O-methylation (Nm) were documented across the rRNA; however, none were localized to the functional domain. Taken together, our results suggest that despite dramatic changes in the nucleolar organization, rRNA transcription and processing persist during lytic reactivation of KSHV. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.The nucleolus is a subnuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during the lytic reactivation of KSHV. This was manifested by the redistribution of key nucleolar proteins, including the rRNA transcription factor UBF. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Significantly, the redistribution of UBF was dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation (Ψ) and 2′- KSHV, also known as human herpesvirus 8 (HHV-8), is a cancer-related gamma-2 herpesvirus which is etiologically implicated in all types of Kaposi’s sarcoma (KS). In addition, KSHV is the causative agent of other disorders, including primary effusion lymphoma (PEL), multicentric Castleman’s disease, and KSHV-inflammatory cytokine syndrome ,4,5,6. AO-methyltransferase Fibrillarin, and the pseudouridine synthase Dyskerin (DKC1). Late rRNA processing, along with assembly and transport of ribosomal subunits, occurs in the GC region, which comprises the remainder of the nucleolus and is enriched in processing and assembly factors such as Nucleophosmin 1 (NPM1/B23) and Nucleolin, as well as ribosomal proteins. The nucleolus is also involved in other cellular processes that may not be directly associated with the biogenesis of ribosomal subunits, including cell cycle control, stress responses, senescence, telomerase activity, protein degradation and sequestration, chromosomal domain organization, and innate immune responses ATP and T4 polynucleotide kinase (Promega) for 30 min at 37 °C. In all cases, membranes were washed twice in 2X SSC, 0.1% SDS at 60 °C for 20 min, and once in 0.1X SSC, 0.1% SDS at 43 °C for 15 min. Then, 18S and 28S rRNA visualized by ethidium bromide was used as loading control.2CO3  at 37  °C for 4 h. The RNA was then fragmented to a size range of 50–150 nt, adaptor was ligated to the 3′ ends, and cDNA was prepared using reverse transcriptase. Then, an adaptor was ligated to the cDNA, and after amplification, the samples were sequenced in Illumina machine in paired end mode. The reads were mapped to human rRNA using Smalt v0.7.5 (default parameters). Each read pair was “virtually” extended to cover the area from the beginning of the first read to the end of its mate. For each base, the number of reads initializing at that location as well as the number of reads covering the position were calculated. A combination of Bedtools and in-house Perl scripts were used to calculate the Ψ-ratio and Ψ-fc (fold change). For each nucleotide, we computed the Ψ-ratio, dividing the number of reads covering that nucleotide by the number of nucleotides initiating at the following base . This was repeated for (−CMC) and (+CMC) samples. The Ψ-fc was computed as the log2-fold change of the Ψ-ratios in iSLK versus the SLK samples. We applied this threshold to each sample. Primer extension was performed as previously described [http://imagej.nih.gov/ij/, accessed on 16 July 2022).Pseudouridine sites were mapped as previously described . RNA froescribed  with or Total RNA (5 µg) was treated with 50% hydrazine (Sigma) for 45 min on ice, and ethanol precipitated . The RNA® MyOne™ SILANE beads , and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase . The RNA was subsequently degraded using 2 µL of 1 M NaOH, and the cDNA was cleaned using Dynabeads® MyOne™ SILANE beads . The cDNA was further ligated to a 3′ adapter using a high concentration T4 RNA Ligase 1  and cleaned of excess adapter using Dynabeads® MyOne™ SILANE beads . The adapter-ligated cDNA was PCR enriched using NEBNext® High-Fidelity  polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel  and size selected at the range of 150–300 bp (containing ~30–180 nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit , and sequenced in a Nextseq system  in paired end mode (20 million reads for each sample).To perform HydraPsiSeq, the fragmented RNA (~800 ng) was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase , cleaned by Agencourt RNA clean XP beads  and ligated to a 3′ linker using high concentration T4 RNA Ligase 1  in a buffer containing DMSO, ATP, PEG 8000, and RNase inhibitor . The ligated RNA was cleaned from excess linker using Dynabeadshttp://www.sanger.ac.uk/resources/software/SMALT/, accessed on 16 July 2022) with default parameters. For each sample, the resulting bam file was sorted and filtered for proper pairs using Samtools v1.9 [https://github.com/FlorianPichot/HydraPsiSeqPipeline, accessed on 16 July 2022), as previously described [The paired end reads obtained from each sample were aligned to the human rRNA sequence using Smalt v_0.7.5  was added, and RNA samples were incubated at 90 °C for 20 min . The hydrolyzed RNA was then used for library preparation. Briefly, 800 ng of the RNA was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase  and cleaned by Agencourt RNA clean XP beads . The RNA was then ligated to 3′ linker using high concentration T4 RNA Ligase 1  in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor  for 1.5 h at 22 °C. The ligated RNA was purified from excess linker using Dynabeads® MyOne™ SILANE beads , and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme  at 55 °C for 45 min. Next, the RNA was degraded using 2 µL of 1 M NaOH, and the cDNA was cleaned using Dynabeads® MyOne™ SILANE beads . The cDNA was further ligated to 3′ adapter using a high concentration T4 RNA Ligase 1  overnight at 22 °C and cleaned of excess adapter by using Dynabeads® MyOne™ SILANE beads . The adapter ligated cDNA was PCR enriched using NEBNext® High-Fidelity  polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel  and size selected for the range of 150–300 bp (containing ~30–180 nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit  and sequenced in a Nextseq system  in paired end mode (20–40 million reads for each sample).Total RNA (5 mg) was denatured at 90 °C for 2 min in a thermocycler. Then, an equal volume of buffer  with the following parameters: --stringency 4 --length 30 --paired --retain_unpaired. The alignment to the reference rRNA sequence was performed by STAR (ver 2.0.6)  [To analyze the RiboMeth-seq libraries, the reads were initially trimmed of adapter sequences using Trim Galore version 0.4.4 (ly 2022) , mapped,ly 2022) . Countinly 2022) .The lytic cycle of KSHV involves the formation of nuclear replication compartments along with changes in nuclear structures . To charO-methytransferase and is involved both in methylation and processing of rRNA, was evident in cells expressing nuclear ORF45 with intact diffused chromatin. Yet, at later stages of the lytic cycle, this protein was localized within 1–3 large peripheral foci surrounded by dense chromatin reminiscent of the nucleolar compartment. NPM1, a multifunctional nucleolar protein that normally resides in the GC substructure, exhibited two major patterns upon induction of the lytic cycle, characterized either by diffuse nuclear staining or marginal nucleolar staining. Similar patterns of UBF and Fibrillarin distribution were documented upon lytic reactivation in iSLK BAC16-infected cells in which the expression of ORF45 was detected with antibodies  which is included in the 47S pre-rRNA transcript but not in the mature rRNA. This analysis employed BAC16-mCherry-ORF45-infected iSLK cells which enabled tracking lytic reactivation in ~70% of cells. As shown in In addition, no accumulation of the selected pre-rRNA intermediates was found by Northern blot analysis, suggesting that the proper processing of pre-rRNA continued . SignifiAlthough the possibility that changes in the half-life of mature rRNAs during lytic reactivation has not been ruled out, when taken together, the lack of accumulation of selected pre-rRNA intermediates suggests that rRNA transcription and processing proceed during lytic reactivation of KSHV, despite the different distribution of nucleolar proteins, which are known to participate in these processes.Recently, it was postulated that rRNA modifications may provide an important source of ribosome heterogeneity, and tune ribosome function in response to various signals ,90,91,92To obtain an independent assessment of the Ψ level, we utilized HydraPsiSeq which relies on the random cleavage of uridine nucleotides upon treatment with hydrazine and aniline. The ability of Ψ sites to resist fragmentation enables stoichiometric quantification of this RNA modification . This meO-methylation (Nm), which is the second most abundant RNA modification, we sought to analyze the stoichiometry of Nms using RiboMeth-seq [To gain insight into the level of 2′-Meth-seq ,73. In tMeth-seq ,73. UsinMeth-seq  and are Meth-seq .The protein content and organization of the nucleolus are dynamic and change under different stress conditions, such as transcriptional inhibition and DNA damage . Major aBy using immunofluorescence microscopy, we documented an extensive and progressive destruction of the nuclear architecture during lytic reactivation of KSHV, with significant condensation of the nuclear chromatin to the periphery of the nucleus. This was associated with extensive redistribution of the nucleolar protein UBF, which normally resides at the nucleolar FC substructure. The RNA polymerase I transcription factor UBF, the large RNA polymerase I subunit RPA194, and the vBcl-2 nucleolar partner PICT-1 , which aThese experiments suggest that nucleolar proteins undergo nonrandom programmed dispersion that maintains certain protein complexes, but disrupts others during productive infection. Furthermore, it is possible that the same mechanisms and functions engage the localization of UBF, RPA194 and PICT-1, which may participate in related processes during infection. It is not currently known whether these proteins acquire additional functions during lytic reactivation, or whether they continue to function according to their known roles. In addition, it is not known whether the dispersion represents a cellular response to viral infection, or whether it takes place due to the formation of replication compartments that generate condensed peripheral chromatin and thus may favor virus replication. Nevertheless, the recruitment of particular nucleolar proteins to replication compartments, such as Nucleolin, which was previously reported to participate in the formation of adenovirus and hCMV replication compartments ,100,101,Why viral proteins localize to the nucleolus and how nucleolar perturbations affect virus infection have not been precisely explained to date. It is likely that viruses utilize the pluri-functional nature of the nucleolus to enhance virus replication or to avoid cell surveillance mechanisms, yet it is also possible that nucleolar alterations signify cellular anti-viral responses. It has also been postulated that maintaining the global production of pre-rRNA avoids activation of nucleolar stress, which can lead to cell cycle arrest and apoptosis . In addiUsing different approaches, we show rRNA levels do not change during the lytic cycle. This finding is in line with previous reports in HSV-1, describing continuous association of UBF with rDNA along with colocalization with the largest RNA pol I subunit RPA43 , and ongThe changes that take place on rRNA during lytic reactivation are intriguing. Alterations in ribosome composition, either by changes the ribosomal proteins or the rRNA modifications, could very well represent examples of epitranscriptomic regulation of mRNA translation ,92,102. Not much is known about the effects of rRNA modifications on ribosome function. A global reduction in Nm by Fibrillarin depletion modulated the ratio between cap-dependent and IRES-mediated mRNA translation . InteresBased on HydraPsiSeq, it was concluded that rRNA pseudouridylation levels differ among human cell lines and during differentiation . VariablIn summary, we describe major changes in the nuclear and nucleolar organization that take place during lytic reactivation of KSHV. These changes may signify cellular nucleolar stress responses, but may also occur due to the recruitment of cellular proteins to replication compartments or association with viral proteins that may have major implications on the translation of viral and host cell proteins during lytic infection. Previous studies showed that the different forms of KSHV infection are controlled through cellular and viral functions, which reprogram host epigenetic, transcriptomic, post-transcriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellular components. The study of rRNA modifications is still in its infancy; however, the notion of altering cell fate by regulating rRNA modifications has recently begun to emerge, and its significance in viral infection is intriguing.


