Using stringent cutoff , we detected 21 down-and 9—up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts’ PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts’ PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts’ health.During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts’ health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60–300 nucleotides) known to guide 2′-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut’s peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts ( During spaceflight, astronauts are exposed to various physiological and psychological stressors, such as microgravity, sleep deprivation, isolation, confinement, and high ionizing radiation , 2. DespExtracellular vesicles (EVs) are secreted from most cells and are enriched in different non-coding RNA (ncRNA) species which serve as significant drivers of intracellular communication in physiology and pathological conditions –7. The mThis study was determined to be a non-human study by NASA’s Institutional Review Board (IRB) and the Icahn School of Medicine at Mount Sinai’s IRB . Samples were collected as part of a NASA flight study . All parg at 4°C. Finally, the supernatant was aspirated, and the beige-colored pellet was dissolved in 100 μl of sterile 1× PBS.Exosomes were isolated from blood plasma samples of five astronauts  at L-10 and R + 3 using the ExoQuick Plasma prep and Exosome precipitation kit , as previously described . BrieflyAccording to the manufacturer’s protocol, total RNA was extracted from astronaut-derived exosomes using PB collected at L-10 and R + 3 using the RNeasy Mini Kit (Qiagen). High-quality RNA was isolated using RNeasy Mini spin columns and eluted using molecular-grade water (RNAse/DNAse free). All samples first undergo quality control assessment to ensure successful library preparation and sequencing. RNA sample quality was assessed by NanoDrop and Agilent 2100 BioAnalyzer. Samples with RNA integrity number (RIN) above 7, OD260/280: 2, and OD260/230 ≥ 2 were used. In addition, RNA degradation and contamination were monitored on 1% agarose gels before small RNA sequencing (sRNAseq).RNA quality was assessed using an Agilent TapeStation , and RNA concentration was quantified by Qubit 4.0 spectrophotometer. The library for sRNAseq was prepared using the Smarter smRNA-seq kit for Illumina . The quantity and quality of amplified libraries were evaluated using Qubit  and Agilent TapeStation high sensitivity D1000 Screen Tape. sRNAseq libraries were sequenced using single-end 75 base pairs sequencing chemistry on NextSeq 500 instruments following the manufacturer’s protocols (Illumina).We isolated total RNA from EVs isolated from PB of independent cohort  and PBMCs  at L-10 and R + 3. According to the manufacturer’s protocol, cDNA synthesis was performed with total RNA using the cDNA synthesis kit . Real-time polymerase chain reaction (RT-qPCR) was performed using TaqMan probes  and QuantStudio™ three Real-Time PCR systems as recommended by the manufacturer. We measured the expression of the following snoRNAs indicated below.1. Subsequently, reads were mapped with Bowtie  < 0.05 and log2 fold change (FC) > 1 were considered significantly differentially expressed genes. Subsequently, we extracted differentially expressed snoRNAs and generated a heatmap using custom R scripts.Sequencing data were processed with Cutadapt  to removh Bowtie  to the hh Bowtie . Then, wh Bowtie  to correh Bowtie . Genes w2.The cryo-EM atomic models of human 80S ribosomes (PDB ID: 6EKO) were used to represent modified nucleotides . Figurest-test for comparisons between means. Statistical analysis was performed using GraphPad Prism 9, version 9.2.0 . Differences were considered statistically significant at p < 0.05.Results are presented as mean ± standard error of the mean (SEM). Data were analyzed using an unpaired p < 0.01), we detected 21 down-regulated snoRNAs and 9 upregulated snoRNAs in PB-EVs at R + 3 compared to L-10. Next, we assessed the top four down-regulated snoRNAs  by RT-qPCR analysis in three independent astronauts . However, only SNORA74A was detectable in the L-10 PB-EVs, whereas SCARNA1, SNORA19, and SNORA47 levels were undetectable in R + 3 and L-10 PB-EVs. Consistent with the sRNAseq results, we found that SNORA74A was significantly down-regulated at R + 3 compared to L-10 (O-methylation (Nm) or pseudouridine (ψ) on rRNA, snRNA, or mRNA , ψ3762, and ψ3764 (in the helix-H69 inter-subunit domain)  .In summary, our study revealed for the first time dysregulation of snoRNAs in PB-EVs and PBMCs from astronauts, blood after a relatively short (median 12 days long) Shuttle missions and underscored the potential translational value of investigating snoRNAs as biomarkers for monitoring astronauts health.This study examined the snoRNA content and space-travel associated changes in their expression in EVs derived from the peripheral blood of five astronauts who flew relatively short (median 12 days long) space Shuttle missions between 1998 and 2001. We provide the first report showing differential snoRNAs packaging in the astronauts’ plasma derived EVs pre- and post-flight.Emerging evidence suggests snoRNA’s involvement in metabolism, diabetes, cancer, and heart development , 23, 34.Recent evidence has supported the notion that rRNA modifications such as Nm and Ψ guided by snoRNAs play an important role in translation by altering ribosome splicing and influencing snRNAs and mRNA stability by altering the binding of RNA-binding proteins , 40. In In summary, our study reports for the first time a dysregulation of snoRNAs in PB-EVs and PBMCs from astronauts’ blood after a relatively short (median 12 days long) Shuttle mission. Thus far, there is no reported evidence of CVD, cancer, or neurodegenerative diagnoses within this given astronaut cohort after approximately two decades. Albeit several limitations, the most considerable limitation of this study is the small sample size due to the rarity of access to astronauts’ PB; despite this limitation, this study demonstrates the feasibility of utilizing retrospectively collected and bio-banked samples for the assessment of EV snoRNA alterations. Furthermore, it highlights the need for further retrospective and prospective longitudinal studies paired with clinical data to assess the utility and validity of the potential translational value of snoRNAs as biomarkers to predict astronauts’ health and/or disease.GSE200079.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: GEO, This study was approved by NASA and Icahn School of Medicine at Mount Sinai’s Institutional Review Board . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DAG, AKR, and VNSG designed the research. AKR, MB, AB, AS, and AE performed the research. AKR, MB, AB, AS, AE, MC, and SU analyzed the data. DAG, AKR, KSR, MB, AB, LH, and VNSG wrote the manuscript. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cell‐free messenger RNA (cf‐mRNA) and long non‐coding RNA (cf‐lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non‐coding RNAs in human biofluids remains challenging.Here, we developed polyadenylation ligation‐mediated sequencing (PALM‐Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell‐free RNA (cfRNA) fragments with 5′ phosphate and 3′ hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs.Using PALM‐Seq, we successfully identified well‐known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf‐mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3′ end and adenine at the 5′ end of cf‐mRNA and cf‐lncRNA, which were correlated with the proportions of short fragments.Together, PALM‐Seq enables a simultaneous characterization of cf‐mRNA and cf‐lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs. PALM‐Seq simultaneously characterizes the abundance and molecular signature of cell‐free coding and non‐coding RNAs. The profiles and end motifs of cf‐mRNA and cf‐lncRNA can distinguish biofluids. Seminal plasma tends to retain longer fragments than other biofluids. Pyrimidine dominates at the 3′ ends of cf‐mRNA and cf‐lncRNA fragments, and its frequency is correlated with the fragment length. Two major approaches have been applied to sequence cfRNAs, including RNA sequencing (RNA‐Seq) for low‐quality RNA samples and for small RNAs. Usually input of millilitres of plasma are required for RNA‐Seq library preparation, and some studies use capture kits of coding RNA to enrich cf‐mRNA.Therefore, we developed polyadenylation ligation‐mediated sequencing (PALM‐Seq) to capture original cf‐mRNA and cf‐lncRNA along with other informative RNA fragments. Through the treatment with T4 PNK and template‐independent addition of poly(A) tail, RNA fragments could be ligated to