brain of the first wapiti revealed severe diffuse perivascular meningoencephalitis with chronic vasculitis, and some cross-sections of nematodes were found in the leptomeninges. A necropsy of the second wapiti revealed severe pachymeningitis and leptomeningitis, where several adult nematode parasites were found. E. cervi was confirmed by molecular methods. The prevalence of E. cervi in the European red deer population is high, but no study has been conducted to assess its prevalence in Slovenia. This was the first confirmation of E. cervi in Slovenia and the first infection with this parasite described in Europe in a wapiti. Elaphostrongylus cervi should also be considered as a differential diagnosis in Europe for all ruminants grazing on pastures frequented by red deer and showing neurological clinical signs. This study describes two female wapitis ( Elaphostrongylus cervi  (E. cervi) is an Old World tissue worm found in the skeletal muscles and central nervous system of cervids, mainly red deer (Cervus elaphus) [E. rangiferi infests reindeer (Rangifer tarandus) [E. alces is common in moose  [E. cervi has expanded to Canada [E. cervi was first reported in Scotland in 1931 [E. cervi in Slovenia is unknown. elaphus) ,2. The carandus) ,4, whiles alces) . The orio Canada  and New o Canada , and it o Canada . In Euro in 1931 ,11,12,13E. cervi usually occurs incidentally in late summer when a deer ingests a terrestrial snail containing the infective third-stage larvae (L3) [An infection with vae (L3) . During vae (L3) . The subvae (L3) ,10,14. Evae (L3) . In the vae (L3) . Native vae (L3) ,17,18.E. cervi. This study describes the first two cases of an E. cervi infection detected in Slovenia in two captive wapitis (Cervus canadensis) imported from Canada. To date, no neurological case in domestic or wild ruminants has been diagnosed in Slovenia due to Cervus canadensis), two female wapitis (case one in January 2020 and case two in March 2021) developed debilitating neurological signs that led to the euthanasia (by harvesting) of both animals. The first wapiti was 8 years old and the second was 5 years old at the time of clinical presentation of the disease. The breeding herd  located in Southeast Slovenia, shares the same enclosure with a herd of 35–40 native red deer (Cervus elaphus). The wapiti herd was first imported from a wapiti breeding farm in Canada to a private zoo in the Czech Republic and then translocated to the Slovenian breeder in 2017. The breeder described both females as being in very good condition (approximately 350 kg) and pregnant. Both wapitis developed head tilt, torticollis, circling, ataxia, and loss of balance. Clinical signs developed and worsened so rapidly that within a few days both animals had to be euthanized. Blood samples, heads, and organs were sent to the Veterinary Faculty of the University of Ljubljana for necropsy and the exclusion of possible zoonoses. Appropriate brain samples were collected and tested for chronic wasting disease (CWD), rabies and Listeria monocytogenes (L. monocytogenes) in the TSE, virology, and microbiology laboratories, respectively. Histopathology of the whole brain and organs with gross lesions was performed after fixation in buffered formalin and paraffin embedding. Slides were stained with hematoxylin and eosin (HE). Blood samples were examined for microelement imbalances and hematological abnormalities. Liver and serum were analyzed for copper, and the intestinal contents were analyzed for gastrointestinal parasites and lungworms. On two separate occasions within the same herd of captive-bred wapitis (9/L), while biochemistry revealed a slightly low hepatic copper level (3.15 µg/kg or 49.6 µmol/kg). The serum copper levels were low, but within the normal range (198 µg/L or 3.12 µmol/L).Hematology revealed a high white blood cell count  that remained undetermined. One of the parasites collected at necropsy and preserved in fixative was female and measured 43 mm , the othE. cervi. Primers NC1 5′-ACGTCTGGTTCAGGGTTGTT-3′ and NC2 5′-TTAGTTTCTTTTCCTCCGCT-3′ [E. cervi, E. rangiferi)  and confirmed the diagnosis of elaphostrongylosis caused by E. cervi.The DNA from the adult worm was extracted using the SmartHelix First DNAid kit  following the manufacturer’s instructions . A polymCCGCT-3′  were usengiferi) . The PCRE. cervi as a most likely cause of neurological disease in two imported wapitis kept in the same enclosure as a herd of native red deer from Slovenia. To the authors’ knowledge, this is the first documented case of an E. cervi infection in Slovenia, the first E. cervi infection of a wapiti in Europe, and the first molecularly confirmed E. cervi infection in a wapiti to date. In our study, we found the presence of L. monocytogenes was indeed found in the brain tissue from the 2021 female wapiti. However, no histopathological lesions were found in the brain that could be associated with this bacterium [L. monocytogenes was not found, had the same clinical signs as the 2021 wapiti. Copper deficiency was another suggested differential diagnosis, as it can cause enzootic ataxia in captive wild ruminants [It was reported that clinical signs developed rapidly in the two female wapitis from this study. There were several differential diagnoses that would fit the observed neurological clinical signs, such as listeriosis, copper deficiency, and extrapulmonary lungworms ,21,22,23uminants . The breE. cervi infection and P. tenuis infection [A necropsy of the 2020 case revealed pathological changes in the liver suggestive of a parasitic etiology but did not immediately indicate that these lesions were related to a neurological disease. When the adult nematode parasites were recovered from the brain samples of the 2021 wapiti, there was sufficient suitable material to apply confirmatory methods. The anatomical location where the worms were found, the histopathological lesions in the brain combined with the clinical signs described in both wapiti led us to two possible diagnoses: nfection ,21.E. cervi is the more likely cause of neurological disorders in small ruminants and cervids in Eurasia [E. cervi as they show almost no clinical signs of infection [E. cervi prepatency period of three to six months [P. tenuis. P. tenuis is also known as meningeal worm and is most common in white-tailed deer [E. cervi [P. tenuis was a possible diagnosis to consider. Epidemiologically,  Eurasia . Furthernfection . Both fex months ,25,26. Oled deer ,27,28. TE. cervi . Based oE. cervi is between 33 mm–58 mm long [P. tenuis is between 40 mm–47 mm long in a wapiti host [P. tenuis [E. cervi follows a porto–hepatic pathway from the abomasum and small intestine to the lungs and then through the general circulation to the central nervous system (CNS) [E. cervi have already been noted in experimentally infected red deer calves, sheep, and goats [E. cervi than the wapiti from 2021, resulting in a mass migration of L3 larvae also through the liver tissue and earlier onset of neurological signs (January versus March). Morphologically, the adult female of  mm long  and the iti host . The adu. tenuis . On the em (CNS) . Lymphohnd goats ,32. Handnd goats  reportedP. tenuis larvae. Post mortem, we detected a dorsally spined larva in the feces of the wapiti from 2021 using Baermann’s