The natural chemical modifications of messenger RNA (mRNA) in living organisms have shown essential roles in both physiology and pathology. The mapping of mRNA modifications is critical for interpreting their biological functions. In another dimension, the synthesized nucleoside analogs can enable chemical labeling of cellular mRNA through a metabolic pathway, which facilitates the study of RNA dynamics in a pulse-chase manner. In this regard, the sequencing tools for mapping both natural modifications and nucleoside tags on mRNA at single base resolution are highly necessary. In this work, we review the progress of chemical sequencing technology for determining both a variety of naturally occurring base modifications mainly on mRNA and a few on transfer RNA and metabolically incorporated artificial base analogs on mRNA, and further discuss the problems and prospects in the field. O-methylation (Nm) . . 123]. DIn this work, we review the current state-of-the-art chemical sequencing methods for detecting both cellular natural RNA chemical modifications and metabolically installed tags on RNA. These technologies have significantly advanced the emerging field of RNA modifications and have prompted our understanding on sophisticated RNA metabolism and functions. Yet some issues remain. First, there are still few RNA modifications that can be detected using the chemical sequencing approach. It is rewarding to develop methods to locate other RNA modifications or discover new modifications. Second, the reliable RNA detection methods for rare and precious samples are lacking. Especially, a robust method for single cell analysis is not available. Third, simultaneous profiling of multiple modifications awaits exploration. ioorthogonal chemical/enzymatic reactions capable of carrying out diverse kinds of bases recoding are required, and are expected to possess higher reaction efficiency and sensitivity. Fourth, the modification stoichiometry should be carefully characterized because it is closely associated with the cellular functions. Fifth, the nucleoside analogs with chemical sequencing power are very rare. If we simultaneously label RNA transcripts in various kinds of cells, different chemical tags are needed. At current, 4SU, 6SG, and aIt should be highlighted that chemical sequencing methods have helped researchers elucidate the mechanism regarding how nucleic acid modification regulates gene expression. The future prospect of chemical sequencing will not only be limited to cellular system, but also be extended to disease diagnosis via the detection of RNA modifications in body fluids. It should be envisioned that the further developments will lead to broader clinical applications and benefit human health.


To establish and validate a novel method for aligning femoral rotation to accurately measure femoral offset for preoperative templating and component sizing, and to identify the physical location of two radiographic lines utilized in the described method.Cadaveric proximal femurs were skeletonized and mounted to a biaxial load frame. Two radiographic lines along the greater trochanter were identified fluoroscopically. The femurs were rotated, and images were taken when the lines appeared superimposed, then in 2-degree increments to 10° of internal and external rotation, and at 30°. Radiographic femoral offset was calculated at each angle, and the maximum and aligned offsets were compared. Bone was removed until the radiographic lines disappeared, then a metal wire was inserted in place of the bone to confirm that the lines reappeared.The physical locations of the radiographic landmarks were on the anterior and posterior aspects of the greater trochanter. The mean true femoral offset was 38.2 mm . The mean aligned femoral offset was 37.3 mm , a 2.4% underestimation. The mean angle between aligned and true offset was 3.6° of external rotation . Intra-rater intraclass correlation coefficient was 0.991.Alignment of the radiographic lines created by the anterior and posterior aspects of the greater trochanter is a reliable and accurate rotational positioning method for measuring true femoral offset when using plain films or fluoroscopy, which can aid surgeons with preoperative templating and intraoperative component placement for total hip arthroplasty. Total hip arthroplasty (THA) is performed on over 370,000 hips per year in the United States . RestoraWhen using plain films to measure femoral offset, the X-ray beam must be perpendicular to the plane determined by the intersection of the center lines of the neck and shaft. All other degrees of rotation will show less-than-true-value offset Fig. . AnteropWhile performing THA with fluoroscopy, the senior author observed that two vertical radiographic lines created by the medial portion of the greater trochanter were superimposed during internal rotation of the femur. It was also observed that when these two lines were superimposed, femoral offset appeared to be at a maximum, representing the true offset.While the posterior line results from the most posterior cortex of the medial greater trochanter, the physical anatomic location correlating with the anterior radiographic line remains unknown. Thus, the purpose of this study was to propose a method for aligning femoral rotation to accurately measure femoral offset for preoperative templating and component sizing, and to identify the physical location of two radiographic lines utilized in the described method. It was hypothesized that the anterior line was created by the convex arc of cortical bone within the fossa of the posterior greater trochanter and that the position for measuring true femoral offset is closely approximated by aligning these two radiographic lines.Eleven unpaired fresh-frozen cadaveric male and female hip specimens  were used in this study. All specimens were dissected free of skin, soft tissues, and muscles to the level of the hip capsule, and then the joint was disarticulated. The proximal femur was then dissected of all soft tissues at the proximal shaft, femoral neck, and intertrochanteric line, leaving only the bone and cartilage intact. The femur was cut 15 cm distal to the lesser trochanter and the distal end was potted in polymethylmethacrylate  in a cylindrical mold with the long axis of the femur along the central long axis of the cylindrical mold.The proximal femur was mounted to a custom-designed fixture attached to the actuator of the dynamic tensile testing machine . Fig. . The rigOne femur was utilized to identify the physical location of the radiographic lines. Lead wire strips were placed on the cortex in the suspected region of the anterior radiographic lines. X-ray images were taken at two precise locations, neutral and 30 degrees of internal rotation, to confirm that this was the area of interest. Bone was removed from the anterior greater trochanteric fossa starting at the area beneath the lead strip and an X-ray image was taken again at neutral and 30 degrees of internal rotation to determine if the radiographic lines disappeared. Bone was removed incrementally, and images were taken until the radiographic lines disappeared. Lead strips were then substituted for the removed cortex, which demonstrated a return of the radiographic lines in the proper alignment Fig. .Fig. 2RaUsing a custom software script written in MATLAB , femoral offset was calculated by defining the proximal femoral axis and measuring the perpendicular distance from this axis to the center of the femoral head. The proximal femoral axis was calculated by creating best-fit lines of 5 points on both the medial and lateral borders of the outer cortex, and then extrapolating the bisector of those lines. The center of the femoral head was calculated by marking 6 points around the spherical femoral head and then creating a best-fit circle from these points. A best-fit circle was also calculated in the same manner about the 1-inch metal calibration sphere to calculate the pixel to mm conversion ratio Fig. . MeasureDescriptive analysis was performed to assess the relationship between rotation angle and femoral offset. To assess intrarater measurement repeatability of the femoral offset measurements, three rounds of measurement were performed by a single rater. A two-way random effects model was used to calculate the single measures, absolute agreement version of the intraclass correlation coefficient (ICC). Non-parametric 95% bootstrap confidence intervals were reported. The ICC values were interpreted as follows: ICC < 0.40 = poor agreement; 0.4 < ICC < 0.75 = fair to good agreement; ICC > 0.75 = excellent agreement –12.External rotation angles were reported as negative values while internal rotation angles were reported as positive values.The posterior line was confirmed to be produced by the most posterior aspect of the medial greater trochanter. The anterior radiographic line was created by the most anterior portion of the fossa of the posterior greater trochanter.The mean true femoral offset was observed to be 38.2 mm , while the mean femoral offset measured at the radiographically aligned position was 37.3 mm . The mean underestimation error  was 0.9 mm, or 2.4% . The mean range between highest and lowest femoral offset measured between − 10 and + 10° of rotation was 2.7 mm .The mean rotation between the true offset position and the radiographically aligned position was − 3.6° . Two specimens had their maximum offset at 0 degrees. Maximum femoral offset was found at − 10° for two specimens, − 8° for one specimen, − 6° for three specimens, − 4° for one specimen, + 2° for one specimen, and + 8° for one specimen. A plot of all specimens’ calculated offsets at each degree of rotation is presented in Fig. Intrarater reliability was excellent among the three rounds of measurement .The present study describes a reproducible method to rotationally position the femur and to measure true femoral offset from plain radiographic images with an mean error of less than 1 mm. Prior research has consistently demonstrated that current radiographic methods of assessing femoral offset are not dependable.Preoperative templating for THA is performed via AP pelvic X-rays taken on a supine patient with their hip internally rotated 10–15 degrees, which accounts for femoral anteversion and more closely approximates true femoral offset than a neutral or externally rotated hip . HoweverAdditionally, the present study is one of the first to provide a radioanatomic description of the posterior contours of the medial aspect of the greater trochanter, defining the relationship between the physical anatomy and the corresponding radiodensities seen on two-dimensional imaging. Of note, the anterior radiographic line is produced by the convex arc of the fossa of the greater trochanter, a surface observed to be approximately perpendicular to the true femoral offset. While a future study with CT validation is necessary to prove this claim, this observation serves as one possible reason why the alignment of these radiographic lines produces the femoral rotation for accurate measurement of femoral offset. In addition to its utility in determining femoral offset for THA preoperative templating of any approach, these radioanatomic landmarks are readily identifiable in a supine patient allowing for intraoperative use of fluoroscopy with an anterior approach. Intraoperative use of fluoroscopy in THA has been shown to improve acetabular component placement  and reduThe authors acknowledge several limitations to this study. First, the study was performed on eleven cadaveric specimens, and a larger sample size may better represent the general population. However, Audigé et al. determined a minimum sample size of ten subjects is sufficient for analyzing a new measurement method . AdditioTo our knowledge, this study presents one of the first quantifiable, reliable, and reproducible method for calculation of maximum offset using two-dimensional imaging. This method can be used in clinical practice by surgeons to accurately identify maximum offset on preoperative templating with X-ray and intraoperative assessment via fluoroscopy, which will aid in THA implant placement, ball-head length, and offset sizing and improve patient outcomes.