adaptors and reversely transcribed to complementary DNA (cDNA) with the removal of abundant RNA by the RNase H method. The sequencing of fragments regardless of length enables the analyses of the profile, fragment length and ends preference. We further applied PALM‐Seq to describe cfRNA in maternal blood plasma during pregnancy and profiled cfRNA in various biofluids. Our study provides an improved method to simultaneously characterize cf‐mRNA and cf‐lncRNA.22.1We recruited healthy individuals and pregnant women to collect body fluids, including blood plasma, urine, saliva, seminal plasma and amniotic fluid. The information of participants is listed in Table g at 4°C. The upper layer liquid was collected carefully and then centrifuged for another 10 min at 14 000 g at 4°C. For seminal plasma, the spermatozoa were removed by centrifuging at 10 000 g for 30 min at 4°C. The supernatant of all biofluids was collected and mixed with TRIzol LS  by vigorous shaking with a ratio of 1:3. For each library, 300 μl of plasma, 300 μl of amniotic fluid, 200 μl of seminal plasma, 300 μl of saliva and 1 ml of urine were used to extract cfRNAs following the manufacturer's manual. Five micrograms of RNase‐free glycogen  was added as a carrier.The fluid biopsies except seminal plasma were centrifuged for 10 min at 1600 2.22.2.1For cfRNAs, 10 μl of cfRNA was mixed with 4.5 μl of DEPC water , 2 μl of 10× RNase H buffer, 2 μl of ATP , .5 μl of T4 PNK , .5 μl of poly(A) polymerase  and .5 μl of an RNase inhibitor  and then incubated at 37°C for 30 min. For Universal Human RNA Reference (UHRR) or Human Brain Reference RNA (HBR), total RNA was mixed with DEPC water, 10× RNase H buffer and ATP to 18.5 μl, incubated at 94°C for 5 min to fracture the long RNAs and then mixed with T4 PNK, PAP and RNase inhibitor and incubated at 37°C for 30 min. For UHRR‐RNase A treatment, .2‐ng RNase A  was added to 100‐ng UHRR, digested at 37°C for 15 min then was extracted by precipitation by adding an equal volume of iso‐propanol. Incubation at 94°C was omitted. For the External RNA Controls Consortium (ERCC) assay, 1 μl of 1:100 ERCC RNA Spike‐In Mix  was added to the UHRR total RNA. A volume of 2‐μl stop‐annealing buffer  was added to stop the reaction.2.2.2A volume of 1‐μl depletion oligo mix composed of .5 μM of each oligo was added. Y RNA and vault RNA (vtRNA) depletion oligos are shown in Table A volume of 4‐μl 10× T4 RNA ligase buffer, 4 μl of ATP, 7 μl of 50% PEG, 1 μl of T4 RNA ligase 1 , .5 μl of RNase inhibitor and .5 μl of 20‐μM 5′ RNA adaptor were added. The samples were then incubated at 25°C for 1 h. Then 2 μl of rSAP  was added and incubated at 37°C for 10 min to remove ATP.2.2.32 was added and incubated at 37°C for 30 min to remove the depletion probe and residual cell‐free DNA. A volume of 50‐μl RNA Clean Beads  were used to purify the modified RNA, and the RNA was resuspended in 12.5 μl of DEPC‐treated water.A volume of 1‐μl RNase H  together with 5 μl of DEPC treated water was added  and incubated at 37°C for 30 min . Then 2 μl of DNase I  together with 3 μl of 30‐mM CaCl2.2.4A volume of 1‐μl 5‐μM reverse transcription (RT) primer was added, and the mixture was incubated at 65°C for 5 min and then placed on ice for 2 min. A volume of 1‐μl 10‐mM dNTP, .5 μl of RNase inhibitor, 4 μl of 5× HiScript II buffer and 1 μl of HiScript II reverse transcriptase  were added and incubated at 50°C for 15 min, and the mixture was then incubated at 85°C for 5 min to inactivate reverse transcriptase.2.2.5A volume of 5‐μl PCR primers (5‐μM each) and 25 μl of 2× Q5 Hot Start PCR Master Mix  were added. PCR was carried out following the manufacturer's manual, with annealing temperature of 56°C, extension time of 1 min and 20 cycles of amplification. The PCR products were purified with 75 μl of DNA Clean Beads  and then circularized.2.2.6Sequencing was performed on BGISEQ‐500RS or MGISEQ‐2000RS to generate 100‐bp single‐end reads. In addition, 100‐bp paired‐end reads were generated in biofluids to evaluate the length of cfRNA fragments. The adaptors and primers are shown in Table Small RNA libraries were constructed by using an MGIEasy Small RNA Library Prep Kit  according to the kit's protocol. An amount of 10‐ng HBR was added to each library.2.3Adaptors and poly(A) sequences were removed using cutadapt (v1.18)2.4q < .01, |log2FC| > 1. Differentially abundant genes in a specific biofluid were obtained by comparing samples belonging to that biofluid against to the rest of the samples (e.g. plasma vs. other biofluids). Differentially abundant genes during pregnancy were identified by comparing samples from first, second and third trimesters with non‐pregnant samples, with a cut‐off q < .05. The number of reads mapping to each ERCC transcript was counted using the samtools idxstats packageWe generated miRNA reference using the name of mature miRNAs and the sequences of hairpin miRNAs. All reads assigned to a given miRNA were checked by comparing the mapping position with a known mature miRNA position in miRNA precursor, and the mapped reads that had an edit distance greater than 3 bp at the start or greater than 5 bp at the end were excluded. Then a custom Perl script was used to count the reads. To make them comparable, we normalized the RNA profiles to the read counts of a target RNA per million mapped reads (RPM). For tRNAs and piRNAs, we performed parallel alignment and allowed the reads to align across different biotypes simultaneously if they shared the same best mapping quality. piRNAs usually had a lower priority than tRNAs to ensure the correct assignment of reads, because the confidence of their annotations was lower. Finally, a custom Perl script was used to count the reads of various small RNAs and scaled to reads of a target RNA per million. For mRNA and lncRNA, the software RSEM (v1.2.9)2.5https://metascape.org/gp/index.html).http://bioinf.xmu.edu.cn/PaGenBase/). We used CIBERSORT and xCell to calculate the immune scores of five body fluids using the abundance matrix with TPM of mRNAs. For CIBERSORT, we run the R code (https://rdrr.io/github/IOBR/IOBR/man/CIBERSORT.html) with the ‘absolute’ mode to perform analysis and the output ‘absolute score’ for further analysis. The ‘ImmunoScore’ of xCell was analysed on the official online website (https://xcell.ucsf.edu) with default parameters.Functional annotation of enriched gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was performed by Metascape . The RT primers and qPCR primers are shown in Table 2.7We extracted the length of mapped reads from aligned bam files to count the fragment length for all types of RNAs. For paired‐end data, we first assembled paired reads and then analysed the merged reads as single‐end using our pipeline. Similarly, the length of the fragment was extracted from alignment files. Next, to evaluate the length of RNAs with insert fragment between sequencing reads, the paired reads were analysed in single‐end mode separately. If the first read had a mapping length of 90 bp, its paired read that mapped to the same transcript was kept. Then, the length of this fragment was calculated as the distance between the mapping loci of the paired reads plus the mapping length of the second read. Finally, we merged the count of each length in the previous two strategies and assigned as the number of fragments with certain length.To reveal the molecular features of cfRNA fragments, we identified the locus that one base before the alignment locus to RNA reference as the 3′ end of mRNA or lncRNA fragments and then calculated the frequency of each type of nucleotide. The mapping locus of RNA reference was identified as 5′ end and the nucleotide was recorded for each fragment. For the 4‐mer end motif, we extracted the nucleotides from minus and plus two sites of the origin mapping region to RNA references. The frequency of nucleotide on each site and all 256 kinds of 4‐mer end motifs was counted and normalized to the value of percentage.2.8t test (two‐tailed). Two‐tailed Student's t test was also used to evaluate the differences between the absolute score of immune cells. The level of significance was identified as following: not significant, *p < .05, **p < .01, ***p < .001.All statistical analyses were performed using R and Python. Principal component analysis (PCA) was calculated using the R package ‘prcomp’ function. Wilcoxon rank‐sum test was used to compare the ratio of sense and anti‐sense fragments. Differences between the preference nucleotides of 3′ end or 5′ end were analysed by matched Student's 33.1As cf‐mRNA and cf‐lncRNA are highly degraded, with low concentration and various modified ends, we used T4 PNK to generate linear cfRNAs with 5′ phosphate and 3′ hydroxyl.The sequencing data of PALM‐Seq libraries underwent a highly stringent bioinformatic pipeline. Generally, the adaptors and poly(A) sequences were trimmed from the end of reads, and only reads longer than 17 bp were retained. The reads mapped to rRNA, vtRNA and Y RNA were then filtered. Next, an iterative alignment strategy was used to map the remaining reads to the RNA references, including miRNA, tRNA, piRNA, mRNA, lncRNA and other non‐coding RNAs Figure .3.2In order to verify the PALM‐Seq strategy for cf‐mRNA and cf‐lncRNA sequencing, we compared libraries with or without PNKT and TD of abundant RNA. We isolated cfRNA from blood plasma and constructed libraries, using 300‐μl plasma for each library. As expected, PNKT alters the frequency of various classes of RNAs. Compared to no_PNKT‐no_TD libraries, PNKT without TD increased the proportions of mRNA (1.9% vs. 1.0%) and lncRNA (1.4% vs. .5%) and r/Y/vt RNA (82.6% vs. 25.8%), as well as the number of detected mRNA (13403 vs. 11178) and lncRNA (4641 vs. 2233) Figure . The appR2 > .94, Figure R2 value of mRNA between technical replicates was .99 and the correlation coefficient between PALM‐Seq and reverse transcription‐quantitative PCR (RT‐qPCR) was comparable  as expected, whereas the length distribution of miRNA and piRNA formed peaks between 20 and 40 nt Figure . We then3.5CGA, CSH1 and CSH2 and the long non‐coding gene PLAC4S100A8, which is related to pregnant immunomodulation,4 was increased significantly in the third trimester , 28.2 in the second trimester (q < .001) and 14.2 in the third trimester (q < .01). Placenta‐specific miRNA genes from the chromosome 19 miRNA cluster (C19MC),q < .001), 23.9 in the second trimester (q < .001) and 36.6 in the third trimester (q < .001)  Table . Differer Figure . For exa) Figure . We veri) Figure . The ideDuring pregnancy, the placenta releases nucleic acids into the blood,3.6It is known that various biofluids contain cfRNAs.TRPM8, which has been proved as a marker for high‐risk prostate cancer and was detected overrepresented in blood plasma,UGT2B7, a non‐coding RNAs marker of liver cancer,FABP1Next, we identified differentially abundant genes among these body fluids Table . Accordip < .05). The immune score, which is a composite score of immune cell types, was calculated by xCell. Similar to the absolute score, plasma and saliva had the relatively high immune score (.605 ± .199 and .773 ± .041), indicating the high immune cell rate in these two biofluids versus other biofluids (p < .001)  Figure . Our res3.7In addition to profiles of amount, we further analysed the length and cleavage sites of cfRNA fragments in different body fluids using paired‐end sequencing data. Although cellular mRNAs and lncRNAs are usually longer than 200 nt,Next, we characterized the features of 4‐mer end motifs of cfRNA fragments among body fluids to propose clues for the process of RNA fragmentation. The frequency of each 4‐mer end motif was calculated using cf‐mRNA and cf‐lncRNA reads Tables  and S7, p < .05), whereas negatively correlated with the percentage of adenine base at 5′ ends . For lncRNA, the percentage of fragments shorter than 50 nt was positively correlated with the percentage of pyrimidine at 3′ ends  and the percentage of adenine base at 5′ ends . These analyses provided a proof‐of‐concept that PALM‐Seq could quantify cell‐free mRNA, lncRNA and small RNAs simultaneously and characterize cell‐free mRNA and lncRNA fragments, providing a way to study the generation processes and functions of cfRNAs.As 3′‐end nucleotides of cf‐mRNA and cf‐lncRNA in biofluids showed a consistent pattern of RNase A–mediated degradation, we further analysed the correlation between the end bases and fragment length Figure . Our res4In this study, we developed a new library preparation strategy, PALM‐Seq, to quantify and characterize cfRNA in multiple human biofluids. PALM‐Seq simultaneously captures cell‐free mRNA, lncRNA and small RNAs regardless of length and efficiently depletes abundant rRNAs. The profiles and characterizations of blood plasma cfRNA by PALM‐Seq showed the contribution of placenta‐derived RNA, when comparing pregnant and non‐pregnant women. We also applied PALM‐Seq to describe the signatures of cfRNA in various biofluids, including the abundance features, fragment lengths, bases at fragmental ends and 4‐mer end motifs, which implied the promising applications of cfRNA investigation.In order to profile and characterize cfRNA, we designed PALM‐Seq through several improvements. Treating cfRNA with T4 PNK could capture more RNA fragments.The existing cfRNA sequencing strategies are focusing on either long or short RNA fragments, and it remains unclear which strategy is more representative. PALM‐Seq libraries were constructed regardless of RNA length and successfully reflected length distributions of cellular RNA biotypes, so we analysed the length of cf‐mRNA and cf‐lncRNA in biofluids. As our data show, cfRNAs were degraded to different extents in distinct biofluids. cfRNAs in saliva, urine and blood plasma were more degraded than seminal plasma,The small RNA‐Seq ; The Science, Technology and Innovation Commission of Shenzhen Municipality ; The Shenzhen Peacock Plan (Grant No. KQTD20150330171505310).The authors declare that they have no competing interests.Supp. Figures InformationClick here for additional data file.Supp. Table S1 InformationClick here for additional data file.Supp. Table S2 InformationClick here for additional data file.Supp. Table S3 InformationClick here for additional data file.Supp. Table S4 InformationClick here for additional data file.Supp. Table S5 InformationClick here for additional data file.Supp. Table S6 InformationClick here for additional data file.Supp. Table S7 InformationClick here for additional data file.


Naturally occurring ribozymes with a modular architecture are promising platforms for construction of RNA nanostructures because modular redesign enables their oligomerization. The resulting RNA nanostructures can exhibit the catalytic function of the parent ribozyme in an assembly dependent manner. In this study, we designed and constructed open-form oligomers of a bimolecular form of an RNase P ribozyme. The ribozyme oligomers were analyzed biochemically and by atomic force microscopy (AFM). Proteins and RNAs are biopolymers with the ability to fold into a variety of three-dimensional tertiary structures and to exhibit biological functions ,2. BeyonThe structures and properties of self-folding RNAs share similarity with proteins in their monomeric states, and RNA molecules can also form quaternary complexes with protein. These RNA–protein complexes exhibit various structural and biophysical properties ranging from liquid-like properties ,8 to nanTetrahymena group I ribozyme to achieve intermolecular assembly . Th. Th33]. ructures ,35,36, t2+, the C-domain RNAs also showed distinct mobilities from the parent Rz-α  were introduced into the Sα RNA and Sβ RNA, the mobilities of the respective RNAs decreased only slightly , four distinct unit complexes could be formed depending on the identity of S-domain RNA and C-domain RNA. Two are shown in e levels . These o2+, under which conditions the mixture of Sβ RNA and αC RNA exhibited no catalytic activity and another mixture (SαRNA and Cβ RNA) had only residual activity [As the interfaces between the S-domain and C-domain were mismatched in unit complexes, the unit complexes were predicted to be catalytically inactive. Activity assays confirmed this prediction because the four complexes showed no detectable pre-tRNA cleavage activity . This waactivity .We then examined the assembly of Rz-α (Sα:αC complex) and Rz-β (Sβ:βC complex) through a single KL interaction (x:x′ or y:y′) to form tetramolecular complexes. We first examined the assembly of Rz-α and Rz-β ribozymes by a single KL interaction between αC RNA in Rz-α and Sβ RNA in Rz-β. To assemble the first set of RNA components , we examined three assembly protocols A. The fiIn protocol iii, assembly of the third component (Sα or βC) to the unit complex  in an alternating manner using two KL interactions C. We comWe then examined the catalytic abilities of Rz oligomers by analyzing the activities of the two ribozymes in the oligomers independently. To distinguish the catalytic activities of Rz-α and Rz-β in the oligomers, it was necessary to differentiate their substrate specificities. We engineered structural elements governing the interaction between RNase P ribozyme and its substrate ,38,39. A2+, with which the component RNAs formed oligomeric forms as determined by EMSA , to which the x:x′ KL interaction was introduced to design a unit complex (hCx′:xSh) A. The re2+ B. A high2+ B, and thubstrate C.2+, AFM images of the sample solution containing the components of the parent bimolecular ribozymes (Sh and hC) exhibited dispersed particles corresponding to the Sh:hC complexes . In the presence of 20 mM Mgomplexes D. On theomplexes E. One imomplexes E, left. ribozyme . On the Tetrahymena group I ribozyme [In this study, we engineered a bimolecular RNase P ribozyme to construct hetero-oligomers consisting of an orthogonal pair of bimolecular ribozymes (Rz-α and Rz-β). We also installed orthogonality into substrate recognition to distinguish the activities of Rz-α and Rz-β. While the ribozyme oligomers derived from RNase P ribozymes were open-forms in this study, polygonal and three-dimensional nanostructures could be designed and constructed using RNase P ribozymes by following the design strategy for ribozyme-based nanostructures using the ribozyme ,20,21,22ribozyme . It is aribozyme ,41,42. Sribozyme ,44, whicTo expand structural design of ribozyme-based RNA oligomers and its reliability, it is important to eliminate undesired interactions and assemblies, which were often formed when additional elements were introduced to the parent ribozymes  , becauseThermotoga maritima RNase P ribozyme (PDB ID: 3Q1Q) and the nuclear magnetic resonance (NMR) structure of the RNAI/RNAII inverse KL complex (PDB ID: 2BJ2) were used for construction of 3D models of a ribozyme oligomer  and nucleoside triphosphates (1 mM each). The DNA template in the solution was removed by treatment with DNase I for 30 min. Transcribed RNA was then purified by 9% denaturing PAGE. The purified pre-RNA and its variants were then labeled with boron dipyrromethene (BODIPY) fluorophore [Plasmids encoding sequences of S-domains and C-domains of bimolecular ribozymes  were described previously . Introduorophore .2). The resulting mixture was incubated at 37 °C for 30 min and then at 4 °C for an additional 30 min. In the case of two-step assembly using in formation of Rz oligomers, RNA solutions prepared in step 1 were incubated at 37 °C for 30 min, mixed in step 2, and additionally incubated at 37 °C for 30 min followed by at incubation at 4 °C for an additional 30 min. After addition of 6× loading buffer containing 50% glycerol and 0.1% xylene cyanol, the samples were loaded onto a 5% nondenaturing polyacrylamide gel (29:1 acrylamide:bisacrylamide) containing 80 mM Tris-acetate (pH 7.5) and 20 mM or 30 mM Mg(OAc)2. Electrophoresis was carried out at 4 °C, 200 V for the initial 5 min, followed by 75 V for 5 h. RNAs were visualized by staining of gels with ethidium bromide after electrophoresis.An aqueous solution containing components of a bimolecular ribozyme, unit complex, or ribozyme oligomers  was heated at 85 °C for 5 min. To this solution was added 10× concentrated folding buffer 2) was added to each solution. The resulting solutions were further incubated at 37 °C for 30 min to fold pre-tRNA or assemble bimolecular ribozyme, unit complex, or ribozyme oligomers. The two solutions were then mixed to initiate the substrate cleavage reaction catalyzed by RNase P ribozymes. Aliquots were taken at given time points and treated with 1.0 volume of stop solution containing 75% formamide, 0.1% xylene cyanol, and 100 mM EDTA. The reaction mixtures were separated by electrophoresis in 9% (w/v) polyacrylamide gels containing 8 M urea. The substrate and reaction products were analyzed using a Pharos FX fluoroimager . All of the ribozyme activity assays were repeated at least twice, and the mean values are shown in the figures with the minimum and maximum values indicated by error bars.An aqueous solution of pre-tRNA and an aqueous solution containing components of a bimolecular ribozyme, unit complex, or ribozyme oligomers were prepared separately. These solutions were heated at 85 °C for 5 min. A 3× concentrated reaction buffer  was performed according to the protocol for EMSA. Concentrations of RNAs in the solution were then adjusted to ~80 nM by addition of sample loading buffer 2, pH 7.5). The resulting sample (2 μL) was deposited onto a mica surface coated with 0.1% (3-aminopropyl)triethoxysilane. After incubation for 5 min, the mica surface was washed gently with sample loading buffer. The samples in the sample loading buffer solution were imaged at room temperature. A silicon nitride cantilever  with a spring constant of 0.1–0.2 N/m and a resonant frequency of 400–1000 kHz was used. AFM imaging was carried out at a scan rate of 0.2 fps.AFM was performed with a high-speed instrument . Assembly of RNA components in buffer , 20 mM Mg(OAc)We engineered a bimolecular RNase P ribozyme to construct its open-form oligomers, in which ribozyme units were connected by KL interactions. The resulting ribozyme oligomers were investigated by EMSA and AFM, which provided experimental data supporting oligomerization of the unit RNAs. Although this is the initial step toward modular structural engineering of RNase P ribozymes, this study expanded the repertoire of structural platforms of modular ribozymes for RNA nanotechnology.


N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites—Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.“Epitranscriptomics” is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There are several RNA base modifications, such as  Plasmodium, Toxoplasma, Eimeria, Sarcocystis, Cryptosporidium, Theileria, Babesia, Trypanosoma, Leishmania, and Trichomonas are known to cause severe diseases. In this review, we will focus on 3 unicellular parasitic protozoa that are representative members of the above 2 phyla: Plasmodium falciparum and Toxoplasma gondii, the 2 model Apicomplexa and the kinetoplastid parasite Trypanosoma brucei. Emerging evidence suggests that posttranscriptional gene regulation is a key component of genetic control mechanisms in these unicellular parasites, particularly in the regulation of their virulence genes [N6-methyladenosine (m6A), are reversible marks that provide a mechanism for the potential dynamic regulation of gene expression [6A and other RNA modifications [6A modification, which has emerged as most prevalent in mRNAs and noncoding RNAs, and discuss how new techniques for defining epitranscriptomic signatures can enhance our understanding of m6A and other RNA modifications.By definition, a parasite is an organism that lives on or in another organism and feeds on it, ideally without killing it, yet often reducing its quality of life while the parasite exists within the host. Many protists are parasites that must infect other organisms to survive and propagate and they are mostly unicellular. There are about 200,000 known unicellular protozoa and about 5% of them have adapted to a parasitic lifestyle . These pce genes , an excipression . Nearly ications –11. HerePlasmodium falciparum is highly pathogenic and the deadliest parasite causing malaria in humans [Anopheles mosquito, carrying the P. falciparum parasite, takes its first blood meal, injecting sporozoites into the human, which very rapidly makes their way to the liver to differentiate in the hepatocytes to reproduce asexually and become merozoites. The parasites now re-enter the bloodstream to invade erythrocytes (red blood cells or RBCs) and begin the intraerythrocytic developmental cycle (IDC) also referred to as the blood stage. The life cycle of P. falciparum is characterized by an exogenous sexual phase in the invertebrates (insects such as mosquito species: Anopheles stephensi) and an endogenous asexual phase (schizogony) in the vertebrate host . P. falciparum’s IDC depends on the varying levels of protein abundance to maintain its pathogenesis inside of the human host [P. falciparum [P. falciparum in human RBCs has a cyclic pattern of steady-state mRNA expression, with more than 75% of the genes achieving high abundance of mRNAs at only 1 time point of their 48-hour life cycle [Among all the parasitic protists identified so far, n humans . The higman host . The IDClciparum –17, numefe cycle . Severalfe cycle –22. WhilToxoplasma gondii parasite, is one of the most common parasitic diseases preying on immunocompromised individuals fitting the mold of an opportunistic pathogen. This obligate intracellular protozoan can infect any warm-blooded mammal but has 2 modes of transmission within its encysted forms, either found in undercooked animal products as tissue cysts or released as oocysts by feline feces [T. gondii were performed primarily on tachyzoites and bradyzoites, which revealed highly heterogeneous gene expression patterns, including the expression of the large family of surface antigen (SAG)-related sequence (SRS) proteins that are known to mediate T. gondii attachment to the host cell, and