method, but it remained indeterminate. This method has proven unreliable for an antemortem diagnosis of an extrapulmonary lungworm infection because larvae are shed intermittently and in low numbers in aberrant hosts, such as the wapiti [The two female wapitis from this study were 8 and 5 years old. Importation from Canada was at least 5 years ago, so the 5-year-old wapiti was most likely born in Europe, and it is very unlikely that the wapiti was infected with e wapiti . E. cervi in the wapiti from 2021. In the wapiti from 2020, no ancillary test was performed to undoubtedly confirm the same diagnosis. However, due to an almost identical similarity to the wapiti case in 2021, we can safely assume that the most likely diagnosis is also elaphostrongylidae caused by E. cervi. The wapiti has been known as a possible host for E. cervi since 1976. At that time, two wapitis were diagnosed as infected with E. cervi based on the morphological characteristics of the parasite [E. cervi infection in wapiti since that time. Finally, molecular methods confirmed the most likely diagnosis of elaphostrongylosis caused by parasite . InteresE. cervi infections of the remaining wapiti herd should be prevented. Deer farmers rely on anthelmintics and changes in management practices to control parasite infections [E. cervi larvae in red deer, and it was suggested that the adult parasites were still alive and reproducing after treatment [E. cervi, and if the wapiti herd had been treated with injectable ivermectin in the fall, when the L3 larvae in the wapiti host are in the early migration phase. According to Mitskevich [E. cervi infection in wapiti. Future fections ,35. Macrfections . The wapreatment . Based oreatment , the waptskevich  and Panitskevich , definitE. cervi infection has been reported in domestic or wild ruminants in Slovenia. However, some studies have reported the disease in grazing small ruminants in Italy and Switzerland [E. cervi infection is misdiagnosed because the clinical signs resemble those of neurological listeriosis. Elaphostrongylosis should be considered as a differential diagnosis when a domestic ruminant or farmed deer exhibits neurological clinical signs and has a history of grazing on pastures frequented by red deer [E. cervi. However, further studies are needed to assess the prevalence in the resident red deer population and the associated risk for grazing domestic ruminants and farmed mixed herds of deer. As far as the authors are aware, no case of an tzerland ,16. It ired deer . Based ored deer ,40,41, tE. cervi in Slovenian wapiti. Both animals, which showed signs of neurological disease, had been imported and shared an enclosure with a herd of native red deer in Slovenia. Elaphostrongylosis was confirmed by necropsy, parasitological and molecular methods. We believe that elaphostrongylosis should be considered as a differential diagnosis for ruminants grazing on pastures frequented by red deer and showing neurological clinical signs.This study confirmed the presence of the nematode


Post-transcriptional and translational control of specialized genes play a critical role in the progression of spermatogenesis. During the early stages, mRNAs are actively transcribed and stored, temporarily bound to RNA binding proteins in chromatoid bodies (CBs). CBs are membrane-less dynamic organelles which serve as storehouses and processing centers of mRNAs awaiting translation during later stages of spermatogenesis. These CBs can also regulate the stability of mRNAs to secure the correct timing of protein expression at different stages of sperm formation. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is an essential regulator of spermatogenesis. GRTH transports mRNAs from the nucleus to the cytoplasm and phospho-GRTH transports mRNAs from the cytoplasm to the CBs. During spermiogenesis, there is precise control of mRNAs transported by GRTH from and to the CBs, directing the timing of translation of critical proteins which are involved in spermatid elongation and acrosomal development, resulting in functional sperm formation. This chapter presents our current knowledge on the role of GRTH, phospho-GRTH and CBs in the control of spermiogenesis. In addition, it covers the components of CBs compared to those of stress granules and P-bodies. Spermatogenesis is a complex, serial and highly specialized differentiation program during which progenitor cells undergo a series of cellular reorganization events, resulting in the production of mature functional sperm ,2,3. ThiThe CB is a perinuclear organelle and a ribonucleoprotein (RNP) granule present in the cytoplasm of male germ cells . The funDrosophila, Polar granule in C elegans, and chromatoid body, Balbiani body, and Cajal body in mice and zebrafish [RNP complexes and granules are powerful composite structures of merged functions and unique properties. RNP granules are formed under physiological conditions. In addition, granule formation can be induced by stress such as heat, starvation, oxidative stress, etc. These granules are more dynamic in nature, and they occur in condensed or diffused states and depend on the cell requirements. Specific mutations occurring in the cell can prevent RNP assembly or disassembly and diminish cellular and organism function, resulting in several pathologies. Examples include fragile X-syndrome, associated primary ovarian insufficiency in human germ cells, and amyotrophic lateral sclerosis , possibly occurring due to the failure of disassembly of stress granules. The importance of RNPs is diverse and the complete functions are still not completely understood. The main functions of RNPs include carrying out intricate tasks in RNA-processing pathways and the post-transcriptional regulation of mRNAs. The association of RNA-binding proteins such as DDX4 and RNA in the germ line of several organisms gives rise to non-membranous structures called germ granules. These are known by several different names in different organisms with minor variations in function, from Nuage, Pole plasm, piNG body (piRNA nuage giant body) in ebrafish . One of The CB is a membrane-less, filamentous-lobular, perinuclear organelle (0.5–1 μm) and an electron-dense structure present in the cytoplasm of male germ cells ,14. It aThe origin of CBs is highly debatable. The most accepted view is that they emerged from small granules (precursors of CBs) which are associated with the nuclear envelope present near the electron-dense inter-mitochondrial cement in the late pachytene spermatocytes . The CB There is limited information about the specific mechanisms of CB function during spermatid elongation. With the identification of small non-coding RNA-mediated gene regulation and other associated mechanisms, the functions of the CB are slowly beginning to be understood. However, several of the molecular mechanisms are still unclear, requiring further in-depth molecular studies.Compartmentalization of molecular processes is accomplished by various intracellular organelles that spatially segregate functionally related molecules. RNPs act as organelles that lack any demarcating membrane and play a key role in mRNA homeostasis. RNP granules formed under physiological conditions in male germ cells are called CBs, while in somatic cells they are called RNA processing