In the era of multi-omic sciences, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been providing new perspectives to see through. In this context, extracellular vesicles (EVs) are offering a new level of complexity, given their role in cellular communication and their activity as mediators of specific signals to target cells or tissues. Indeed, their heterogeneity in terms of content, function, origin and potentiality contribute to the cross-interaction of almost every molecular process occurring in a complex system. Such features make EVs proper biological systems being, therefore, optimal targets of omic sciences. Currently, most studies focus on dissecting EVs content in order to either characterize it or to explore its role in various pathogenic processes at transcriptomic, proteomic, metabolomic, lipidomic and genomic levels. Despite valuable results being provided by individual omic studies, the categorization of EVs biological data might represent a limit to be overcome. For this reason, a multi-omic integrative approach might contribute to explore EVs function, their tissue-specific origin and their potentiality. This review summarizes the state-of-the-art of EVs omic studies, addressing recent research on the integration of EVs multi-level biological data and challenging developments in EVs origin. In the era of multi-omic sciences, which continuously provide a more comprehensive overview of human pathogenesis, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been welcomed to look at pathologies through new perspectives ,2,3,4,5.As intricated heterogeneous structures, EVs’ first approach is their content profiling, characterizing as much as possible their biomolecules, such as proteins, RNAs, metabolites and lipids. Indeed, their proper characterization is the fundamental requisite for understanding the EVs’ functions. However, EVs content characterization is beyond the focus of this review, rather it is to consider omic sciences potentialities in EVs functions and applications.EVs proteomics explicates its potentiality as it might suggest numerous answers to tricky questions, following well-defined protocols and taking advantage of many different approaches, such as gel-free and gel-based .As commonly accepted, EVs’ heterogeneity in term of subtypes is far beyond the classical classification by size or biogenesis and the difficulty to discriminate them within a whole biological sample represents a limit in analyzing their protein cargoes, how these change across the subpopulations and how these associate with a pathogenic process . In the Of equal importance, mass spectrometry by shotgun approach might provide a wide and highly sensitive range of information on EVs proteomic content, contributing considerably to their characterization and profiling ,61,62,63EVs RNA content has been considerably investigated in the past years, reporting the presence of prevalently mRNAs, miRNAs and long non-coding (lnc) RNAs. However, the development of more advanced high-throughput RNA sequencing methods has led to the detection of various other RNA species, such as small nuclear (sn) RNA, small nucleolar (sno) RNA, non-coding (nc) RNA, long intergenic non-coding (linc) RNA, piwi-interacting (pi) RNA, vault RNA, small non-coding (Y)-RNA, small conditional (sc) RNA, circular (circ) RNA, signal recognition particle (SRP)-RNA and 7SK-RNA, as well as fragments originating from rRNA, tRNA, mRNA and lncRNAs ,78,79. GDespite growing potentiality of metabolomics, EV databases, such as Vesiclepedia, ExoCarta or EVpedia mainly contain protein, mRNA and miRNA entries with less lipid and metabolite data ,88,89. AAnother branch of EVs omic research addresses their genomic cargo, including single strand (ss) DNA, double strand (ds) DNA, mitochondrial (mt) DNA, plasmid DNA, along with nuclear protein histones as well. In addition, DNA could be found either enclosed within EVs or attached to the EVs’ outer surface, or both . The mosAmong omic sciences, surfaceomics has been attracting much attention recently. Surfaceome consists of the collection of membrane proteins of which parts are exposed to the extracellular space, for this reason it might be considered responsible for the interaction between the cells and the surrounding environment. Its growing interest is associated to the several functions that the surfaceome carries out and that might be target of biotechnological innovations: first, it regulates the selective permeability of the plasma membrane, which is of particular importance for efficient drug delivery systems to cells; second, the surfaceome is rich in receptors that capture and mediate specific external signals to the internal compartment of the cell, triggering a specific response. Moreover, the surfaceome is also composed of proteins that mediates both a defense and offense against harmful attacks. Due to these features, the surfaceome is a big source of knowledge of physio-pathological processes, as well as of potential pharmaceutical targets . For insAs previously discussed, omic biological data represent the most promising source of information of EVs. However, one of the most important points of EVs is still challenging to be determined: their origin. Being able to identify the specific tissue and/or cell type from which EVs in a certain biological fluid originate has many advantages: first, EVs from different sources own a unique signature content, at each omic level, and specific surface proteins which not only may allow to date back to their progenitor tissue or cell type, but also to determine specific biomarkers of local and systemic pathologies just by easily accessible biological samples, such as blood and plasma. Secondly, it may provide additional information on tissue crosstalk via EVs, contributing to the understanding of EVs functions at a paracrine and endocrine level . FurtherAlong the years numerous efforts have been made to map human genes and to analyze human metabolism, regulatory networks and protein–protein interactions especially, in physiological and pathological conditions. Remarkably, these analyses produced impressive lists of disease–associated molecules, each coming from a single layer of high-throughput “omic” analyses. Indeed, nowadays several EVs databases are available and publicly accessible such as EVpedia, , VesicleGiven the numerous potentialities of EVs in terms of source of biological knowledge and of translational applications, omic studies represent the optimal approaches to know deeper these nanoscale biological systems. Surely, the forward steps will be directed to the development of innovative computational and bioinformatic tools that would provide the means to integrate and biologically interpret EVs heterogeneous content. Still, many issues are encountered in each step of EVs multi-omic analysis, starting from EVs isolation to the data integration methods, suggesting that this field is at its naïve state and requires further improvements.


The impedance modulus could still reach 4.54 × 104 Ω·cm2 after self-healing, showing excellent self-healing properties for anti-corrosion protection. Synchronously, the SFPUHE-HTF-CNTs exhibited a satisfactory damage sensing performance, enabling the real-time monitoring of fractures at different sizes. This work realized the effective combination of self-healing with corrosion protection and damage detection functions through a bionic design, and revealed the green, and low-cost preparation of advanced composites, which have the advantage of scale production.Self-healing anti-corrosion materials are widely regarded as a promising long-term corrosion protection strategy, and this is even more significant if the damage can be monitored in real-time and consequently repaired. Inspired by the hierarchical structure of human skin, self-healing, solvent-free polyurethane/carbon nanotubes composites (SFPUHE-HTF-CNTs) with a skin-like bilayer structure were constructed. The SFPUHE-HTF-CNTs were composed of two layers, namely, a hydrophobic solvent-free polyurethane (SFPUHE-HTF) containing disulfide bonds and fluorinated polysiloxane chain segments consisting of a self-healing layer and CNTs with good electrical conductivity consisting of a corrosion protection layer, which also allowed for the real-time monitoring of damage. The results of corrosion protection experiments indicated that the SFPUHE-HTF-CNTs had a low corrosion current density (8.94 × 10 Metal corrosion is endangering various industries and compared with earthquakes, tsunamis, and other natural disasters, metal corrosion is silent but equally destructive, causing substantial economic losses every year . AccordiCurrently, there are two common methods for preparing self-healing anti-corrosion coatings. The first involves adding microcapsules loaded with healing agents or corrosion inhibitors to the polymer matrix . The loaMore significantly, self-healing anticorrosive coatings also face a pivotal problem that is often overlooked; that is, although self-healing can reduce the maintenance costs and improve the service life, usually in the early stages of product damage, the resulting micro-crack is tiny and hardly detectable. To avoid a micro-crack from growing into a fatal defect, there is an urgent need to develop smart, anticorrosive coatings with damage monitoring capabilities . SeveralPolyurethane exhibits outstanding flexibility, good mechanical strength and chemical resistance stability ,25. SolvPolyethylene adipate diol (CMA-1000), polytetrahydrofuran ether diol (PTMG-1000), and isophorone diisocyanate (IPDI) were procured from Yantai Daocheng Chemical Co., Ltd. , and polyol was used after vacuum drying at 110 °C for 4 h. 2-hydroxyethyl diisocyanate Sulfide (HEDS) was obtained from Shanghai McLean Biochemical Technology Co., Ltd.  Dibutyltin dilaurate (DBTDL) was purchased from Shanghai Aladdin Biotechnology Co., Ltd.  1,4-butanediol (BDO) was supplied by the Tianjin Fuchen Chemical Reagent Factory . Hydroxyl-terminated fluorine-containing polysiloxane (HTF) was produced by Shanghai Silicon Mountain Polymer Materials Co., Ltd.  A CNTs aqueous dispersion (TNWDM-M2) was provided by Chengdu Organic Chemistry Co., Ltd. , the Chinese Academy of Sciences, and WPU (XWB3030) was purchased from Asahikawa Chemical (Suzhou) Co., Ltd .First, CMA-1000 , PTMG-1000 , IPDI , and DBTDL (2.0 μL) were sequentially added to a three-neck flask, and the mixture was heated to 80 °C and reacted for 3 h to obtain the prepolymer (Pr-PU). Then, HEDS  was employed to the reaction system to expand the chain for 30 min, and the mixture was taken out and evenly coated on the release paper (with a coating thickness of 60 μm). Considering that the molding of solvent-free polyurethane involves a chain growth and cross-linking reaction, direct high-temperature heating tends to lead to a too-fast cross-linking speed, which then leads to an uneven film formation. Consequently, a staged heating process was implemented: the mixture was (i) heated at 60 °C for 1 h; (ii) heated to 90 °C for 2 h; (iii) heated to 120 °C for 4 h; then (iv) lowered to 70 °C for 8 h, and then taken out to obtain the disulfide-bonded, self-healing, solvent-free polyurethane (SFPU-HE). The SFPU-HE0 was prepared by replacing the HEDS with BDO as a control group. Meanwhile, 12% of the Pr-PU mass of HTF was added to replace part of the HEDS, and the reaction was stirred for 20 min at 80 °C to obtain a self-repairing, solvent-free polyurethane containing HEDS and HTF (SFPUHE-HTF), then scraped onto the release paper and placed in the oven at 60 °C for 1 h for cross-linking. Finally, the CNTs aqueous dispersion was mixed with an appropriate amount of polyurethane emulsions and ultrasonically dispersed for 30 min, and sprayed evenly onto the incompletely-solidified SFPUHE-HTF coating to improve the adhesion between the CNTs layer and the polyurethane layer. Then, the obtained material was dried at 80 °C for 30 min, cycling the spraying/drying process several times to prepare a composite coating with different CNT contents and abbreviated as SFPUHE-HTF-CNTs-x, where x denotes the weight content of the CNTs .