thus, trigger host immune responses [T. gondii [T. gondii [Toxoplasmosis, which results from infection with the ne feces . Once inne feces . It is nne feces  as the uesponses ,25. Post. gondii , includi. gondii . Transla. gondii .Trypanosoma brucei is a species that belongs to an ancient group of protozoans in the Trypanosomatidae family and thought to be evolutionarily divergent comparatively to other species within this family that also includes Leishmania and Trypanosoma cruzi [T. brucei falls under the kinetoplastid group of parasites and survives by shuttling back and forth from a human host, as a “bloodstream form,” and an insect host (via a tsetse fly), as a “procyclic form,” the 2 most proliferative forms of this parasite. During the life cycle inside of the human, it lives extracellularly, initially, creating inflammatory chaos within the circulatory system and lymph causing trypanosomiasis, or sleeping sickness [T. brucei, antigenic variation is manifested by sequential expression of immunologically distinct variant surface glycoproteins (VSGs), its major surface antigen [T. brucei has a large VSG gene pool [VSGs in expression sites (within 2 kb from the telomere) can be monoallelically expressed [Trypanosoma spp. playing a role in the regulation of life stage differentiation; however, exuberant understanding still lacks within this organism’s epitranscriptome regulation [ma cruzi . T. brucsickness . The folsickness . Interessickness . In T. b antigen ,32. T. bene pool , but onlxpressed ,35. VSG gulation .Plasmodium spp., enteric coccidian parasite Toxoplasma spp., and kinetoplastid parasites, Trypanosomes or Leishmania, have evolved with both generic and specialized RNA processing pathways that provide an extraordinary glimpse of early eukaryotic evolution and the basis of phylogenetic diversity in RNA metabolism. Importantly, all above parasites develop through different life stages predominantly in mammalian or insect vectors, which require timely expression of genes/proteins for their growth and metabolisms, most notably, their virulence-associated genes essential for parasite survival. Emerging evidence indicates a significant proportion of spatiotemporal gene regulation in parasitic protists is dependent on posttranscriptional regulation at the RNA level [Co-transcriptional RNA processing events are critically important for the maturation of pre-mRNAs and noncoding RNAs. Particularly, mRNA maturation requires several posttranscriptionally regulated processing events, such as 5′ capping, splicing, 3′ poly (A) tail addition (polyadenylation) and mRNA decay ,38, whicNA level ,40. HoweWithin the past half century, a multitude of RNA modifications have been identified and contribute to posttranscriptional regulation of cellular RNAs. The addition of chemical modifications presents an additional layer of regulation of all major classes of RNA. Highly abundant species such as rRNA and tRNA have been known for decades to be heavily modified, but it has not been until recently that cellular mRNAs have been shown to be susceptible to chemical modification. However, it is now recognized that mRNAs contain a diverse and dynamic “epitranscriptome” characterized by a growing list of chemical modifications present at varying abundances ,42.6A has been characterized as the most abundant internal mRNA modification in almost all living organisms. m6A was first detected in animal mRNAs [6A is a reversible modification that is deposited by the METTL3–METTL14 methyltransferase complex and removed by demethylases FTO and ALKBH5  domain family proteins, which contain a highly conserved YTH domain that specifically recognizes m6A. Several RNA processing events are impacted by m6A, including miRNA biogenesis, pre-mRNA splicing, polyadenylation, cellular localization, mRNA stability, and translation [6A-dependent changes in RNA structure can influence gene expression through the control of translation or through impacting RNA:protein interactions [Of the mRNA modifications that have been identified in mRNAs, mal mRNAs ,44 and lal mRNAs , bacterial mRNAs , plants,al mRNAs ,44,47,48d ALKBH5 . In addinslation , and reanslation ,50. In aractions .6A can dynamically alter RNA structure [6A-U Watson–Crick base pairing through 2D NMR studies, the methylamino group energetically favors syn geometry, which places m6A in the major grove in the context of double-stranded RNA, destabilizing duplex structures [6A has also been reported to stabilize m6A-U pairing when neighboring a 5′ bulge, indicating that it is not universally disruptive to RNA structures [This distinct chemical modification transforms the bioenergetics regarding RNA base pairing ,53. It hructures . Single-ructures . However6A and other RNA modifications [Oxford Nanopore Technologies (ONT) has arisen as a third/fourth generation sequencing technology capable of directly sequencing DNA and RNA samples without the need of cDNA conversion. Recently, direct RNA sequencing using ONT sequencers have been used to detect mications ,56. In tications ,58. A coThe extracted cDNA molecules are injected onto a flow cell channel connected to an ONT reader device. The flow cell contains hundreds of small nanopore proteins (green), with a nearly 1 nm aperture, embedded in an electro-resistant membrane (black). When the motor protein (yellow) on the cDNA (blue) comes into contact with the nanopore, the difference pulls the connected RNA (red) through the pore, while the tether protein (violet) blocks the cDNA from entering. As the molecule traverses the roughly 5 nucleotide length channel, it disrupts the electrical differential of the membrane. This disruption is captured by a small electrode near the base of the pore and transmits the change in current to the receiver in the sequencing device. This process continues until the RNA has completely passed through the pore, then the motor protein dissociates, allowing another motor protein to attach and begin the process anew.The current recorded by the electrode is recorded as a squiggle. Each change in potential is characteristic of a particular 5-mer, the 5 nucleotides contained in the pore, and can be analyzed using machine learning models. This process of reading a squiggle to predict the nucleotide sequence is called base-calling. After base-calling, the resulting long-reads can be used for numerous downstream analyses: de novo genome assembly, transcript isoform discovery, differential gene/transcript expression analysis, or mapping nucleotide modifications at single nucleotide and single molecule resolution –61.P. falciparum (approximately 90% AT-richness in the intergenic regions).Since the development of nanopore sequencing, numerous different base-callers have been created, not only to predict the 4 constituent bases, but also to predict specific base modifications. This is possible due to the distinctly different change in current caused by a 5-mer containing a modified base, relative to a 5-mer with an unmodified base. The characteristic difference in current due to RNA base modifications results in base-call errors in those regions, leading to a preponderance of base-call errors at loci encoding for modified bases . Since O6A is deposited by the METTL3 methyltransferase enzyme, which interacts with a complex of accessory proteins including METTL14, WTAP, VIRMA, RBM15/15B, ZC3H13, and HAKAI that function to guide METTL3 to target RNAs and promote the stability and localization of the complex to nuclear speckles [6A mark, which includes methyltransferase-like protein 3 (METTL3) [mspeckles ,65. In m(METTL3) . This ac(METTL3)  The meth(METTL3) ,68. In a(METTL3) .6A has recently been uncovered as an abundant modification in P. falciparum, relative to its approximately 85% AT-rich genome [P. falciparum, the annotation differs, but the main writer proteins, WTAP, MT-A70, and MT-A70.2, are homologous to the evolutionarily conserved RNA methyltransferase core proteins found in other eukaryotes. In P. falciparum, the mammalian ortholog of METTL3 has been identified and designated as PfMT-A70 (PF3D7_0729500) [Toxoplasma, in which the additional proteins VIRMA and TGGTI_275990 have been identified [mh genome . In P. fentified . HHPHREDentified ,72.6A appears in essential genes encoding for VSGs in the T. brucei bloodstream form proliferative stage, particularly in the poly(A) tail [6A in the 3′ UTRs of VSGs, accelerated deadenylation occurred leading to the degradation of mRNA [6A incorporation in the poly(A) tail [In Trypanosomatids, direct orthologs of the methyltransferase writer complex have not been identified . However(A) tail ,74. It w6A reader proteins. These proteins contain a highly conserved YTH (YT521-B