bodies or P-bodies. CBs’ overall functions fall between P-bodies and stress granules and are in concert with the maintenance of RNA regulation. Stress granules and processing bodies are also membrane-less RNA granules that dynamically sequester translationally inactive messenger ribonucleoprotein particles (mRNPs) into compartments which are distinct from the surrounding cytoplasm ,20. ThesIn contrast to CBs and stress granules, P-bodies are not associated with the regulation of translation initiation; instead they serve as a site for mRNA degradation, translation repression, storage of non-translating mRNAs, and RNA-binding proteins  10,20,2,220,22. During spermatogenesis, the early precursors of sperm utilize small regulatory RNAs such as microRNAs to control the expression of an array of genes at transcriptional or post-transcriptional levels during complex and specialized processes of sperm production. The role of CBs in small RNA-mediated gene control and the associated mechanisms are not clearly delineated at present. The repertoires of microRNAs and piRNAs have been identified and both serve as important regulators of male germ cell differentiation.miRNAs are small in size (21–23 nucleotides) and belong to the class of noncoding RNAs which act as endogenous gene regulators and participate in a wide array of biological functions, by promoting target mRNA degradation and inhibition of translation ,29,30. ETestis-specific miRNAs such as miR-469, testis-preferred miR-34c, miR-470 and others such as let-7 family members , and miR203 were upregulated in round spermatids of GRTH KO mice. Furthermore, the enzyme complex (Drosha-DGCR8) required for the processing of miRNA was also upregulated in the KO mice. MiR-469 target TP2 and Prm2 mRNA by binding to their coding regions and thereby preventing translation of these essential mRNAs to proteins . GRTH nepiRNAs comprise the biggest and most complex class of small non-coding RNAs. Unlike miRNAs (22 nt long), piRNAs are slightly longer, in the range of 26–32 nucleotides, and the biosynthesis of piRNAs is different from that of miRNAs and siRNAs ,34. DuriGRTH, first identified in our laboratory, is a multifunctional RNA helicase and is a member of the Glu-Asp-Ala-Glu (DEAD)-Box family of proteins which plays an essential role in the process of spermatogenesis . GRTH diGRTH acts as a negative regulator of steroidogenesis (in Leydig cells) and mitochondrial, death receptor, and nuclear factor k-B (NF-kB) pathways, and plays a central role in preventing germ cell apoptosis . In addiGRTH knock-out mice are sterile with halted spermatogenesis (step eight of round spermatids stage) with no elongated spermatids, and the sperm and lumen of the epididymis contain only degenerating spermatids. These mice have normal levels of circulating gonadotropins and testosterone with normal sexual behavior. The CBs of GRTH KO mice are highly condensed and markedly reduced in size, with a lack of the “nuage” appearance which is usual at all steps of round spermatids. These changes in the CB of null mice are consistent with their lack of GRTH-dependent nuclear-cytoplasmic transport of messages concerned with the progress of spermatogenesis. Microarray differential gene expression analysis of polysome-bound RNA revealed a genome-wide perspective of GRTH-regulated genes, with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFkB/TP53/TGFB1 signaling networks .242H) in the gene expressing GRTH, which resulted in the lack of phospho-GRTH (pGRTH). GRTH knock-in (KI) transgenic mice (human mutant GRTH gene with R242H) lack the 61 kDa phospho-species in CBs and cytoplasm, while the non-phospho form is still present in the cytoplasm, nucleus, and CBs of germ cells [In germ cells, there are two species of GRTH: a 56 kDa non-phosphorylated form, predominantly found in the nucleus; and the 61 kDa phosphorylated form (pGRTH), present exclusively in the cytosol and found to be associated with polyribosomes. Previous studies from our laboratory have shown that 5.8% of Japanese infertile men (non-obstructive azoospermia) have a specific missense mutation R22H in therm cells . The levThe absence of the phospho form of GRTH in the CB of GRTH KI mice has direct impact on the structural integrity of CBs in RS, as phospho-GRTH is one of the abundant RNA binding proteins along with other CB proteins such as MVH, MIWI, etc. Previous studies from our laboratory demonstrated that GRTH protein, through its conserved binding motifs, bind to the 3′ UTR region of Tp2 mRNAs . In a reThe loss of phospho-GRTH in GRTH KI mice serves as a very good model to study the relevance of GRTH phosphorylation in relation to CB transcriptomic/mRNA storage profiles and healthy sperm formation. The CBs obtained from the GRTH KI mice were markedly smaller in size compared to WT mice which were analyzed using electron microscopy B. FurtheThe CBs of mutant mice germ cells are small in size and store very few mRNAs which are essential for sperm formation in addition to the marked decrease in half-lives of mRNAs. The levels of non-phospho GRTH were unaltered, while pGRTH protein was completely absent in the CBs of GRTH KI. This model clearly illustrates the functions mediated by phospho-GRTH in round spermatids.Phospho-GRTH is one of the important structural proteins of CBs  along wiFurthermore, GRTH protein binding to specific germ cell mRNAs  in the CBs was highly diminished in the absence of phospho-GRTH in GRTH KI mice. This also clearly shows that the GRTH binding to important mRNAs is the phospho-form of GRTH.Since the mRNA binding function in the CBs was impacted significantly in GRTH KI mice (lacking pGRTH), this resulted in impaired chromatin compaction and spermatid elongation, and stalled spermiogenesis at step eight due to the failure of the translation machinery assembly and the loss of mRNAs to decay inside the CBs . The traeIF4e2, eIF4ebp2, eIF3l and eIF3m) together with mRNAs related to 60S subunit (Rpl10l/Rplp0) were increased and accumulated in the CB, resulting in the disruption of translation machinery of germ cells which caused spermatogenic arrest and loss of sperm in GRTH KI mice.During the process of spermiogenesis, the RS undergoes the elongation process where the histones are hyperacetylated and replaced by the highly basic transition proteins TNP1/2, which constitute 90% of the chromatin basic proteins, followed by the deposition of protamine PRM1/2 ,42. ThesCBs are highly critical organelles in the developing sperm which control the spermatid elongation processes precisely by mediating the post-transcriptional and translational control. In the absence of phospho-GRTH there is a loss and degradation of mRNAs (in CBs) coding for essential proteins which are involved in chromatin condensation, spermatid elongation, and spermatozoa formation, resulting in a lack of sperm and infertility. Our studies in this line of work unraveled the role of phospho-GRTH on cell-specific regulation occurring in the CBs of germ cells. Additional functional analyses would reveal more specific mechanisms governing functional sperm production and male fertility. Furthermore, more studies will unravel the molecular shuttling events of mRNA transport to the CB and to the polysomes by phospho-GRTH, and its role in translation.


Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated. Transposable elements (TEs) are a heterogeneous collection of DNA sequences that, when active, are capable of movement to different positions within a genome, often through a replicative mechanism. During evolution, TEs have increased their copy numbers through extensive transposition and duplication and now make up nearly 40% of mammalian genomes ,2. HowevThe RNA sequences of long-noncoding RNAs (lncRNAs) are rich in TE fragments , and whiTo explore the contribution of TE sequences to the transcriptome in a normal non-diseased state, we took advantage of the large number of hPSC short read RNA-seq samples, which we supplemented with long read RNA-seq. Our analysis shows that TEs are incorporated into both coding and noncoding RNAs, and their presence is correlated with lower levels of steady-state transcript accumulation compared to TE-free transcripts. The presence of TE sequences within RNAs also led to effects on the distribution of coding and noncoding transcripts between the nucleus and cytoplasm, RNA half-life, and differential binding of RBPs to transcripts. Whilst TE sequence fragments could be found inside predicted ORFs (open reading frames), ribosomes were bound to the RNA and peptides were detected in only a few cases. The one exception was the relatively large number of endogenous retroviral protein-derived peptides. Finally, using single-cell RNA-seq we show that transcripts containing different TE-types are present in distinct subpopulations of hPSCs. The hPSC-state is dominated by HERVH and LTR7-containing transcripts, and upon differentiation, HERVH-containing transcripts decline, and SINE and LINE-containing transcripts become more common.SOX2 and NANOG. Long read sequencing for the two cell lines was performed in duplicates. For PCR of selected transcripts, 1 ug of extracted RNA was reverse-transcribed using the PrimeScript RT Master Mix (Taraka: RR036A). Thereafter, cDNA samples were amplified by real-time PCR using TB Green™ Premix Ex Taq™ II (Taraka: RR820A) to saturation (40 cycles) with the primers listed in The cell lines used in this study were hESCs (H1 and WIBR3 line) and iPSCs . These The short reads (SR) were obtained from the Sequence Read Archive (SRA) or from the European Nucleotide Archive (ENA). In total, we found 317 publicly available SR datasets of wild-type unperturbed hESC and iPSC samples, of which 150 could pass stringent quality control criteria (see results). The accession numbers, number of reads, read lengths, inset sizes and library sizes (in base pairs) for each sample is presented in ccs  data for the RBPs DDX6, ILF2, FUS, DCP1B and matching input data  were dowhttp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeRikenCage). The reads were aligned to the human hg38 genome with HISAT2. The same pipeline for SR RNA-seq quantification using StringTie, described above, was then used for quantification. We then computed the relative concentration index (RCI) for each transcript using a previously reported formula , the relative RNA abundance was computed as the log2 fraction of the TPM at time t versus the 0 h TPM.Data was downloaded from the SRA (GSE156671). The data included RNA-seq data at 0, 1, 2, 4 and 8 h after treatment with actinomycin D. Adapters were removed by fastp. The reads were aligned to the human hg38 genome with HISAT2. The same pipeline for SR RNA-seq quantification using StringTie was used for quantification. For each transcript at each time point (The PhyloP  track fohttp://fantom.gsc.riken.jp/5/datafiles/reprocessed/hg38_latest/basic/human.timecourse.hCAGE/)  . The thr.hCAGE/) . Then th.hCAGE/)  was retr.hCAGE/) . The aliZ-score was then computed for the expression level of each hPSC transcript using the mean and the standard deviation computed from panel of somatic samples. The top 25% of transcripts with the highest Z-score were classified as ‘enriched’, with a Z-score of at least 0.66. Transcripts with a Z-score <–0.66 were classified as ‘depleted’, while those with –0.66 ≤ Z-score ≤0.66 were classified as ‘nonspecific’.We downloaded representative RNA-seq samples from human somatic cell types and tissues from the SRA or the ENA databases. We used only samples with paired-end reads, and at least 10 million reads and with an alignment rate of at least 70%. In total, 174 samples from 63 different human tissue and cell types were used see . The expThe coding potential of the transcripts was assessed with FEELnc . The traTo analyze the mass spectrometry (MS) data, we used the set of transcripts that contained a TE and had a predicted coding sequence (CDS), and then used criteria to exclude known proteins or fragments of known proteins. FEELnc predicts whether a transcript has a putative CDS, but does not predict the most likely ORF in the RNA sequence. Hence, we measured the longest ORF for each transcript. Many ORFs are incomplete and lack a STOP codon . Hence, E-value < 1e–20) for the full-length protein. Next, to restrict the search to only novel peptide sequences, we masked out any peptide sequence fragments in a predicted ORF with >90%  identity with any fragment of a protein from GENCODE. This would remove instances of ORFs that match part of a known protein but have novel peptide sequences inside. We further deleted any proteins with <20 unmasked amino acids, as short peptides are unlikely to be detected in the MS data, and any proteins that did not contain at least one K or R amino acid, as the search algorithm only considers a peptide match with at least one cleaved terminus. The raw MS data from the HipSci project was first converted to centroid data using msconvert, a part of the MSGF+  was performed on the 10× chromium according to the manufacturer's instructions, using one sample from H1 hESCs and one sample from c11/S0730 iPSCs. We supplemented this data with sc-RNA-seq data from WTC cells from E-MTAB-6687 , and twoets from , processhttps://github.com/oaxiom/hesc_lincrna. Example software code for key parts of the analysis can also be found in the Supplementary Methods. The code requires glbase3 (https://github.com/oaxiom/glbase3) to run  to run .R2 > 0.6) with other hPSC samples, (ii) must express hPSC-marker genes or (iii) must have low levels of differentiation-specific genes  paired-end-only hPSC RNA-seq data samples. To quality control the data, we began by mapping the hPSC samples to the hg38 genome assembly using HISAT2 . As a loic genes . This leic genes . We usedic genes ,37 ; variant (with an exon overlapping any exon from a GENCODE transcript but not necessarily matching splice sites) and novel (with no exon overlapping any GENCODE exon) Figure . Whilst To define coding and noncoding transcripts we used FEELnc to computationally predict coding potential. FEELnc is a machine learning algorithm that uses sequence signatures in the RNA to assess coding potential, particularly k-mer frequencies in complete and incomplete ORFs inside transcripts . FEELnc Z-score, transcripts were divided into hPSC-enriched (top quartile), hPSC-depleted (bottom quartile), or hPSC-nonspecific  , we compared our transcript assembly to a panel of non-embryonic somatic RNA-seq datasets (detailed in ) Figure . This ap4 Figure . There w4 Figure . As expe4 Figure . Finally4 Figure . Using LTE-derived sequences are found in the untranslated regions (UTRs) of coding transcripts , and conWe next explored if there were differences between TE-containing and TE-free transcripts. We looked at several transcript properties, including total transcript length, intron length, exon length and the number of exons . OverallTEs have nonrandom distributions in the genome . This isWe next asked whether the frequency of TE sequences inside mRNAs varied by TE family. LINE:L1, LINE:L2 and SINE:Alu elements were specifically enriched in both UTRs, and particularly in the 3′UTRs Figure . LINEs aWe next looked at how the presence of TE fragments affected steady-state RNA levels. There was no significant difference between the average RNA levels of TE-containing and TE-free transcripts Figure . To explAs we have shown that TE sequences inside coding transcripts are associated with changes in the steady-state levels, we next sought to explore the mechanisms behind how TEs affect mRNAs. One way to modulate RNA function is to alter its subcellular distribution. We reanalyzed hPSC subcellular RNA-seq data for the cytoplasmic and nuclear fractions , and calWe next