−1. The Raman spectra were obtained using a Raman spectrometer  with 514 nm of laser excitation. An XRD was performed using an X-ray diffractometer  at a tube pressure of 40 kV, a scanning range of 2θ from 10°~60° and a scanning speed of 5°/min. A TGA was carried out on a thermogravimetric analyzer  over the temperature range from 30 °C to 800 °C at a heating rate of 5 °C/min under a nitrogen atmosphere. A DSC was performed on a DSC-Q2000 instrument  using a heating rate of 10 °C/min and purging nitrogen at a flow rate of about 50 mL/min. The self-healing process of the specimens was investigated under a polarized optical microscope (POM) equipped with a programmed warming heating station to observe the cracks (made by blades). A KH-8700 ultra-depth-of-field 3D microscope  was used to observe the self-healing process of the samples in three dimensions. To test the UV self-healing performance of the SFPUHE-HTF-CNTs, a film with a length, width and height of 3 cm × 2 cm × 0.4 cm, respectively, was cut with a sharp knife, and then laid on a PTFE sheet and placed in a UV chamber. The UV irradiation was performed at an energy value of 200 and the repair of the fracture surface was observed at regular intervals. According to the GB/T 528-1998 standard, the SFPUHE-HTF-CNT film was cut into a dumbbell shape (40 mm × 10 mm × 0.8~1.0 mm) and tested for tensile strength using a universal tensile machine  at a rate of 100 mm/min, followed by cutting out a constant width crack with a blade and then testing the tensile strength of the sample after repairing, using Equation (1) to characterize the self-repair efficiency of the polyurethane samples. The water contact angle of the material surface was measured using a Dataphysics video optical contact angle measuring instrument, with the volume of water droplets set at 5 μL, while the average value was taken at five random points on the membrane.A Fourier-transform infrared (FTIR) spectrophotometer  was applied to record the FTIR spectra at the wavenumbers of 4000~400 cmTo characterize the surface morphology of the SFPUHE-HTF-CNTs, a scanning electron microscope (FEI Verios) was applied at different magnifications of the coating surface at the accelerating voltage of 20 kV. The samples for the SEM observations were coated with gold on the surface. The self-healing properties of the SFPUHE-HTF-CNT films were tested by an electron microscope . The electrochemical corrosion behavior in a NaCl solution (3.5 wt%) was measured by an electrochemical workstation . In addition, the optical microscope was used to observe the corrosion on the surface of the bare and coated steel sheet immersed in a NaCl solution  before and after the coating was damaged (made of blades) at a magnification of 1000. The surface chemical compositions of the SFPUHE-HTF-CNTs before and after self-healing were carefully detected by energy-dispersive spectroscopy . A SZT-2B four-probe tester  was used to measure the electrical conductivity of the SFPUHE-HTF-CNTs coating before and after the damage. The sensing performance of the SFPUHE-HTF-CNTs coating was analyzed by an I-t curve test in a PARSTAT4000 electrochemical workstation.Inspired by the multi-layered structure of human skin, the bilayer skin-like coating of SFPUHE-HTF-CNTs was constructed by the scraping and spraying techniques in this paper. As shown in −1, 3331 cm−1, 3328 cm−1 and 3317 cm−1 corresponded to the stretching vibrations of the N-H bond in the SFPU-HE0, SFPU-HE, SFPUHE-HTF and SFPUHE-HTF-CNTs, respectively. It was evident that the N–H absorption bands shifted to lower wavenumbers, indicating an increase in the degree of hydrogen bonding with the addition of HEDS, HTF and CNTs. Moreover, the absorption bands at 2945 cm−1 and 2862 cm−1 were attributed to the stretching vibration of –CH3 and –CH2– bonds in the self-healing polyurethanes. The absorption bands appeared at 1705 cm−1 and 1232 cm−1 belonging to C=O and C–O–C bonds, respectively. As shown in the spectrum of the SFPUHE-HTF and SFPUHE-HTF-CNTs, the appearance of a new minor peak at 795 cm−1 was assigned to Si–CH3. The appearance of all these above absorption bands proves the successful preparation of self-healing polyurethanes. Moreover, there were no new absorption bands in the spectrum of SFPUHE-HTF-CNTs, indicating that there was only a physical mixing between the CNTs and polyurethane without chemical bonding interactions. −1 and 640 cm−1, respectively. Furthermore, it was found that after some of the disulfide bonds were replaced by HTF, the intensity of the bands corresponding to disulfide bonds in the SFPUHE-HTF spectrum decreased relative to that of the SFPU-HE, indicating that the disulfide bond had been successfully cross-linked to the polyurethane molecular chain. In addition, it is worth mentioning that since the detection depth of the Raman laser was only about 10 nm, the SFPUHE-HTF-CNTs did not show the characteristic Raman shifts of sulfur-containing bonds, but showed the D and G peaks corresponding to CNTs at 1353 cm−1 and 1583 cm−1  sample, with the self-healing efficiency calculated by Equation (1) . As pres−1, the C=O group with ordered hydrogen bonding at 1693 cm−1, and the C=O group with disordered hydrogen bonding at 1719 cm−1, in general agreement with the literature [−1, and the free C=O group appeared at 1731 cm−1, basically consistent with the literature [The self-healing mechanism of the SFPUHE-HTF-CNTs is shown in terature ,43. In aterature . GeneralTo explore the self-healing mechanism of the hydrophobic property of the SFPUHE-HTF-CNTs, an EDS test was carried out on the SFPUHE-HTF-CNTs before and after the repair. It can be seen from corr) and corrosion potential (Ecorr) of SFPUHE-HTF-CNTs with different CNT contents were obtained from the polarization curves, as shown in corr and Ecorr of 1.47 × 10−6 A·cm−2 and −0.68 V, respectively. After coating with SFPUHE-HTF-CNT-0, the Icorr decreased to 5.24 × 10−7 A·cm−2 and the Ecorr increased to −0.54 V. Past research has shown that a smaller Icorr and more positive Ecorr represented lower corrosion trends and dynamic corrosion rates [corr of the coated steel sheet showed a trend of decreasing and then increasing, while the Ecorr was the opposite. The SFPUHE-HTF-CNTs-3-coated steel sheet had a relatively excellent anticorrosive effect, and its Icorr could reach 8.94 × 10−9 A·cm−2, reduced by two orders of magnitude relative to the SFPUHE-HTF-CNTs-0; the Ecorr was −0.38 V and was shifted by 0.16 V in the positive direction relative to the SFPUHE-HTF-CNTs-0. It was thus clear that, due to the barrier and shielding effect of the CNTs layer, the bilayer SFPUHE-HTF-CNTs had a better anti-corrosion performance compared with the single-layer coating. However, the corrosion protection ability of the SFPUHE-HTF-CNTs decreased when the CNTs content was further increased to 4 wt.%, which might have been due to the tendency of agglomeration when the CNTs content was too high, thus, reducing the interaction with the polymer chain segments and making it easier for the corrosive media to penetrate into the steel substrate.The skin-like bilayer structure design grants a coating the same self-healing function as a human epidermis and has shielding and anticorrosive effects. The anti-corrosion protective layer of a SFPUHE-HTF-CNT coating consists of CNTs, which can form zigzag barrier channels to shield corrosive media due to their large aspect ratio . Here, ton rates ,49, indis), the polarization or charge transfer resistance of the substrate (Rct), the coating resistance (Rcoat), the capacitance to protect the coating (Qcoat), and the double capacitance (Qdl) associated with the capacitance at the electrode/electrolyte interface [ct is closely related to the corrosion process. It can be seen from ct was up to 2.51 × 104 Ω·cm2; however, the Rct after soaking for 24 h, 7 d and 14 d dropped to 1.71 × 104 Ω·cm2, 6.91 × 103 Ω·cm2 and 1.65 × 103 Ω·cm2, respectively. This may have been caused by the gradual penetration of the corrosive medium into the coating with the extension of the soaking time; however, compared with the bare steel substrate, the coated SFPU-HTF-CNT-3 steel still exhibited a good corrosion resistance, which is subsequently confirmed in 5 Ω·cm2, 1 h) after immersion in a NaCl solution. It was shown that the skin-like bilayer structure of the SFPUHE-HTF-CNTs had effective anticorrosive properties, which can be attributed to the good nano-size effect of CNTs prolonging the path of corrosive media  into the steel substrate surface [4 Ω·cm2, 1 h) after self-healing, which proves the excellent anticorrosive self-healing properties of the composite coating. Similarly, the Tafel curve  was used to evaluate the responsiveness of the composite coating, where ΔR is the change in resistance in the pressure state and R0 is the original resistance in the unstressed state. 0 of 25.03%, 34.23%, 44.82%, 54.32%, and 65.90% when subjected to different levels of pressure, respectively, with a linear increasing trend and good repeatability. 0 as the scratch size increases. Usually, damage is caused by external forces. Considering that SFPUHE-HTF-CNTs-3 has relatively excellent conductive self-healing properties, the pressure sensing performance of SFPUHE-HTF-CNTs-3 was examined by simulating pressure damage to the coating during an actual application by the pressure generated by weights. The normalized relative resistance change (ΔR/R0) of 6400 Ω, and that the electrical signal remained relatively stable under constant pressure. In contrast, the R0 of the broken SFPUHE-HTF-CNTs-3 increased to 7595 Ω and the electrical signal was unstable due to the destruction of the conductive network , a positive corrosion potential (−0.38 V), and a high impedance modulus (|Z| = 4.79 × 105 Ω·cm2). The impedance modulus still reached 4.54 × 104 Ω·cm2 after self-healing, showing an excellent anti-corrosion self-repair performance. Morever, due to the formation of a conductive network by the CNTs, when the coating was damaged, the average distance between the CNTs changed and their conductivity changed; thus, the SFPUHE-HTF-CNTs also exhibited good damage-sensing performance and could achieve the real-time monitoring of damage with different pressures and sizes. This work not only provides a simple and effective avenue for preparing innovative, polyurethane-based, anti-corrosion coatings with real-time monitoring, but also combines it with advanced functions to expand the application to intelligent sensing, which is expected to be used in self-healing wearable-retractable electronics.In summary, a skin-like bilayer, self-healing SFPUHE-HTF-CNT smart anti-corrosion coating with disulfide bonds and fluorinated polysiloxane chain segments was reported. The SFPUHE-HTF-CNTs could self-repair cracks by heating at 60 °C for 16 min or irradiating under UV light at the energy of 200 for 48 h. The coating could be self-healed by thermal stimulation under mild conditions, which avoids the risk of material degradation due to a local concentrated heat. Additionally, the outer CNT corrosion protection layer possessed a superior barrier and excellent conductivity, with a low corrosion current density (8.94 × 10