homology) domain that is responsible for m6A recognition. Humans possess 5 YTH domain-containing proteins either characterized as YT521-B homology (YTH) domain family proteins (YTHDF) or as YT521-B homology (YTH) domain-containing proteins (YTHDC). Mammalian YTHDF and YTHDC proteins, Arabidopsis CPSF30, budding yeast Pho92, and fission yeast Mmi1 contain this m6A reader domain, although the Mmi1 protein does not preferentially bind m6A [6A readers and have been implicated in the regulation of mRNA stability, translation, and localization [YTH domain-containing proteins are the most well-characterized mbind m6A . YTHDC1 bind m6A –79. YTHDbind m6A ,80,81. Tlization ,82–85. Alization –88. Intelization . FurtherP. falciparum: PfYTH.1 (PF3D7_1419900) and PfYTH.2 (PF3D7_0309800) [Plasmodium species, the PfYTH.2 protein contains an aromatic amino acid cage that forms the methyl-binding pocket. More specifically, the phenylalanine in the 98th position plays a significant role in successful m6A binding to PfYTH2 [6A recognition and affinity. YTHDC1 contains hydrophobic amino acids, W377, W428, and L439 that act as an aromatic cage that is stabilized through the methyl-π interaction [6A complex through the cation- π of the C+1 and positively charged R475 on YTHDC1 [6A, W411, W465, and W470 [Two orthologs of YTH domain-containing proteins have been identified in o PfYTH2 . Structueraction . The adjn YTHDC1 . HsYTHDFn YTHDC1 ,93 and cand W470 .6A methyltransferase complex, but readers recognizing m6A on the 3′ end of the transcript, have brought rise to investigating alternative lengths and instability mediated by Cleavage and Polyadenylation Specificity Factor-30 (CPSF-30) [6A plays a role in determining the fate of transcripts, therefore, protein abundance, in some of the most pathogenic species within infectious disease, such as P. falciparum and T. gondii [P. falciparum protein, PfCPSF-30 (renamed to PfYTH.1) contains tandem zinc finger domains adjacent to a YTH domain. This plant-like polyadenylation specificity factor with an m6A reader domain provides a unique opportunity to target this protein family in parasites to develop antiparasitic drugs and thus avoid unwanted human interactions.Interesting comparisons between metazoan and protozoan species distinguishing not only the mCPSF-30) . This al. gondii . The arc6A. These include fat mass and obesity-associated protein (FTO) and ALKBH5, members of the AlkB family of proteins that are nonheme Fe (II) and α-ketoglutarate-dependent dioxygenases [6A demethylase, and indeed, subsequent studies found that FTO acts on m6A, m6Am [6A demethylase to be discovered, and unlike FTO, it does not have activity toward m6Am [6A in key positions resulting in an accumulation of shorter transcripts [6A can be reversed in cells suggests the possibility for dynamic regulation of m6A. However, these proteins do not appear to act globally on all m6A residues, but rather can remove m6A from specific transcripts in distinct cell types or cellular environments. Moreover, m6A profiling in chromatin-associated and nucleoplasmic mRNA has shown that the majority of m6A sites are conserved in cytoplasmic mRNA, arguing against the idea of widespread demethylation [6A sites are targeted by FTO and ALKBH5 in different cell types.Two demethylase proteins (“erasers”) have been identified that can remove the methyl group from mygenases ,97 FTO lygenases . An impoygenases . This ch6A, m6Am . ALKBH5 nscripts . The idehylation . FurtherPlasmodium or Toxoplasma species and proteins such as FTO remain exclusively found in the vertebrate lineage. One reason for this could be unicellular, protozoan organisms advance through their life cycles requiring differentiation for transmission and overall survival. As a result, there are noticeable, routine phenotype changes. With ambitious requirements from such small genomes, there would be no reason to eliminate a modification that aligns with diversifying the range of expression these organisms can obtain. However, ALKB family protein, TbABH, has been identified in T. brucei that showed amino acid identity with known FTO [Notably, there have not been homologous eraser proteins detected in nown FTO .6A, alternative proteins can directly bind to this modified base that allows them to fall under the “reader” category. A translation initiation factor, eIF3 [6A motif [6A additions provide recognition of RNA binding motifs for hnRNP-C to facilitate alternative splicing at the target mRNA [6A switch.” In the absence of m6A, RNA hairpins bury the U-rich stretch that inhibits recognition [6A-mediated increased accessibility of RNA leading to alternative splicing has also been recognized by hnRNP-G. HnRNP-A2B1 elicits similar alternative splicing and processing roles, however, has been found predominantly interacting with microRNA facilitation [Although YTH domain-containing proteins contain the highest affinity for binding to mor, eIF3 , has beeor, eIF3  proteinsor, eIF3 , and a h6A motif . It has get mRNA , which iognition . The m6Alitation .Plasmodium or Toxoplasma spp. However, hnRNP F/H homologs have been found in Trypanosoma through proteomic analysis that is differentially expressed throughout their life cycle being more ubiquitous during the human bloodstream form parasite [No hnRNP orthologs have been identified in parasite . Within N6,2′-O-dimethyladenosine (m6Am) is an extension of the 5′ cap that can occur on the first transcribed nucleotide if it is an adenosine. This addition is made by the PCIF protein [6Am has been shown to impact both mRNA translation and stability [The co-transcriptional 5′ 7-methylguanosine cap is required for mRNA stability and translation initiation while the transcript enters the cytoplasmic environment. There are known events that take place in the 5′- UTRs of certain viral strains that do not require a 5′ cap when large structural elements are present, known as internal ribosomal entry sites (IRESs). Eukaryotic organisms require a high level of complexity at the 5′ ends to safely exit the nucleus, proceed with protein translation, and be identified as endogenous.  protein . The addtability ,111.6A modification has been shown to facilitate cap-independent translation under some conditions [6A can recruit the translation initiation factor eIF3 to select mRNAs and facilitate cap-independent translation during stress [6A sites within the 5′ UTR of Hsp70, protecting it from demethylation of FTO [6A mediated 5′ cap-independent translation initiation contributes to the parasite translation apparatus. During interactive proteomic assays, researchers reported elongation initiation factor 3 (eIF-3) existing in the same complex as PfYTH.2, presenting possibility for this avenue in Plasmodium and permitting further exploration related to this mechanism [6A demethylase enzymes to require protection under similar conditions.Although most translation is achieved through recognition of the 5′ cap, the mnditions ,112. Forg stress . Addition of FTO . Althougechanism . Althougcis elements) as well as protein factors. The 3′-end cleavage and polyadenylation reaction is directed by sequence elements within the untranslated region (UTR) of the pre-mRNA , which includes an “Adenosine”-rich, hexameric AAUAAA polyadenylation signal (PAS). This molecular process is located at the 3′ end of the newly RNA Pol(II) synthesized RNA that traditionally requires the polyadenylation polymerase to make the addition of a poly(A) tail, accompanied by a poly(A)-binding protein, once cleavage occurs 25 to 30 nucleotides downstream from the polyadenylation signal motif [Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Particularly, 3′-end processing promotes mRNA stability and translation. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA  site choice, consistent with this possibility. This evidence suggests an intimate link between polyadenylation machinery and the epitranscriptome in the mechanism of 3′ end processing in eukaryotes. Similarly, to plants, YTH-family reader proteins constitute part of the machinery involved in processing the cleavage and polyadenylation in apicomplexan parasites, such as Toxoplasma and Sarcocystis species [Interestingly, in plants, adenosines within the multi-partite plant polyadenylation signal could themselves be methylated, raising the possibility that recognition of m, CPSF30 , may con species ,118.Arabidopsis suggesting the polyadenylation pathway’s involvement with m6A in the 3′ end of the transcript [Plasmodium falciparum, Babesia bovis, Eimeria tenella, Toxoplasma gondii, Neospora caninum, Chromera velia, Vitrella brassicaformis, and Arabidopsis thaliana [Theileria parva, Cryptosporidium parvum, Perkinsus marinus, Tetrahymena thermophila, Chlamydomonas reinhardtii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens [6A is nonexistent along key sites in the 3′ untranslated regions created chimeric mRNA events, potentially offering an alternative isoform opportunity through polyadenylation (polyA) site choice, adding yet another tool in this organism’s arsenal to expand on its minimal genome requirement. Alternatively, the polyadenylation signal (PAS), AAUAAA, being A-rich, in addition to intergenic poly(A) stretches, offers an opportunity for m6A to interact with RBPs that are recruited to these locations. With the disproportion of adenines along the 3′ ends of P. falciparum transcripts, there is possibility of erroneous cleavage leading to the truncation of proteins in addition to leaving mature mRNA without a 3′ UTR. As seen in P. falciparum that exists in T. gondii. Rather than allowing for cellular damage, protozoans may have adapted to find utilization in chemical modifications, especially m6A, to act as a supplemental signal to benefit by their A-rich nature.A YTH domain has been discovered to be contained within CPSF30  in anscript . Once ththaliana . Organis sapiens . In factPlasmodium spp.), mammalian cells could use this modification to overthrow its degradation mediated by interfering RNA. Although there is no evidence suggesting homology between CPSF30-YTH, in Plasmodium (PfYTH.1) and Toxoplasma, and proteins involved in polyadenylation in humans, HsYTHDF1 appears to show consistent YTH containing C-terminal domains with a flexible N-terminus [6A associated methyltransferase protein, VIRMA [6A in the 3′ UTR and near the stop codon facilitating alternative polyadenylation in Toxoplama spp. [6A enriched [6A mediated polyadenylation occurring in mammals [6A signatures with the cis-acting elements of the polyadenylation process indicates there is potentially important molecular choreography between m6A modifications in addition to the polyadenylation machinery being a key process in maturation of mRNAs in parasitic protists. Compelling experiments performed by independent groups further explored m6A’s importance in the viability of transcripts facilitated by appropriate 3′ end processing in T. gondii [6A installment (METTL3 and WTAP) resulted in a complete arrest of parasite replication and impairs appropriate 3′ end formation, which makes perfect sense being that m6A distribution along the transcriptome was primarily found to reside near 3′ transcript ends [Arabidopsis thaliana, being that the shared YTH domain is conserved in polyadenylation specificity factors (CPSF4) in both A. thaliana and T. gondii [6A enrichment via METTL3 knockdown [Important regulatory sequences are housed in the 3′ end of mRNAs, such as microRNA and long noncoding RNA complementary sites. As RNA silencing pathways do not exist in some of the unicellular eukaryotes , and m5C can significantly affect eukaryotic mRNA stability [P. falciparum, m5C affects mRNA stability and contributes to its sexual stage development [In mammals, mRNA stability is intimately linked to mtability ,122. Corradation ,123. Thitability . Notablyelopment .6A. Initially, a study found mRNA enrichment following a MeRIP experiment in procyclic form (355) versus bloodstream form (95) suggesting m6A takes a stage specific role [6A containing transcripts [6A being located in the poly(A) tail with a 16-mer motif located upstream in the 3′ UTR [6A in the 3′ end and degradation was addressed in the context of VSGs transcripts [6As are located within the poly(A) tail of VSGs and are crucial for transcript stability [Trypanosomes contain a unique feature being the poly(A) tail itself has been found to contain mfic role . This grnscripts . Furthere 3′ UTR . The linnscripts . They obtability . Removaltability .Like protein coding RNAs, noncoding RNAs (ncRNAs) undergo extensive processing. In humans, the coding RNA accounts for approximately 1% to 2%, while in protists, the number slightly increases to around 3% to 4%; however, the great majority of RNA species remains noncoding, making noncoding RNA’s importance quite clear in cellular processes. NcRNAs are generally classified as small RNAs that are <200 nucleotides in length or long RNAs that are >200 nucleotides long. Potential for how the cell utilizes modified ncRNA opens the gates for further exploration in all organisms once the hurdle of characterization has been accommodated.6Am addition involving splicing, reports highlight the addition of m6A on microRNA [6A within key functional stem loops resulted in a lack of stabilization and increased degradation suggesting an m6A dependent modality [6A sites have been found to cause cellular dysregulation resulting in cancer and disease. Additionally, dysfunctional m6A regulators  contribute to tumorigenesis and cellular proliferation in multicellular organisms. Understanding the mechanistic regulation of m6A in cancer therapy response and resistance could indicate potential targets for precise therapeutics [6A additions on circRNA can recruit eIF proteins, when the reader protein is utilized as an adaptor, further indicating translation involvement [T. brucei, snoGRUMPY, was recently found to play a role in cell differentiation and is a C/D box snoRNA family [Recently, METTL4 was found to take on a novel catalyzing role and in addition to its mmicroRNA . Long nomicroRNA . THOR ismicroRNA . In lncRmodality . Mutatioapeutics . Circulaapeutics . Additioapeutics ,129. m6Aolvement . So far,A family . The typA family . RemarkaA family .6A modification particularly in human TR (hTR) [6A removal that is consistent with the loss of trimethylguanosine synthase 1 (TGS1) resulting in a dysfunctional hTR from mislocalization [P. falciparum [T.brucei [6A-TR interaction and their functional consequences remain uncharacterized. The other RNA, known as telomeric repeat-containing RNA or TERRA plays important roles in telomere biology, including regulation of telomerase activity and heterochromatin formation at chromosome ends. This G-rich RNA is known to form G-quadruplexes (G4) that contain stacked Hoogsteen-bonded G-quartet motifs stabilized by monovalent cations [T.brucei [6A abundance in these 2 RNAs and their specific role in unicellular parasitic protists remains unexplored.Long noncoding RNAs are known to play a crucial role in genome integrity, particularly for the maintenance of chromosome ends, known as telomeres . One of TR (hTR) . ALKBH5 lization . Althouglciparum  and T.brT.brucei  are know cations . Recentl cations . AlthougT.brucei , m6A abuP. falciparum, emerging evidence suggests tRNAs behave differently depending on the presence of particular RNA modifications in key structural elements [P. falciparum, following tRNA modifications exist: ncm5U, mcm5U, mcm5s2U, ψ, s2U, Gm, Cm, I, and m5C [P. falciparum defined dynamic tRNA modifications as the parasite undergoes 3 stage-specific “reprogramming” events highlighted by the up-regulation of Am and 2-thiouridine modifications in the ring stage, an elevated level of a set of different methyl modifications that happened during the ring-to-trophozoite transition period and the dynamic changes affecting specific wobble tRNA modifications in the late-stage parasites. These “reprogramming” events are coupled to codon-biased protein expression system to fine-tune parasite gene expression across IDC. In this parasite, m6A is not found to disrupt Watson–Crick base pairing, therefore, will not contribute to wobble modification facilitated translational modulation [Asp(GTC) regulation through cytosine methylation at the 38th nucleotide position [2U) has proven importance in all living organisms contributing to aminoacylation, structural stability, and the recognition of codons [T. gondii contained a tRNA thiouridylase enzyme, TgMnmA [TgMnmA led to abnormalities in apicoplast biogenesis [Transfer RNAs (tRNAs) are required for the synthesis of proteins as they are the adaptor molecule between the messenger RNA and the growing string of amino acids. Just like other eukaryotes, tRNAs in protozoan organisms are heavily modified. In elements . In P. f and m5C . An elegdulation . PfDNMT2position . A PfDNMposition ; 2-thiouf codons . Before , TgMnmA . This grogenesis  and beca6A and other RNA modifications have emerged as important regulators of cellular translation [6A modifications in P. falciparum coding regions over the course of blood-stage development showed that m6A is highly developmentally regulated in P. falciparum, and m6A levels is relatively higher than any known eukaryotic species [6A sequencing reads mapped to protein-coding regions; however, there was a negative correlation of m6A RNA modification with translation efficiency, suggesting that CDS m6A is likely enriched in transcripts with inactive translation in P. falciparum.mnslation ,84,119.  