analyzed TE sequences inside the CDSs of coding transcripts, to explore if TEs can directly contribute to the proteome. We divided the transcripts into classes based upon the effect of the TE sequence on the CDSs or predicted ORF Figure . We did Discrimination between coding and noncoding transcripts is not always clear-cut and is a challenging problem ,80,81. FTo provide further evidence for translation, we reanalyzed hESC TrIP-seq (Transcript Isoforms in Polysome-sequencing) which sequences RNAs from monosome, low (2–4 ribosomes) and high (4+ ribosome) polysome fractions based on their elution from a sucrose gradient . As expegag, pol and env but not pro, of a putative progenitor HERVK (PCAT14 gene (prostate cancer-associated transcript 14) that has hitherto been considered noncoding. We observed 14 unique HERVK peptides derived from PCAT14, and the predicted ORFs in PCAT14 code for a near-complete product of gag and fragments of pol . We noticed that TE-containing coding transcripts had only slightly reduced the RNA half-life compared to TE-free coding transcripts Figure . NoncodiTE sequences have complex patterns of evolutionary conservation, and they can show signs of evolving under purifying selection, which implies function . To explR = 0.37), and most  lncRNAs showed no primate evolutionary conservation for either the TE-containing or TE-free parts of the lncRNA  plot showed no clear separation between hESCs and iPSC samples, and no bias in the biological replicate samples , indicatL Figure .TOP2A, MALAT1, PIF1     .CDH1 and EPCAM were downregulated and the mesenchymal genes CDH2 and VIM were up-regulated  and cluster 4 (0.5%). GO analysis of genes specific to cluster 3 suggested that these cells were spontaneously differentiating, as indicated by the overrepresentation of terms related to kidney epithelium, osteoblast differentiation, and synapse formation Figure . Clusteregulated . This isegulated . The maiegulated . These degulated : ClusterIn addition to gene expression heterogeneity in single cells, there is evidence that TEs are heterogeneously expressed and mark subpopulations of cells . HoweverTEs constitute a major proportion of the DNA sequence of the human genome . The vasThe binding of RBPs to TEs is a potential mechanism to regulate TE-containing transcripts. In our reanalysis of the RBP data from Di Stefano , the difenv protein. Interestingly, HERVK env proteins were specifically detected in the neurons of amyotrophic lateral sclerosis (ALS) patients, and transgenic mice overexpressing HERVK env suffered from neurodegeneration caused by toxicity to the env protein . One class of TE-containing coding transcripts that produced detectable peptides were those that included sequences derived from fragments of or intact HERVK viral proteins. We detected peptides from a near-complete viral  protein . It is i cancers , where Tor SALL4 ,104,105.or SALL4 . Intriguanscript . This suanscript , showingThere were substantial differences between the TE-free and TE-containing coding and noncoding RNAs, particularly, steady-state levels, RNA half-life, RBP binding, and subcellular distribution of RNAs. Coding transcripts with TE sequences in the 5′UTR and CDS tended to have lower RNA levels, whilst TEs seem to be tolerated in the 3′UTRs, and correlated with higher RNA levels. For noncoding transcripts, the presence of TEs was correlated with lower RNA levels, and the higher the number of TEs in the transcript, the lower the level of RNA. These effects have been hinted at before , but we Overall, our analysis demonstrates that TE sequences are incorporated into the RNAs of hPSCs and have a greater impact than previously appreciated. Utilizing ultra-deep short read sequence data and guided by long read RNA-seq we assembled transcribed TEs in their transcriptomic context and explored how TEs can impact steady-state RNA levels, half-life, subcellular distribution and RBP binding patterns. Our data suggests that TEs have important roles in regulating RNA metabolism, and that TEs are a major component of the normal transcriptome of hPSCs.The long read RNA-seq was deposited in the Sequence Read Archive (SRA) with the accession number: PRJNA631047, and the sc-RNA-seq data with the accession number: PRJNA631808.gkab710_Supplemental_FilesClick here for additional data file.


RNA editing leads to post-transcriptional variation in protein sequences and has important biological implications. We sought to elucidate the landscape of RNA editing events across pediatric cancers.Using RNA-Seq data mapped by a pipeline designed to minimize mapping ambiguity, we investigated RNA editing in 711 pediatric cancers from the St. Jude/Washington University Pediatric Cancer Genome Project focusing on coding variants which can potentially increase protein sequence diversity. We combined de novo detection using paired tumor DNA-RNA data with analysis of known RNA editing sites.n = 706) or C-to-U sites (n = 14). RNA editing was enriched in brain tumors compared to other cancers, including editing of glutamate receptors and ion channels involved in neurotransmitter signaling. RNA editing profiles of each pediatric cancer subtype resembled those of the corresponding normal tissue profiled by the Genotype-Tissue Expression (GTEx) project.We identified 722 unique RNA editing sites in coding regions across pediatric cancers, 70% of which were nonsynonymous recoding variants. Nearly all editing sites represented the canonical A-to-I (In this first comprehensive analysis of RNA editing events in pediatric cancer, we found that the RNA editing profile of each cancer subtype is similar to its normal tissue of origin. Tumor-specific RNA editing events were not identified indicating that successful immunotherapeutic targeting of RNA-edited peptides in pediatric cancer should rely on increased antigen presentation on tumor cells compared to normal but not on tumor-specific RNA editing per se.The online version contains supplementary material available at 10.1186/s12885-021-08956-5. Post-transcriptional modification of RNA sequences, termed RNA editing, occurs in many species . In humaDespite clearly established mechanisms for canonical RNA editing, there is a lack of consensus regarding its prevalence. Some consider RNA editing to be a rare event across the transcriptome –13, whilThese controversies highlight the importance of accurate RNA-Seq mapping to the human transcriptome , which c, we also analyzed known RNA editing sites reported in the RADAR [We applied StrongArm along with a post-processing pipeline to identify novel and known single nucleotide variant (SNV) RNA editing events in protein-coding regions that show differences in matched RNA-Seq and DNA sequencing (WGS or WES) from 711 pediatric cancer samples from the St. Jude/Washington University Pediatric Cancer Genome Project (PCGP) . As low-he RADAR  databasehe RADAR . In all,https://pecan.stjude.cloud/permalink/rnaediting) [Details regarding RNA and DNA isolation from PCGP samples have been reported in a series of PCGP-related papers –31. All editing) .PolyA-enriched mRNA-seq of PCGP samples was performed using the Illumina TruSeq V2 RNA library preparation kit, with a starting input of 1μg of total RNA according to manufacturer’s protocol. The number of cycles of library amplification was reduced to 10 cycles to reduce PCR duplicates. The resulting data files were converted to FASTQ files using CASAVA 1.8.2. All reads were 101 bp in length. For discovery of RNA editing events, we initially used 717 pediatric cancer samples’ RNA-Seq from the PCGP, including only samples for which tumor-normal DNA-Seq (WGS or WES) was also available . We used several input BAMs for each sample, each with two million reads. The whole workflow of StrongArm is illustrated in Fig. StrongArm accepts reads stored in unaligned BAM format , which mIn the first phase, reads were aligned using five combinations of mapper and database. The aligners we used include BWA  and STARAfter initial mapping, the pipeline chooses a record for each read from among the five available. The best record is chosen according to the following algorithm: (1) mapped records are always better than unmapped; (2) among mapped records, those with more matches are always better than those with fewer; and (3) among mapped records with the same number of matches, records with fewer indels are always better than those with more.At this stage, there may still be several records tied for the