Saccharomyces cerevisiae is the organism in which ribosome biogenesis and the role of RNA helicases in this process is best studied, we focused our review on insights from this model organism, but also make comparisons to other organisms where applicable.Eukaryotic ribosome biogenesis involves the synthesis of ribosomal RNA (rRNA) and its stepwise folding into the unique structure present in mature ribosomes. rRNA folding starts already co-transcriptionally in the nucleolus and continues when pre-ribosomal particles further maturate in the nucleolus and upon their transit to the nucleoplasm and cytoplasm. While the approximate order of folding of rRNA subdomains is known, especially from cryo-EM structures of pre-ribosomal particles, the actual mechanisms of rRNA folding are less well understood. Both small nucleolar RNAs (snoRNAs) and proteins have been implicated in rRNA folding. snoRNAs hybridize to precursor rRNAs (pre-rRNAs) and thereby prevent premature folding of the respective rRNA elements. Ribosomal proteins (r-proteins) and ribosome assembly factors might have a similar function by binding to rRNA elements and preventing their premature folding. Besides that, a small group of ribosome assembly factors are thought to play a more active role in rRNA folding. In particular, multiple RNA helicases participate in individual ribosome assembly steps, where they are believed to coordinate RNA folding/unfolding events or the release of proteins from the rRNA. In this review, we summarize the current knowledge on mechanisms of RNA folding and on the specific function of the individual RNA helicases involved. As the yeast  For several assembly factors, the approximate stages of ribosome maturation at which they are required have been determined and their structures when bound to pre-ribosomal intermediates were revealed by cryo-EM. However, the molecular mechanisms how assembly factors facilitate ribosomal restructuring and maturation steps still remain unknown in many cases  and a large subunit  form the mature (80S) ribosome that consists of 79–80 ribosomal proteins (r-proteins) and four rRNAs. The ribosomal subunits are assembled in an extremely complex biogenesis pathway that takes place in the nucleolus, the nucleoplasm, and finally in the cytoplasm. An efficient and accurate assembly is accomplished by the coordinated activity of at least 200 non-ribosomal assembly factors and around 80 small nucleolar RNAs (snoRNAs), which drive a complicated cascade of rRNA processing, r-protein incorporation, and ribosome maturation steps. Among these assembly factors are, besides several structural proteins, a variety of enzymes such as AAAesis see ).Saccharomyces cerevisiae. Despite some differences and additional features owed to increased complexity in higher eukaryotes, there is a high degree of conservation of the assembly pathways from yeast up to humans regarding principal mechanisms and key factors involved .,86.23,86Subsequently, U3 is released by the Dhr1 helicase (see below), allowing for the central pseudoknot (CPK), a long-range tertiary interaction representing a key structural feature of the 18S rRNA, to form ,102. LasThe 25S rRNA folds into six distinct secondary structure domains (named in the 5’ to 3’ direction as domains I to VI), and a seventh domain (domain 0) from which these domains originate  85]. Mo. Mo85]. Bacterial LSU assembly was not characterized to the same extent as SSU assembly in vitro. Still, a hierarchical binding map of r-proteins was also established for the LSU ,104. Mor2 pre-rRNA, is highly flexible and that this flexibility is greatly reduced upon transition to the 27SB pre-rRNA [2 pre-rRNA containing pre-60S particles, whereas domains III, IV, and V adopted a similar to mature conformation only in the later 27SB containing pre-60S particles [2 containing pre-60S particles have been solved until now. The earliest pre-60S particles, for which a cryo-EM structure could be solved, contain 27SB pre-rRNA and display stably folded cores of 25S rRNA domains I (with the 5.8S rRNA) and II, as well as ITS2 (together with its associated factors forming the prominent pre-60S foot structure), in state 1/A particles .https://people.biochem.umass.edu/fournierlab/3modmap/main.php).snoRNAs are short non-coding RNAs that assemble with a distinct set of proteins into snoRNPs. Most snoRNPs introduce modifications into the rRNA. There are two main classes of snoRNPs: H/ACA snoRNPs, which catalyse pseudouridylation, and C/D box snoRNPs, which promote 2’-O ribose methylation . In bothImportantly, methylations and pseudouridylations can alter the properties of RNA by either favouring or blocking the formation of base-pairs, and can consequently have local effects on RNA folding ,116. MorBesides the effects of the introduced modifications on the rRNA structure, a probably more important role of snoRNAs in rRNA folding comes from their base-pairing with rRNA elements which are double-stranded in the mature ribosome. Hence, the hybridization of a snoRNA to a given region most likely prevents the folding of the respective region.It is remarkable that almost all so far known/predicted snoRNP binding sites on the 25S rRNA  map to oAlso, the binding of numerous snoRNAs to domains IV and V, which are the two last 25S rRNA domains to be finally folded, might be indicative of a role of snoRNAs as RNA chaperones coordinating these folding events by preventing premature base pairing events. An example for a critical region in the rRNA for which this may be relevant is the PTC, comprised of 25S rRNA helices H89 to H92 in domain V, which is targeted by nine different snoRNAs  method suggests that some snoRNAs have, beside their hybridization to the site where they guide modifications, additional binding sites in the rRNA ,121. An 0, A1 and A2 [The U3 snoRNA is a large C/D box snoRNA. It is one of the few essential snoRNAs and does not catalyse any rRNA modification but is instead required for early pre-rRNA processing steps at sites A1 and A2 . The U3 1 and A2 ,19,21–231 and A2 ,123 and 1 and A2 ,19,22. I1 and A2 . Cryo-EM1 and A2 ,19,22. T1 and A2 . Notably1 and A2 . Moreove1 and A2 . These aRegardless of whether or not additional rRNA elements apart from H1 and H27 are bound by U3, the binding of the snoRNA in this important region is thought to function in holding these subdomains of the 18S rRNA apart, preventing their premature interaction and hence regulating the timing of CPK formation (see Dhr1 section below).0, A1 and A2 in the pre-rRNA [1 amenable to cleavage by the Utp24 endonuclease [A second major function of U3 snoRNA is connected to its base-pairing with the 5’ ETS: the coordination of early folding and cleavage events at sites Apre-rRNA ,131,132.nuclease ,19,22.0, A1 and A2 [snR30 is a H/ACA snoRNA and represents another essential snoRNA that is required for early rRNA cleavages at sites A1 and A2 . snR30 b1 and A2 ,135. As 1 and A2 ,22, snR31 and A2 .0, A1 and A2 [snR10 is non-essential, but its deletion leads to a cold-sensitive phenotype . It has 1 and A2 . snR10 w1 and A2 . Moreove1 and A2 .0, A1 and A2. Like snR10, U14 potentially has two different functions: it has a non-essential region that mediates methylation in 18S rRNA H14, whereas the second region is essential and binds to H6 of the 18S rRNA [U14 is, beside U3 and snR30, the third essential snoRNA. It is a C/D box snoRNA required for early pre-rRNA cleavages at sites A18S rRNA ,139–141.18S rRNA .Although snR190 is predicted to guide methylation at G2395 in 25S rRNA, this modification has never been detected; therefore, snR190 was suggested to function in rRNA folding instead . In addiGiven that snoRNAs often prevent rRNA folding events, rRNAs have to be liberated from the snoRNAs again at some point to allow them to fold into their destined conformation. It is only poorly understood how snoRNAs are released, however, several mechanisms can be envisaged: snoRNAs may be displaced by similar mechanisms as upon remodelling of mispaired rRNA segments; binding of assembly factors or r-proteins may compete snoRNAs away from their binding site; last but not least, several RNA helicases have been implicated in actively mediating snoRNA release .Figure S2). While most motifs required for ATP-binding and hydrolysis  are found in the N-terminal RecA-like domain (RecA1), the C-terminal RecA-like domain (RecA2) mainly harbours motifs for substrate RNA-binding. Regarding the structural conformation of the helicase core two major states are distinguished, while also several transition states are suggested to exist [RNA helicases are enzymes that utilize NTPs (mostly ATP) to bind and remodel their RNA substrates. Their activities are essential for almost every cellular process involving RNA and include, among others, the disassembly of RNA duplexes, RNA strand annealing, re-arrangement of RNA-protein complexes, and unwinding of sn(o)RNAs ,144. RNAto exist ,154–156.An overview of all RNA helicases in ribosome biogenesis, including their (putative) functions, is provided in Figure S2) [A second important RNA helicase group for ribosome biogenesis is the DEAH-box family, including assembly factors Prp43, Dhr1, and Dhr2. The helicase core of this group, which is named after the Asp-Glu-Ala-His signature in motif II, contains in large parts a functionally similar set of conserved motifs as found in DEAD-box helicases, however, with a clear difference in their primary amino acid sequences ,149. Anogure S2) ,155. By gure S2) . At the gure S2) ,172–176.0, A1 and A2 processing steps [Fal1 is a nucleolar DEAD-box helicase with homology to translation initiation factor eIF4A that is required for 40S synthesis. Its depletion, as well as overexpression of a dominant negative mutant blocks the early Ang steps . Fal1 bing steps ,181,182 ng steps . The MIFng steps . While tng steps . Conside0, A1 and A2 [Dbp8 is another DEAD-box helicase that localizes to the nucleolus . Dbp8 di1 and A2 ,185. The2 pre-rRNA [dbp4 motif I  and motif III (SAT) mutants the ATPase activity was strongly reduced, these mutants are not dominant upon over-expression [Dbp4  was found associated with 90S pre-ribosomes purified via Pwp2-TAP ; howeverpre-rRNA ,187. Thepre-rRNA ,189. Whipression . Thus, ADepletion of Dbp4 results in strongly increased co-sedimentation of the U14 and, to a lesser extent, of the snR41 snoRNAs with 90S fractions after sucrose gradient centrifugation . Since U–Enp2 to the 18S rRNA 5’ domain to which also U14 hybridizes [–Bfr2–Lcp5 module and the Dbp8 co-factor Esf2 [A yeast two-hybrid assay revealed interaction of Dbp4 with assembly factor Bfr2 and the helicase co-sedimented in a complex (without U3 and U14) with Bfr2 and Enp2, which are part of the Kre33 module on compacted 90S pre-ribosomes after integration of the 18S rRNA 5’ domain ,193. Potbridizes . Consisttor Esf2 .Considering all data, it remains doubtful that Dbp4 would directly facilitate U14 snoRNA release. However, Dbp4 may promote folding around the U14 binding region in the 18S rRNA 5’ domain, resulting in integration of this domain into the 90S scaffold, which was shown to occur only after initial integration of the 3’ and central domains . Additio1 and A2 [Rrp3 is a DEAD-box protein that shows in vitro ATPase activity in the presence of single-stranded RNA and was shown to unwind short RNA duplexes with single-stranded extensions in vitro in an ATP-binding, but not hydrolysis-dependent manner ,189. Its1 and A2 ,194. Rrp1 and A2 ,179,182,Rok1 is a DEAD-box helicase and shows ATPase activity in vitro ,195. Rok0, A1 and A2 [Rok1 is a nucleolar protein and is required for early pre-rRNA processing steps at A1 and A2 . Rok1 bi1 and A2 .ROK1 gene is a high copy suppressor of rrp5 mutants [The  mutants , suggest mutants . Both Rr mutants ,201.2 cleavage and to subsequently carry out its function in pre-60S maturation [Rrp5 directly interacts with Rok1 via its C-terminal domain  and is rturation .Besides that, Rok1 also has a snoRNA related function: Rok1 crosslinks to snR30 , as doesrrp5 mutant could also be explained retaining the model that Rok1 releases snR30: Rrp5 is a cofactor of Rok1, and was reported to provide specificity to Rok1 by changing its conformation [snR30 also accumulates in ribosome bound-form upon expression of a Rrp5-mutated variant that binds less efficiently to Rok1 and is consequently less efficiently released from 90S particles. Based on that result, and the observation that Rok1 was still recruited to pre-ribosomal particles in that mutant, the authors claimed that Rok1 only has an indirect role in snR30 release by promoting Rrp5 release, which they in turn postulated to be a prerequisite for snR30 release . Howeverormation . Thereforok1 mutants were found to be synthetic lethal with mutations of the H/ACA snoRNA snR10 and of the gene encoding H/ACA snoRNP component Gar1 [Apart from snR30, Rok1 also crosslinks to the U14, U3 and snR10 snoRNAs . Notablyent Gar1 . Despiteent Gar1 . Hence, 0, A1 and A2 [Dhr2 is a DEAH-box helicase that is localized in the nucleolus. Its depletion, as well as overexpression of a dominant-negative mutant inhibits pre-rRNA processing at sites A1 and A2 ,204. Mor1 and A2  and to i1 and A2 ,206. Suc1 and A2 . These d1 and A2 ,208.Dhr1 is a DEAH helicase and shows RNA-dependent ATPase and RNA unwinding activity in vitro . ATP binBesides its requirement for pre-rRNA processing in 90S particles, Dhr1 is responsible for release of the U3 snoRNA from pre-ribosomal particles, and thereby promotes rRNA re-arrangements leading to the formation of a key structural element of the SSU, the CPK. The mechanisms of Dhr1 recruitment and function are summarized below.1, suggesting that Dhr1 is recruited to 90S particles at an early stage, before A1 cleavage. Nevertheless, Dhr1 failed to be visualized in cryo-EM structures of pre-A1 90S particles, suggesting it is bound to flexible elements in these particles [Biochemical as well as structural data indicate that Dhr1/DHX37 initially binds to 90S particles and remains bound during the transition to the earliest pre-40S particles ,23,102. articles . While tarticles ,214 the articles . Utp14 aarticles . As howearticles .1 90S particles [1 90S structures [The first 90S structures in which Dhr1/DHX37 could be visualized are post-Aarticles ,23  90S particles occupied by Pno1. Hence, the Dhr1 helicase core and C-terminal domain have to be at another position in pre-A1 particles and likely move to the site previously occupied by Pno1 in the course of transition from pre- to post-A1 90S particles. Considering the multiple RNA contacts of the Dhr1/DHX37 N-terminal extension, it is tempting to speculate that the N-terminal part of the protein represents an initial anchoring point, whereas the helicase core and C-terminal domain move in the course of 90S maturation.In contrast to the extensive rRNA contacts formed by the N-terminal extension, the Dhr1/DHX37 helicase and C-terminal domains are not in contact with RNA in post-A1 particles, the catalytic and C-terminal domains of Dhr1 are distant from the U3 substrate, while part of the N-terminal domain of Dhr1/DHX37 is positioned in proximity to the helicase target site in the region where 18S rRNA H27 base-pairs with box A’ of the U3 snoRNA  ubstrate ,23.1 and A2 [1 cleavage has not occurred [1 cleavage.The catalytically inactive conformation of Dhr1 in 90S particles suggests that Dhr1’s role in 90S particles is a structural one that does not rely on its catalytic function. Dhr1 depletion inhibits pre-rRNA cleavages at sites A1 and A2 ,204 and 1 and A2 ,86. Enp1occurred . Hence, 0 cleavage, a part of the 5’ ETS clamped between Utp7 and Pwp2 has to be set free. This happens by a change of the orientation of the two proteins, leading to a ‘ring-opening’. Additionally, a repositioning of Pno1 and the associated 18S rRNA H45 to a site previously occupied by Krr1 and Faf1 takes place . The re-positioned Pno1/H45, additionally bound by Dim1, is then stabilized by the Dhr1 N-terminus [1 processing.This goes in line with structural data: in the course of exosome-mediated 3’ to 5’ removal of the 5’ ETS after Aterminus . These rterminus ,216. Hendhr1 mutant is able to perform the 90S to pre-40S transition (with the mutated Dhr1 remaining bound to pre-40S particles), and accumulates 21S pre-rRNA, an intermediate in which A1 cleavage has occurred, while A2 cleavage was skipped [2 cleavage to occur. This observation is further complemented by data showing that 90S, but also pre-40S particles purified by split-tag purification via both Noc4 and Dhr1 contain a substantial fraction of 21S pre-rRNA [2 cleavage normally occurs within 90S particles, but potentially, when Dhr1 does not exert its function in A2 cleavage in time , some particles may escape this step, resulting in pre-40S particles with the pre-rRNA not yet cleaved at site A2.In contrast to the consequences of complete absence of Dhr1, an ATP-binding incompetent  skipped . Hence, pre-rRNA . A2 cleadhr1 catalytic mutants and dhr1 cold-sensitive mutants. Moreover, the growth defects of the cold-sensitive mutants can be rescued by mutations in the U3 region base-pairing with 18S rRNA, suggesting that weakening of the interaction of U3 with 18S rRNA can partially compensate for the absence of active Dhr1 [dhr1 ATP-binding deficient mutant still remains base-paired with the 18S rRNA [dhr1 mutant, further underscoring that the CPK does not form in the absence of the unwinding activity of Dhr1 [Dhr1 as well as its human counterpart DHX37 directly bind to the U3 snoRNA, as revealed by co-IP, CRAC analyses and cryo-EM ,204,211.ive Dhr1 . Further18S rRNA . As deta of Dhr1 .As described above, Utp14 is re-positioned in the course of 90S to pre-40S maturation. In early pre-40S particles, the Dhr1 C-terminal domain docks to the repositioned region of Utp14 and is thereby targeted into closer proximity of the U3-18S rRNA hybrid  complex [Mtr4 is another helicase that is acting on both 90S and pre-60S maturation intermediates. As a processive helicase it shows ATPase and 3‘-5‘ RNA unwinding activity that is stimulated by structured RNA species such as tRNA in vitro . It is a complex . In cont complex ,252–254  complex ,256. The complex . Mtr4 is complex . Moreove complex ,261. Upo complex ,86,101.0-cleaved 5’ ETS RNA fragment (black) is channelled through the helicase core to the nuclear exosome core (blue) docked to exonuclease Rrp44 (violet). While the Mtr4 RecA-like domains contact Utp6, the arch domain is close to an N-terminal part of Utp18 (orange). Note that the AIM-containing region within the Utp18 N-terminus is not resolved in the structure. Utp14 is indicated in cyan. (B) The 3’ extension of the 5.8S rRNA (black) is channelled through the helicase core, while the arch domain is interacting with 25S rRNA H16 and r-protein Rpl8. The exosome components Rrp6 and Rrp47 are depicted in cyan and light blue, respectively. Note that AIM-containing Nop53 is not visible in the structure.Mtr4 (red) is associated via its arch domain with 90S (A) and pre-60S (B) intermediates . (A) The A1 cleavage and is released again upon A1 cleavage and subsequent 5’ ETS degradation [1 cleavage. After A0 cleavage and upon exosome recruitment via Mtr4, the 5’ ETS-A0 fragment might be, starting with the 3’ end, channelled through the helicase core into the exosome cavity. On recent cryo-EM structures, the helicase is resolved on 90S particles in proximity to the 3’ end of the 5’ ETS at the base of dislodged H9 at a composite surface formed by Utp6, the Sof1 module (Sof1–Utp7–Utp14), Fcf2, Utp14, and the AIM-containing Utp18 N-terminus [–Mtr4 interaction. Mtr4 binding may potentially be stabilized also by α-helical elements of Utp14, a co-factor of the helicase Dhr1 [Mtr4 binds to 90S particles already at a step prior to Aradation ,86,101 , Npa2 , Nop8, and Rsa3, as well as the r-protein Rpl3 . ImportaDBP9 can act as a dosage suppressor of some dbp6 mutations, suggesting that Dbp6 and Dbp9 have partially overlapping functions, and that in the case of not fully functional Dbp6, Dbp9 can at least partially take over Dbp6’s functions [Compared to Dbp6 and Dbp7, less information is available about Dbp9. unctions .dbp7-1 mutation all lead to similar rRNA processing defects, including the accumulation of 35S and 32S pre-rRNA and consequently a reduction of 27SA2 pre-rRNA and later precursors in the 60S maturation pathway [2 pre-rRNA, suggesting that the primary effect of absence of Dbp7 is a delay in maturation of 27SA2 pre-rRNA to subsequent species [Dbp6 and Dbp9 depletion, as well as the  pathway ,267,269. species .2 pre-rRNA. Additionally, small amounts of 35S and 32S pre-rRNAs co-purify with both proteins, suggesting that they additionally associate with 90S particles and that they are present during the 90S to pre-60S transition [2 pre-rRNA has been converted to later forms [At what maturation stage are the Npa1 complex and Dbp6, Dbp7 and Dbp9 bound to pre-ribosomal particles? Both Dbp6 and Dbp7 co-purify predominantly 27SAansition ,265. Laser forms ,265. A ser forms . These dSo, what is the actual function of these helicases within early pre-60S particles? Generally, the Npa1 complex is believed to act as a chaperone in early rRNA folding . The larDBP7 is not essential, however its deletion results in a strong growth defect. This phenotype can be alleviated by mutations in SNR190 predicted to reduce base-pairing with its rRNA binding sites, but also by mutations in 25S rRNA H73 of domain V base-pairing with snR190 [Dbp7 crosslinks to H89 and H90 in domain V, and to H94 and H98 in domain VI of the 25S rRNA  Figure . The H94h snR190 . Hence, h snR190 ,265.Apart from this strong functional connection to snR190, Dbp7 binds also to snoRNAs snR10, snR42, snR5, and snR61, which all base-pair with rRNA elements in proximity to Npa1’s rRNA binding sites. It is up to now unclear whether Dbp7 is also