best. If there are multiple candidates, and the reads are paired, then the best pair is chosen based on the following rules: 1) pairs on the same chromosome are always better than pairs on different chromosomes; (2) pairs that are closer  are then preferred; (3) pairs in forward-reverse orientation are then preferred; (4) pairs from the same mapper and database combination are finally preferred; and (5) if ties remain, then the choice is made based on the priority of the mapper and database combination used  if the read was originally unmapped, then it is an improvement; (2) the alignments are scored using + 1 for alignment, − 1 for gap open, − 1 for gap extend, and + 5 for any splice of length < 100 kb, and if the scores are unequal, then improvement is determined based on the scoring; (3) if the reads were previously on different chromosomes, but sim4 places them on the same chromosome, then it is an improvement; (4) if the reads were previously not in forward-reverse orientation, but sim4 places them in forward-reverse, then it is an improvement; and (5) otherwise, it is not an improvement.At this point, the pipeline also soft-clips the alignment of poly-A tails, which may contain one or more spurious splices to poly-A runs in the reference genome. The new individual aligned BAM files are sorted and merged, duplicates are marked, and the files are indexed, as for the unrefined BAM, to create the final BAM file.StrongArm alignment was compared to TopHat and STAR alignment for 15 PCGP samples’ RNA-seq data. TopHat version 2.1.1 using Bowtie version 2.1.0 was run in both default mode with parameter “-m 2″, and annotation-based mode with parameter “-m 2 --transcriptome-index GENCODE.v19.index”. STAR mapping was performed with STAR version 2.7.1a in two-pass mode. The GRCh37 reference genome was used. The RNA editing VAF of the 722 sites analyzed in the study were determined using the Ace2.ReadReport utility within Bambino  for the , we reviewed the evidence of coding editing sites that were included in the RADAR database with ≥3 mutant reads in ≥2 PCGP samples . The RADAR [https://darned.ucc.ie/download/. REDIportal [http://srv00.recas.ba.infn.it/atlas/download.html. The RNA editing VAF of the 722 sites were calculated using the Ace2.ReadReport utility within Bambino [After mapping is performed with StrongArm, the RNA editing detection pipeline starts with variant calling using Bambino  followedhe RADAR  version he RADAR  hg19 ediDIportal  hg19 edi Bambino .https://github.com/ucscGenomeBrowser/kent/blob/master/src/hg/makeDb/outside/gtexHub/metadata/sraToSample.tab.GTEx RNA-Seq files were previously downloaded from dbGaP accession phs000424 and reads were previously mapped using STAR version 2.7.1a in two-pass mode to hg38 for another study. To avoid remapping these 5454 BAM files to hg19 using StrongArm for this study  we instead used the existing hg38-aligned files and analyzed the RNA editing levels for each of 722 RNA editing sites identified in pediatric cancer (converted to hg38 coordinates), using the Ace2.ReadReport utility in Bambino . To veriIn Fig. Neoepitope prediction was performed using neoepiscope  run on aCurrent RNA-Seq alignment tools have varying properties that can affect the detection of RNA editing. For example, STAR has a high mapping rate because it permits incomplete alignments which increase the sensitivity for detecting RNA editing events, but the fraction of fully-mapped reads is lower than other tools which can lead to false-negatives. Annotation-based TopHat2 has the opposite characteristics . As exprTo facilitate the detection of RNA editing and other variants using RNA-Seq data, we designed an exhaustive mapping pipeline called StrongArm. This pipeline selects the mapping location of a read-pair based on mapping to five different reference database/mapper combinations, from which the best mapping is chosen Fig. A. To anaWe analyzed RNA editing in 954 pediatric cancer samples from PCGP  in its multi-exonic paralog GLUD1 near an exon-intron boundary  Variants at the 3′ side of homopolymers on the antisense strand, suggesting an error introduced during reverse transcription , we identified 722 unique RNA editing sites in coding regions post manual curation  cancers  was edited specifically in blood cancers , we first identified the reads for which the read or its mate pair were mapped to splice junctions. These “spliced reads” were considered as a signal of mRNA which were then used to calculate the (spliced) mRNA editing level Fig. A. The diNARF  different editing between mRNA and pre-mRNA using polyA-enriched RNA-Seq  in 10% of acute myeloid leukemia (AML), 18% of T-lineage acute lymphoblastic leukemia (T-ALL), and 2% of B-ALL, but virtually no expression in other cancerous tissues  as shown for  cancers , an editThese findings indicate that the RNA editing profile of each pediatric cancer type is similar to its corresponding normal tissue of origin. Thus, the somatic mutations present in pediatric cancers do not appear to promote tumor-specific RNA editing events which can be immuno-therapeutically targeted. Rather, RNA editing profiles in pediatric cancer likely result from the transcriptional state the cancer inherits from the original normal tissue. However, RNA editing events occurring in cancers with overexpression of the edited transcript may provide therapeutic targets, which merits further study. These results also provide a map of the coding RNA editing events across pediatric cancers, and identify novel RNA editing events whose function should be explored in future studies.Additional file 1: Supplementary Fig. 1. RNA editing analysis workflow and samples included. Workflow showing the discovery process and the criteria for sample inclusion at each step. To discover de novo potentially novel RNA editing sites (top), SNV calling was performed on samples with tumor RNA-Seq, and germline plus tumor DNA-Seq (either WGS or WES), including 717 samples . After filtering and discovering RNA editing events in these 717 samples as shown in Fig. n = 722 sites total) was analyzed in 954 pediatric cancer RNA-Seq samples from the PCGP, whether or not DNA-Seq was available (middle). Finally, certain samples with batch effects in RNA editing VAFs and relapsed or duplicate samples were filtered out, resulting in 711 pediatric cancer diagnosis samples with one diagnosis sample per patient (bottom). This set of 711 samples was the primary sample set shown in analyses in this study. Supplementary Fig. 2. Examples comparing alignment between StrongArm vs. STAR mapping. (A) Splice junction-adjacent reads that failed mapping by STAR (blue) but mapped by StrongArm (red) as viewed in the BAM alignment. This shows a view of one sample’s BAM files aligned by the two tools, with each row representing one read, and part of the COPA gene is shown. Soft-clipped read regions have a darker gray appearance. Gray arrows (>) indicate that part of the read is aligned elsewhere (to another exon). Non-reference sites are shown in red, including an RNA editing site (T > C). Only reads containing the edited allele are shown. (B) Read with RNA editing at the end that failed mapping (soft-clipped) by STAR but mapped by StrongArm. Coloring and other features are as in (A), except that the MRPS27 gene is shown. This shows a view of one sample’s BAM files aligned by the two tools. Only reads containing the edited allele  are shown. Supplementary Fig. 3. Mapping rate and soft-clipping comparison between StrongArm and other tools. (A) Mapping rate comparison between StrongArm, STAR, TopHat2 and annotation-based TopHat2. The mapping rate of the same sample in different aligners is connected by a dotted line. Y-axis represents the percent of mapped reads, and 15 selected pediatric cancer samples from PCGP are analyzed. (B) The percentage of reads with soft-clipped nucleotides in each aligner. The same sample is connected by a dotted line. TopHat2 does not map soft-clipped reads. (C) The percentage of reads (y-axis) with different numbers of soft-clipped bases (x-axis). The unclipped reads (0) are separated due to different axis scale. This compares STAR (blue points) and StrongArm (red points) and does not include TopHat2, since TopHat2 does not map soft-clipped reads. Each point represents one of the 15 PCGP samples shown in (A) and (B). Boxplot shows median (thick center line) and interquartile range (box). Whiskers are described in R boxplot documentation (a 1.5*interquartile