involved in release of some of these snoRNAs .2, and most prominent the 27SA3 pre-rRNA species, which could indicate a role of Dbp3 in facilitating A3 cleavage by the RNase MRP [DBP3 is not essential, with a dbp3Δ strain showing a cold-sensitive slow-growth phenotype, and the lack of Dbp3 or expression of a catalytically inactive mutant did not clearly impair production of the mature 25S rRNA, only a minor contribution of this helicase to rRNA maturation was assumed [dbp3Δ cells, potentially by bypassing the requirement of the helicase for snoRNA recycling [3 cleavage.The non-essential DEAD-box protein Dbp3 shows ATPase activity stimulated by RNA and unwinds short RNA duplexes in vitro ,189,272.Nase MRP . As DBP3 assumed . Neverthecycling ; thus, sThe DEAD-box helicase Drs1 is associated with early to intermediate nucleolar 60S precursor particles  and is released again before late nucleolar  maturation steps ,67,275 . When prdrs1 mutant that was still capable of pre-60S binding, 25S rRNA synthesis was impaired and the 27SB pre-rRNA accumulated [drs1 alleles  further showed synthetic lethality when combined with mutant alleles of the gene encoding the foot-factor Nop7 [de novo rRNA synthesis [–Erb1–Ytm1 subcomplex in vitro [–Erb1–Ytm1 from pre-60S particles lacking Drs1 during sucrose gradient centrifugation indicates that the helicase might be required for stable incorporation of this module . In addition to that, Nsa1 affinity-purifications upon Drs1 depletion revealed a massive amount of free bait protein, suggesting a (possibly indirect) role of Drs1 for Nsa1 assembly as well .In cells harbouring a cold-sensitive umulated ,277. Distor Nop7 . Moreoveynthesis  and intein vitro . In linedrs1 mutants that were capable of pre-60S binding, depletion of Drs1 leads to accumulation of 27SA2 and 27SA3 and, notably, 35S pre-rRNAs [2 cleavage could shift more to a post-transcriptional ITS1 processing [In contrast to the 27SB pre-rRNA accumulation in cold-sensitive re-rRNAs . Furtherocessing . Semiquaocessing   module and, independent of this interaction, contributes to processing of the 47S pre-rRNA [Taken together, the physical presence of Drs1 could be required at an early maturation stage to allow Apre-rRNA .mak5 G218D (motif I) allele [A point mutation within the ATP-binding Walker A motif I of Mak5 causes deficiency in maintenance of the M1 dsRNA virus yielding a killer minus phenotype . Further) allele . This sudbp2Δ cells and less pronounced in catalytic Walker A motif I mutants, the levels of both mature 25S and 18S rRNA are decreased and the 35S and to some degree the 27S pre-rRNAs accumulate, whereas polysome profiles possibly indicate a slight 60S synthesis defect [Dbp2 is a DEAD-box helicase that has several non-essential cellular functions including its contribution to ribosome assembly . Both Dbs defect . Mass sps defect ,289. Intdbp2Δ cells [Xenopus oocytes showed that U8 base-pairing with the 5’ end of the 28S pre-rRNA is required for 28S and 5.8S production [While the role of yeast Dbp2 in ribosome biogenesis is poorly characterized, there are data available for its human homologs DDX5 and DDX17 that fulfil a redundant function for cell proliferation and viability and, at least in case of DDX5, can complement the 60S biogenesis defect of 2Δ cells ,290. A 3oduction ,292. It oduction . A similoduction . While toduction . The heloduction . As a ro2 [The essential DEAD-box helicase Dbp10 is required for processing of the 27SB to the 25.5S and 7S pre-rRNA species  and inte2 . The hel2 ,236,297 2 . In addi2 . Exactly2 . Of note2   to 23S rRNA H92 [DbpA, the bacterial homolog of Dbp10 in rRNA H92 ,302–304,rRNA H92 . The dirrRNA H92 ,306.dbp10 mutants (which efficiently associate with pre-60S particles), Nop2 is still recruited, Spb1 fails to efficiently bind to pre-60S particles. Interestingly, also the recruitment of the GTPase Nug1 is fully abolished in these dbp10 mutants , which is surprising as vice versa Dbp10 was also not efficiently assembled upon Nug1 depletion [Assuming a similar role of Dbp10 in conformational restructuring of the immature PTC in eukaryotes, its activity could be important for the access of the two methyltransferases Nop2 and Spb1 to modify their target nucleotides C2870 (at the base of H89) and G2922 (in H92), respectively ,308. In epletion . Furtherepletion ,61  could make this region accessible for Spb1- and potentially Nop2-mediated base methylation. However, it remains unclear at which exact maturation stage the two methyltransferases are triggered to modify their rRNA targets.spb4 catalytic mutant backgrounds, processing of the 27SB precursor is blocked [–Erb1 removal by the AAA+ ATPase Rea1, corresponding to structural state E [The DEAD-box helicase Spb4 is essential for cell viability and for biogenesis of the 60S subunit and is mainly bound to 27SB pre-rRNA containing pre-60S particles ,310. Upo blocked ,309,310  blocked . More sp state E . Such pa state E , it acts state E ,55,64. A state E ,54.A second major crosslink site was found at H99/H100/H101 (25S rRNA domain VI) , which wIn the course of ribosome biogenesis, pre-rRNAs are transcribed by RNA polymerase I and III and successively fold into the tremendously complex three-dimensional rRNA structure of mature ribosomes. rRNA folding comprises numerous individual (and sometimes interdependent) folding events, for the majority of which the exact choreography and mechanisms remain elusive. RNA helicases are believed to actively mediate such folding and unfolding events in ribosome biogenesis. However, it has to be acknowledged that due to the sheer multitude of structural changes occurring during this process, the only 19 RNA helicases participating in ribosome biogenesis in yeast can only be responsible for a minority of these events, while most restructuring events can likely proceed without the help of enzymes. Nevertheless, the fact that many RNA helicases are known to act on particularly important or complex RNA folds, like the CPK or the PTC, suggests that RNA helicases promote key restructuring events in ribosome biogenesis.In line with the observation that most rRNA folding events occur in the very early steps of ribosome biogenesis, almost all RNA helicases in ribosome assembly function in the nucleolus.Although the naive idea that RNA helicases function in directly unwinding RNA duplexes may be correct in several instances, and putative functions in such RNA remodelling events have been proposed for many RNA helicases in ribosome biogenesis, there are only few examples of RNA helicases for which the precise substrate and function is definitely proven. The prime example for a well-understood helicase is Dhr1, for which ample biochemical, genetic and structural data demonstrate the function in release of the large scaffold snoRNA U3 from the 18S rRNA. In turn, the liberated single-stranded rRNA elements can undergo interactions with their cognate hybridization partner sequences, leading to the formation of a central structural and functional element of the 18S rRNA, the CPK. As the CPK is a composite structure formed by rRNA elements which are more than 600 nucleotides apart, it is not surprising that coordination and enzymatic assistance is required to establish this complex fold.A function in snoRNA release was also found for Dbp7 and proposed for Dbp4, Rok1, Prp43, Has1, Dbp7, Dbp3, and Dbp2, and it is tempting to speculate that in all these cases, the release of the respective snoRNAs and the resulting liberation of rRNA elements triggers strategically important rRNA folding events. Of note, all the snoRNAs that are known to have mainly structural roles in ribosome biogenesis are believed to be released by RNA helicases, suggesting that in contrast to the more transiently binding modification-guiding snoRNAs, more effort is needed to remove the snoRNAs with chaperone function.Other RNA helicases have been postulated to directly act on rRNA helices and mediate their restructuring, i.e. Mak5, Dbp10, Spb4, and Prp43. However, in lack of molecular mechanisms, detailed insights into such restructuring processes remain mainly elusive. Last but not least, at least one RNA helicase, Mrt4 does not target rRNA but spacer elements, and the unfolding mediated by this RNA helicase does not serve the goal to eventually generate a new rRNA fold, but instead makes these spacer elements susceptible to exonucleolytic degradation.Despite common enzymatic mechanisms, the functions and exact sites of action of RNA helicases in ribosome biogenesis are diverse. All of them share the domain architecture typical for DEAH/DEAD box helicases, however, many of them carry N- and/or C-terminal extensions specific to the respective proteins, which may be involved in determining the helicase binding sites and different unique functions. Additionally, some of them have specific co-factors ensuring that the helicases become active at the correct time. Many helicases associate with pre-ribosomal particles only transiently and dissociate again after having performed their functions, while some of them are more stably bound to pre-ribosomal particles and could even be visualized in a pre-ribosome bound stage by cryo-EM. These likely have, in addition to their catalytic function, also a non-catalytic, structural function in ribosome biogenesis.Coming back to the title of this article and reconsidering the role of the function of RNA helicases in RNA folding, it remains unknown how many RNA helicases directly mediate rRNA folding. Such direct rRNA folding activity and promotion of rRNA duplex formation may be feasible for helicases that possess RNA strand annealing activity (e.g. Rok1). In contrast, many RNA helicases appear to promote rRNA folding indirectly by removing ‘folding inhibitors’ like snoRNAs in the case of processive RNA helicases (DEAH and Ski2-like family) or local melting of rRNA–snoRNA base pairs in case of DEAD-box helicases. These actions then likely promote the folding of the now liberated rRNA elements. In addition to snoRNAs, also proteins can act as folding inhibitors, and it is conceivable that helicases might also be engaged in the release of proteins from rRNA, as has been postulated for example in the case of Rok1 (Rrp5 release) and Has1 (Rrp36 release).As detailed in this article, the definite function of most helicases is not yet clear and remains subject to speculation, and in several instances, conflicting data exist on the putative function of a helicase. Moreover, some RNA helicases have overlapping binding sites, and it is unclear how their actions are coordinated. Some helicases even remain almost uninvestigated. Major challenges in studying RNA helicases are the often-transient nature of their interaction with their substrates, and the technical difficulty to reconstitute snoRNA release and rRNA folding events mediated by RNA helicases in vitro. Also, structural investigations are often challenging owing to flexible helicase domains and their association with flexible elements of pre-ribosomal particles. Considering their presumed key functions in rRNA folding, the functional characterization of RNA helicases in ribosome biogenesis will without doubt represent an important research line in the ribosome biogenesis field in future years.Click here for additional data file.