range rule is used). Supplementary Fig. 4. Comparison of RNA editing detection between STAR- and StrongArm-mapped RNA-Seq data. This analysis includes 15 PCGP samples’ RNA-Seq data mapped by both StrongArm and STAR . (A) More RNA editing sites are evaluable by StrongArm than by STAR mapping. Boxplot shows the number of RNA editing sites  that are informative (at least 10 reads of coverage) only by STAR or only by StrongArm mapping in each sample. Boxplot shows median (thick center line) and interquartile range (box). Whiskers are described in R boxplot documentation (a 1.5*interquartile range rule is used). (B) High concordance of RNA editing VAFs derived from STAR-mapped and StrongArm-mapped BAM files, when analyzing RNA editing sites with at least 10 reads of coverage with both STAR and StrongArm. See table at top-left of each graph to see the number of variants falling into this category (the “Both (shown)” category). Each graph shows one leukemia patient. Each point represents a single RNA editing event positioned by its RNA editing VAF by StrongArm (x-axis) or by STAR (y-axis). Dotted line represents the identity line (x = y). r value is by Pearson correlation. Supplementary Fig. 5. False-positive RNA editing examples requiring manual removal. (A) Example false RNA editing due to the presence of a germline SNP in one of two paralogs. GLUD1 and GLUD2 are paralogs; GLUD1 has introns while GLUD2 is mon-exonic. The coding regions (CDS) have 98% identity between the two genes. Left, in patients with GLUD1 SNP rs9421572 (a SNP which is near a splice junction in exon 7), DNA (WGS) reads will correctly map the SNP to GLUD1 instead of GLUD2, as the intronic region of reads containing the SNP will map uniquely to GLUD1 but not to intron-less GLUD2. Right, in RNA-Seq, by contrast, neither GLUD1 nor GLUD2 mRNAs contain introns, and therefore the mapping will prefer GLUD2 since mapping that does not require the read to span a splice junction is preferred, and GLUD2 lacks introns making it preferred. The GLUD1 SNP is therefore aberrantly considered to be a GLUD2 RNA editing event. Such events must be removed by manual curation. (B) Example of false RNA editing due to the presence of a homopolymer genomic region using an example in the RNF19A gene. False-positive editing events frequently occurred within one base position of homopolymer sequences on the 3′ side of the homopolymer along the antisense strand, suggesting the error was introduced during reverse transcription. Such events must be removed by manual curation. (C) Example of false RNA editing due to mapping to the wrong splice variant. The PHB2 gene includes a very small (or “nano”) exon in one of the UCSC transcripts (uc021qug.1). However, none of the RefSeq transcripts used for mapping includes this exon; therefore, reads which include the nano exon are instead incorrectly mapped to NM_001144831 which lacks the nano exon. Thus, RNA-Seq reads for many samples, including the example adrenocortical cancer sample SJACT001, incorrectly map to NM_001144831 and appear to have an RNA editing event in a region that should in fact map to the nano exon. Supplementary Fig. 6. An example of outlier expression of an RNA-edited gene in a specific tissue type. (A) Boxplot showing CD6 expression in TPM across PCGP pediatric cancer tissues. Parentheses indicate the number of samples analyzed. See Fig. B) As in (A), except that the expression of CD6 is shown in normal GTEx tissues. Due to different RNA-Seq library preparation and sequencing methods, the data in panels (A) and (B) may not be directly comparable to one another.Additional file 2: Supplementary Table 1. 722 RNA editing sites identified in pediatric cancer samples. Supplementary Table 2. Deep amplicon sequencing of RNA vaildation of 10 RNA editing events. Supplementary Table 3. Percentages of cancer and normal tissue samples with RNA editing at each site. Supplementary Table 4. VAFs of RNA editing events in each sample. Supplementary Table 5. Neoepitope predictions for 722 RNA editing sites. Supplementary Table 6. Abbreviations for cancer and normal tissue types.


Retrotransposons are genetic elements present across all eukaryotic genomes. While their role in evolution is considered as a potentially beneficial natural source of genetic variation, their activity is classically considered detrimental due to their potentially harmful effects on genome stability. However, studies are increasingly shedding light on the regulatory function and beneficial role of somatic retroelement reactivation in non-pathological contexts. Here, we review recent findings unveiling the regulatory potential of retrotransposons, including their role in noncoding RNA transcription, as modulators of mammalian transcriptional and epigenome landscapes. We also discuss technical challenges in deciphering the multifaceted activity of retrotransposable elements, highlighting an unforeseen central role of this neglected portion of the genome both in early development and in adult life. LTR  retrotransposons comprise about 8% and 10% of the human and mouse genomes, respectively [LTRs are autonomous elements only active in mice and other species [LTR has LTRs at both ends and a central region carrying three retroviral open reading frames (ORFs): gag, pol, and truncated/mutated Δenv. Mouse and human LTRs can be further classified into several super families that include endogenous retroviruses (ERV) [Retrotransposons, which account for more than 40% of the human and mouse genomes, propagate themselves through transcription and reverse-transcription machinery based on a "copy-and-paste" mechanism , 2. Thisectively . LTRs ar species , not hum species . A typices (ERV) .LTRs in both human and mouse genomes [long interspersed nuclear elements (LINEs) are the major order. These autonomous transposable elements contain two ORFs: ORF1, and ORF2. ORF1 and ORF2 are both required for retrotransposition. ORF1 encodes a ~ 40-kDa protein with nucleic acid-binding and chaperone activity. ORF2 encodes a ~ 150-kDa protein with endonuclease and reverse-transcriptase activity [LINE-1 RNA. The L1 transcript is then exported to the cytoplasm and translated into ORF1p and ORF2p. The binding of the two proteins to the L1 RNA produces a ribonucleoprotein (RNP) complex, which is imported back into the nucleus. Here, ORF2p uses a free 3′–OH produced through its endonuclease activity on the genomic DNA strand as a primer to reverse transcribe L1 RNA. Then, L1 RNA is removed from the intermediate DNA:RNA hybrid, and a second DNA strand is synthesized by ORF2 to generate a novel L1 insertion. This molecular process is known as target-primed reverse transcription (TPRT) [Among non-LTR retrotransposons, which are more abundant (17–20%) than  genomes , long inactivity –11. A con (TPRT) . ORF0, cn (TPRT) .Short interspersed nuclear elements (SINEs) are another order of retrotransposons. As SINE elements utilize L1-encoded machinery to complete their propagation, they are usually called non‐autonomous retrotransposons. In humans, active SINEs include Alu and SINE‐R‐VNTR‐Alu (SVA) retrotransposons [SINEs in mice contain the 7SL‐derived B1 elements and the tRNA‐derived B2 elements [LTRs and LINEs are transcribed by RNA polymerase II [SINEs depend on polymerase III [Of note, although the human and mouse genomes contain a high number of L1 copies, accumulating evidence indicates that only around 100 copies and 3000 copies, respectively, are capable of retrotransposition –16. Shornsposons –19, actielements . While Lerase II , 21, trarase III .LINE-1 elements [ERV-derived enhancers [ERV-encoded proteins accumulation [Retrotransposons may be a source of mutations leading to genomic instability and devastating consequences for the host . Therefoelements –38. Howeelements , neuroloelements , male stelements  or fetalelements . Howeverelements , ERV-dernhancers  and ERV-mulation , or a TEmulation , 47. Themulation , 49.cis-regulatory element [Several decades ago, Britten and Davidson proposed the "gene battery" theory in which repetitive sequences can be used as a pool to deposit regulatory sequences in specific genomic positions to bring multiple genes under the same regulatory network , 51. By  element , 55, as  element –60.LTRs co-opted by host genomes, e.g., MT-C [MLT2B3 LTR [MERVL derived LTR, suggesting that this co-opted sequence has played a leading role in cell-fate regulation in placental mammals [L1 provides alternative promoters for many neuronal protein-coding genes [Among g., MT-C  and the T2B3 LTR , most ac mammals , 64  and trophoblast stem cells (TSCs), respectively. However, only a minor dysfunctional effect on transcription was observed when these elements were perturbed by CRISPR-mediated transcriptional inhibition [HERVK element LTR5HS present in the human genome by combining CARGO (Chimeric Array of gRNA Oligos) with CRISPR technology and showed that activation/silencing of LTR5HS consistently modulated the expression profile of hundreds of human genes [SVA, HERVK, and HERVH TE subgroups (TEENhancers) in naive hESC and demonstrated that these elements markedly influence transcription during human embryonic genome activation (EGA) by acting as stage-specific enhancers [cis-regulatory elements was also found in the innate immunity response by demonstrating their contribution in shaping the evolution of the IFN-response transcriptional network in human [Identification and functional characterization of enhancers elucidate several common features, including unique epigenomic signature, ncRNA transcription, and TF binding sites –72. Comphibition . This rehibition . Therefoan genes . Pontis nhancers . A role in human . On the in human .In addition to immobilized retroelements that modulate transcriptomic homeostasis, our team's recent work has also demonstrated that developmentally regulated de novo insertions in somatic cells contribute to shaping the tissue-specific transcriptional landscape. When mouse fibroblasts were reprogrammed into dopaminergic neurons (iDA), reactivation and retrotransposition of L1 elements occurred. De novo insertions mostly landed near neuronal genes and affected proximal chromatin accessibility, thus creating new transcriptional units, especially of lncRNA .LINE-1 RNA came from a seminal study from Torres-Padilla's group on early mouse embryogenesis, where they demonstrated how early-stage transcription of L1 is important for blastocyst formation as it is the key driving force modulating global chromatin structure in cis [LINE-1 highly expressed in pre-implantation embryos and downregulated as the differentiation proceeded [LINE-1 RNA resulted in global chromatin accessibility impairment and reduction of blastocyst formation [L1 RNA did not revert the effect of nuclear L1 RNA silencing [L1 transcripts at early development stages in controlling chromatin opening, most probably in cis, independent of retrotransposition  were found to be involved in maintaining nucleolar structure and rRNA synthesis [HERVH-derived transcripts provide functional binding sites for a combination of naive pluripotency transcription factors such as LBP9 and OCT4. Their interaction activates hESC-specific alternative and chimeric transcripts, including long non-coding RNAs that modulate pluripotency and maintain human embryonic stem cell identity [In situ analysis of several mammalian cell lines revealed a class of repeat-containing RNAs, mostly ructures . In addiunctions . LINE-2 an brain , and Aluynthesis . HERVH-didentity .SINE B2 repeats in mouse and SINE Alu repeats in human, control gene expression by binding RNA polymerase II and suppressing transcription [SINE Alu and B2 RNAs bind and inhibit RNA Pol II [B2 RNAs and Alu RNAs are self-cleaving; upon stress, these RNAs become destabilized and release stalled Pol II to activate stress response genes [SINE RNAs appear to play a key role in stress response by suppressing or activating transcription based on their processing status  and nucleolus , respectively. This spatial segregation is highly conserved across all eukaryotes and is required to maintain euchromatin/heterochromatin compartmentalization [L1 RNA preferentially binds to L1-enriched genomic loci in ESC by ChIRP-seq [L1 RNA triggered a dramatic re-location of L1 enriched chromosomal regions from the nucleolus and inactive LADs to the nuclear interior and, in turn, their de-repression [L1 DNA and RNA induce HP1α phase separation promoting heterochromatin compartmentalization [L1 RNA in nuclear organization, where L1 RNA binding to L1 DNA silences L1-enriched genes by directing genomic regions to inactive compartments  engaged in long-range intra-chromosomal interaction and looping formation in specific chromatin compartments [HERV-H form TAD boundaries that are specific to pluripotent stem cells and their frequency weaken during differentiation to cardiomyocites [SINE elements are highly enriched for CTCF binding and over-represented at TAD boundaries [SINEs containing CTCF binding sites that are not evolutionary conserved [Retrotransposons act as either boundary elements or docking sites to facilitate folding and compartmentalization of the genome, whose CTCF binding site bordered topologically associated domains (TADs) are important 3D units. Using Capture-4Tran and 4Tran-PCR, Raviram et al. discovered several young mouse ERV families  and mouse  genomes, respectively [LINE-1 loci is fundamental to manipulating these elements' expression through CRISPR-based technologies and testing the effects on genome functions and dynamics.Improved genome editing technologies have allowed modulation of expression/repression of genomic loci, including transposable elements –115. In elements , 81. Chuem cells . As mentInterestingly, Ranjan and Gene used a system based on the fusion of an RNA endonuclease with a nuclease-dead Cas9 (dCas9) to target and eliminate toxic microsatellite repeat RNA , a transLINE-1 was found to be preferentially bound to LINE-1 DNA-rich regions of the genome [Mounting evidence suggests that retrotransposon-derived transcripts may behave like lncRNAs. Global RNA-DNA/chromatin interaction profiles have been mapped using RNA-centered genome-wide analysis. For instance, e genome . To undee genome –122 or Re genome –125. Howe genome , 127.Applying these techniques to in vivo studies in animal models or human biopsies is exceptionally challenging because of the limited amount of starting material. The evolution of protein mass spectrometry instruments, like the recent release of 4D  proteomics platform, has significantly reduced the peptide detection limit, making it possible to study RNA–protein interactions quantitatively with low input samples –130.Another unresolved and controversial question is whether transcription of retrotransposons or retrotransposon-derived transcripts is a real driver of biological processes , 88. ChrInitially, retrotransposons were exclusively considered hazardous genetic elements that required strict epigenetic mechanisms for silencing. Now, largely thanks to the development of novel technologies, mounting evidence unveils an essential role of retrotransposon dynamics in fundamental physiological cell functions, from co-option as regulatory elements to orchestrate cell type specific transcriptional programs, chromatin remodeling, 3D genome organization up to immune systems involved in tissue homeostasis and defense mechanisms. A deeper integration of retrotransposons and host genome in which the dynamic nature of mobile elements includes not only DNA but also RNA impacting both nuclear and cytoplasmic functions may be envisaged.How have these processes evolved? The relatively abundant amounts and quality of ncRNAs produced by these elements in response to environmental signals and the emerging role of RNA in nuclear structure and function, may indicate how these two apparently separated portions of the eukaryotic genome began talking to each other. The two systems could have merged into a unified functional unit, and perhaps further evolved based on epigenome-mediated somatic phenotype variation. As summarized above, retrotransposon-derived RNAs have a strong regulatory potential due to their interaction with proteins involved in chromatin and transcriptional regulation, indicating an exciting new frontier in epigenome biology, developmental processes and control of functional tissue homeostasis. Indeed, finely regulated expression of retrotransposons is essential for proper genome function, as their aberrant expression is strictly correlated to embryo development failure and the onset of several pathologies associated with accelerated aging, cancer, and chronic inflammation.The development of new technologies, and improvements of the available one, will deepen our understanding of how the host genome balances the detrimental and beneficial effects of retroelements in evolution, development, and pathological conditions.