The proliferation of mobile devices and the continuous development of online technologies has led to an increasing variety of channels, leaving customers with a choice of channel alongside the choice of product, service, or retailer. Any attempt to optimize customer experience and engage in successful omnichannel management will require a complete, multifaceted understanding of the processes around channel choice of customers. To date, the many existing studies around multi- and omnichannel research have failed to yield an integrated, comprehensive synthesis of factors involved in customers´ channel choice. Our study conducted a systematic literature review to the end of identifying the factors involved in channel choice which appear in the scientific literature on this topic over the last two decades. We retrieved 128 papers from three bibliographic databases  and carried out descriptive analysis on them. Qualitative thematic analysis inductively extracted 66 different factors of channel choice, each assignable to five broader categories, from the studies included in the review. The findings indicate that perceived channel characteristics, customer needs and situational or contextual factors influence customers´ channel choice directly, and customer characteristics and characteristics of products or services influence it indirectly. Alongside its presentation of an integrated conceptual framework comprising these relationships, our study details a comprehensive research agenda with regards to theories, contexts, and methods and, in particular, with regards to factors influencing customers´ channel choice. Our findings advance the academic understanding of channel choice behavior and provide researchers and practitioners in this area with information on important implications for omnichannel management. The proliferation of mobile devices and the ongoing development of online technologies has led to a constantly increasing variety of channels, such as mobile apps and social media  the offering of merchandise and services through a multitude of available channels, (2.) integration of channels into a unified system from a retailer´s point of view (i.e. enabling the same task-fulfillment on every channel), and (3.) seamless inter-channel interaction from a customer perspective   and research designs and via the use of theoretical foundations and models.The review is structured as follows. After discussing the methodology used for the SLR, the paper provides a descriptive overview of the articles retrieved for the literature review and then proceeds to analyze factors of channel choice as represented in the literature identified. Following this, a section proposing potential areas for future research on channel choice behaviors sets out a comprehensive research agenda. The concluding section points to implications and limitations of this study.The relevance of SLRs to research in the area of business is currently higher than it has ever been Snyder . In collMany guidelines on conducting SLRs recommend the development of a detailed systematic review protocol to ensure transparency and reproducibility of the review process (Kitchenham and Charters Search terms and relevant databases: A search string requires the use of words and phrases (i.e. search terms) directly related to the research question (Snyder choice OR choose* OR select* OR use OR usage OR utili* OR adopt* OR prefer*) AND . To the end of comprehensively covering the relevant literature, we searched within three electronic literature databases: EBSCO Host, Scopus, and Web of Science  were read in detail and assessed for content and method eligibility. This process resulted in the exclusion of thirteen studies due to inapposite content, mainly because the articles examined the consequences of channel choice rather than the reasons behind it , followed by the International Journal of Retail and Distribution Management (n = 12) and the Journal of Business Research (n = 8). Most of the studies do not indicate the application of specific theories to explain customers´ channel choice. However, some studies are explicitly grounded in popular theories of consumer and technology research, such as the theory of planned behavior , with surveys and company database analysis the most commonly used methods Table . QualitaThis section summarizes the factors of channel choice identified within the articles reviewed. In total, we extracted 66 distinct factors from the studies and grouped them into 14 subcategories and five main categories  and as socio-demographically similar customers (homophily)  receives the most mentions in the literature as a factor in channel choice, occurring in almost 40% of the articles reviewed (n = 47). The extent of existing channel experience lessens emphasis on the channel’s perceived disadvantages, such as perceived risks  do not result in clear evidence. The result is a need for future research to examine possibilities for higher-quality segmentation criteria with regard to channel choice for a range of types of product and service  framework introduced by Paul et al. . It firsIn line with Mishra et al. , we findThe major portion of the articles reviewed is from the U.S. (n = 34), China (n = 13), and Western Europe . The complete absence of studies from Africa or South America , primarily using surveys; a number of articles (n = 25) call for the validation of their findings via longitudinal studies and an extended data collection period (see, for example, Hu and Tracogna The present study conducted a systematic literature review to the end of identifying the factors involved in channel choice which appear in the scientific literature on this topic over the last two decades. The review sought to enhance our understanding of customer behavior in multi- and omnichannel environments – a vital factor in successful omnichannel and customer experience management (Verhoef et al. The integrated conceptual framework of customers´ channel choice proposed in this review and our recommendations for potential future research contribute to the literature in this field by advancing knowledge around customer behavior for academia and practitioners, providing a systematic overview of the impact of various factors on channel choice, and precisely formulating over 50 unanswered questions for research going forward (see Tables This review is the first study to date to provide a systematic overview of the currently existing body of knowledge on customers´ channel choice; more generally, it is one of the first SLRs on multi- and omnichannel-related topics to center the customer´s point of view. Notwithstanding this contribution to the research landscape, the review is subject to some limitations. First, we had to restrict the search strings to specific terms such as “channel,” although articles falling within our criteria may have used other denotations (an example might be “interaction choice” (Barwitz and Maas


Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among  Arabidopsis and rice, respectively [Arabidopsis and rice, ABCG transporters have been widely studied and linked with anther and pollen development, cuticle formation, defense response, hormone transport, and seed germination [ATP-binding cassette (ABC) transporter, one of the largest proteins family, widely exists in bacteria, animals, and plants . ABC traectively . The outectively . In Arabmination . The submination . HoweverArabidopsis and rice [It has been reported that 54 ABCG members exist in maize , but onland rice ,6, and tArabidopsis and rice, and their implications for maize orthologs. The functions of maize ABCG genes can be predicted by homologous and bioinformatic analyses. Furthermore, we summarize the current progress and challenges in the identification of ABCG substrates, and present the future research directions and potential applications for ABCG proteins in plants.Here, we present a consolidated nomenclature system for the maize ABCG subfamily, and focus on functional research progress of the reported ABCG genes in https://maizegdb.org/ (accessed on 8 May 2022)), a total of 51 ABCG genes were identified in maize, and named as ZmABCG1 to ZmABCG51 according to their domain organizations  and chromosome locations in maize (A consolidated nomenclature for maize ABCG transporters will provide much needed clarity and a framework for future research. Based on BLAST searches against the maize B73 reference genome (B73 RGV4.0) in the MaizeGDB database ), are listed in Notably, the total number (51) of maize ABCG genes is different from that (54) in the previous report , which mZmABCG12 . FurtherArabidopsis, the maximum likelihood method and the MEGA7 program were used to construct the phylogenetic tree, based on the multiple alignment of all the ABCG protein sequences in these tree plant species. All the identified ABCGs were divided into two subgroups in the phylogenetic tree . Among them, some ABCGs have been reported to be involved in anther and pollen development, such as AtABCG9 [Arabidopsis, which can be further classified into three clades (Clades 2-1 to 2-3). Considering that some of the ABCGs have been functionally characterized in Arabidopsis and rice and several maize orthologs of Arabidopsis and rice, known ABCGs have been reported to play similar roles during anther development and male fertility, such as the orthologous ZmABCG27 (previously named as ZmMS2/ZmABCG26) [In order to analyze the evolutionary relationship of ABCGs in maize, rice and tic tree . Subgrou AtABCG9 , AtABCG2 AtABCG9 ,13,14, O AtABCG9 ,16 and Z AtABCG9 ,10 in Cl AtABCG9 , OsABCG2 AtABCG9 ,19, ZmAB AtABCG9 ,21,22, a AtABCG9 ,24 in ClmABCG26) ,10, OsABmABCG26) ,16 and AmABCG26) ,13,14, tmABCG26) ,19 and AmABCG26) . TherefoArabidopsis and 11 ABCG genes in rice have been functionally characterized, but only three ABCG genes have been identified in maize. These reported ABCG transporters play essential roles in various biological processes, such as anther and pollen development, vegetive and female organ development, biotic and abiotic stress response, and phytohormone transport and signaling , and plays multiple roles in flower cuticle formation, root suberin metabolism, and proper vascular development. The abcg11 mutant displays dwarfism, male sterility, post-genital organ fusions, and reduced cutin load in flowers [Arabidopsis AtABCG1 and AtABCG16, and their rice ortholog OsABCG3, are required for the transport of nexine and intine precursors for male gametophyte development in the post-meiotic stages, and for pollen tube growth; the atabcg1atabcg16 double mutant and osabcg3 mutant show defective nexine and intine formation, and thus male fertility [Arabidopsis [Arabidopsis [Arabidopsis).To date, at least 12 ABCG genes have been reported to be required for anther/pollen development and male fertility in multiple plant species . For exa flowers ,25,26. O flowers ,18,19. A flowers ,15,16. Aertility ,22,23,24bidopsis . AtABCG2bidopsis . TogetheArabidopsis [Arabidopsis cell suspensions during tracheary element differentiation [Arabidopsis AtABCG32, rice OsABCG31 [Arabidopsis ABCG transporters, AtABCG1, AtABCG2, AtABCG6, and AtABCG20 [Additionally, several ABCG transporters are reported to be involved in the formation of diffusion barriers, such as cuticle, suberin, and lignin, during the vegetative and female organ development in plants B. For exbidopsis . AtABCG1bidopsis , similarbidopsis . AtABCG2ntiation . FurtherOsABCG31  and maizOsABCG31  are alsoAtABCG20 ,27, as wAtABCG20  are respAtABCG20 . AtABCG2AtABCG20 . CollectAlternaria brassicicola and Botrytis cinerea [Botrytis cinerea [Another important function of ABCG transporters is in the protection against biotic and abiotic stresses C. Many A cinerea , and AtA cinerea . AtABCG3 cinerea . The hal cinerea . Increas cinerea , AtABCG1 cinerea , AtABCG2 cinerea ,68, AtAB cinerea , and AtA cinerea , while O cinerea . Overexp cinerea ,52. Furt cinerea ,55,56,60Arabidopsis and rice  formation [Multiple studies have shown that some ABCG orthologs of bidopsis ,14,15,19and rice ,19. Hereand rice , which wormation ,21,22,24The expression analysis based on RNA sequencing (RNA-seq) data provides a new window to predict gene function. Based on anther RNA-seq data, there are 62 putative maize genic male-sterility (GMS) genes, 125 putative lipid metabolic GMS genes, and 112 putative sugar metabolic GMS genes which have been predicted in maize ,73,74. AArabidopsis ortholog (AtABCG31) of ZmABCG34 have been reported as being required for pollen wall development and male fertility [http://www.maizegdb.org/ (accessed on 20 April 2022)), and they are orthologs of AtABCG28 required for pollen tube growth and male fertility in Arabidopsis [Arabidopsis and rice, such as ZmABCG27/ZmMS2 orthologous to AtABCG26 and OsABCG15 [Here, we carried out transcriptomic analyses of all maize ABCG members based on the RNA-seq data from four maize inbred lines W23, B73, Oh43, and Zheng58 developing anthers A. Based ertility ,21, indibidopsis   and metabolite analysis. However, the phenotypic effects resulting from knockout studies might be pleiotropic, thus hindering the identification of the actual substrates.N. benthamiana) protoplast system and yeast strains (YMM12 and BY-2) cell lines [Arabidopsis ABCG transporters have been identified by employing radioactivity labeled compounds, i.e., by using isotope labeling experiments in vivo  for plant cuticle formation, based on the export assay using the protoplast system of N. benthamiana [The most powerful approach to identify the substrates of ABCG transporters is to use a transport assay which demonstrates translocation activity across a membrane in a strictly ATP-dependent manner. The prerequisite for the transport assay is the overexpression of the ABCG protein in a proper expression system, such as tobacco  ABCG transporters involved in various biological processes influencing male fertility and plant fitness, it is plausible that the corresponding ABCG orthologs might also play similar roles in other crops, including maize. This can be verified by the use of reverse genetics , and thus deepen our understanding of the functional mechanism of ABCG transporters in multiple plants. The derived mutant lines with elite characters, such as male sterility or higher fitness, have potential applications in hybrid crop breeding and seed production in the future.In summary, given the tremendous progress in defining the critical roles of plant (mainly in