species . Importa6A helps to reinforce the rRNA structure; however, its addition is not made by the conventional mRNA catalyzing methyltransferase [6A particularly on the 28S rRNA [Plasmodium, are found to be heavily modified, with most known 2′-O-ribose methylation and pseudouridine [P. falciparum ribosomes, reducing protein synthesis from translationally active transcripts [P. falciparum transcriptome is highly structured [P. falciparum, which are essential for parasite development and pathophysiology, have extensively structured coding regions (CDS) [P. falciparum mRNAs could provide stability and that engagement of PfYTH.1 to m6A expedites recruitment of translational machinery, including ribosomes, which are then responsible for the unfolding of CDS RNA structures by their intrinsic helicase activity during translocation. Alternatively, mRNAs with low structures result in a further decrease of m6A enrichment and PfYTH.1 interactions, leading to degradation of mRNAs. These mechanistic possibilities are elaborated in Ribosomal RNA (rRNA) plays a crucial role in the ribosomal machinery necessary for protein translation . Fully mnsferase . ZCCHC4 28S rRNA . Ribosomouridine ,143, espouridine . Specifiouridine . The locouridine . Accumulnscripts . Preexisructured ,146. Manns (CDS) . Proteom6A reader protein that contains a helicase domain belonging to DExH family proteins, which promotes RNA helicase activity particularly acting in the 3′ to 5′ direction [6A locations [Plasmodium, PfDDX17 is an ATP-dependent RNA helicase found most abundantly in the trophozoite stage, suggesting its involvement in resolving mRNA structures for modulating protein expression in other asexual, blood stages [YTHDC2 is an mirection . Throughocations . In Plasd stages .6A plays a role in resolving secondary structures in Plasmodium, Toxoplasma, and Trypanosoma; however, its enrichment has been found within the coding sequence implying its placement being intentional. Intriguingly, it has been recently demonstrated that P. falciparum contains more secondary structures (approximately 71.4%) in protein coding mRNAs compared to human, yeast, and metazoan species (approximately 37% to 60%) [6A modifications will be critical as it is known to regulate protein translation in other organisms [It remains ultimately unknown if m to 60%) . The timrganisms ,149.6A was first detected in polyadenylated RNAs in the 1970’s [6A-containing RNAs remained a challenge due to a lack of methods for distinguishing m6A from unmodified A. Unlike other RNA modifications, the m6A modification does not interfere with nominal RNA base pairing properties, making it difficult to detect through traditional sequencing techniques [6A mapping have relied on immunoprecipitation of methylated RNA using m6A-detecting antibodies. These include MeRIP-Seq [6A-Seq [6A-CLIP [6A-REF-Seq [6A SAC-seq, and DART-Seq [6A mapping from low amounts of RNA and even in single cells [6A sites but also quantifying m6A stoichiometry.me 1970’s ,150. Howchniques ,152. MoseRIP-Seq , m6A-Seq [6A-Seq , and m6A[6A-CLIP . However-REF-Seq , MAZTER-DART-Seq . These ale cells  and they6A and other modifications is the availability of sensitive, quantitative, and high-throughput techniques to survey modifications transcriptome-wide. RNA contains about 7 times the amount of methyl modifications compared to those found on DNA [6A and other methyl modifications have been developed [6A detection and uses m6A antibodies to immunoprecipitate methylated RNAs [6A site or multiple sites [6A-enriched transcripts has many alternative applications [A key bottleneck in the detection and analysis of md on DNA ; howevereveloped . MeRIP-sted RNAs ,158. Howted RNAs . Thus, ile sites ,159. Addications .6A in P. falciparum’s asexual developmental stages in human RBCs [6A using C12 and C13 isotopes [Escherichia coli, or other prokaryotic species, as a viable candidate to act as a normalizing control.LC-MS/MS was among the first strategies to shed light on the abundance of mman RBCs , in addiisotopes  using Es6A without the need for antibodies. For example, the DART-seq method uses a fusion protein consisting of the YTH domain tethered to the cytidine deaminase, APOBEC1, to guide C to U editing at cytidines that invariably follow m6A sites [6A mapping methods. First, since the YTH domain does not recognize m6Am (ref), DART-seq enables selective identification of m6A and overcomes the issues of cross-reactivity that challenge antibody-based approaches. Second, antibody-based methods require a large amount of input RNA, which could be a major limiting factor for hard-to-grow cells in large quantities, particularly in the case of P. falciparum. However, DART-seq can be used in vitro to profile m6A using as little as 30 nanograms of total RNA and has recently been used to map m6A in single cells [6A profiling from limited quantities and is therefore a particularly attractive approach for studies involving P. falciparum.In the last few years, several alternative methods have been developed for mapping m6A sites  Fig 3)  6A witho6A sites . DART-sele cells ,161. The6A and other RNA modifications [Oxford Nanopore Technologies (ONT) has arisen as a third/fourth generation sequencing technology capable of directly sequencing DNA and RNA samples without the need of cDNA conversion. Recently, direct RNA sequencing using ONT sequencers have been used to detect mications ,56. In tications ,58. A coThe extracted cDNA molecules are injected onto a flow cell channel connected to an ONT reader device. The flow cell contains hundreds of small nanopore proteins (green), with a nearly 1 nm aperture, embedded in an electro-resistant membrane (black). When the motor protein (yellow) on the cDNA (blue) comes into contact with the nanopore, the difference pulls the connected RNA (red) through the pore, while the tether protein (violet) blocks the cDNA from entering. As the molecule traverses the roughly 5 nucleotide length channel, it disrupts the electrical differential of the membrane. This disruption is captured by a small electrode near the base of the pore, and transmits the change in current to the receiver in the sequencing device. This process continues until the RNA has completely passed through the pore, then the motor protein dissociates, allowing another motor protein to attach and begin the process anew.The current recorded by the electrode is recorded as a squiggle. Each change in potential is characteristic of a particular 5-mer, the 5 nucleotides contained in the pore, and can be analyzed using machine learning models. This process of reading a squiggle to predict the nucleotide sequence is called base-calling. After base-calling, the resulting long-reads can be used for numerous downstream analyses: de novo genome assembly, transcript isoform discovery, differential gene/transcript expression analysis, or mapping nucleotide modifications at single nucleotide and single molecule resolution –61.P. falciparum (approximately 90% AT-richness in the intergenic regions).Since the development of nanopore sequencing numerous different base-callers have been created, not only to predict the 4 constituent bases, but also to predict specific base modifications. This is possible due to the distinctly different change in current caused by a 5-mer containing a modified base, relative to a 5-mer with an unmodified base. The characteristic difference in current due to RNA base modifications results in base-call errors in those regions, leading to a preponderance of base-call errors at loci encoding for modified bases . Since O6A identification and increase throughput, these strategies are exciting new additions to the m6A detection toolkit. Direct RNA sequencing gives rise to higher read quality by avoiding polymerase slippage in AT dinucleotide repeats, highlighting its importance in analyzing data obtained from unusual genomes of parasitic protists [Plasmodium genes contain introns yet the full characterization of splicing variants remains unknown [Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) [Researchers have increasingly turned to direct RNA sequencing using the PacBio and Oxford Nanopore platforms as an alternative to these approaches –164. Altprotists . Additio unknown . Through(PfEMP1) . These h5C), and m6A [6A, many different tools have been developed to identify the modification using nanopore data  than other sequencing platforms ,169. Add and m6A –174. In ore data . Despite6A prediction at a single nucleotide resolution was MINES, or m6A Identification using nanopore sequencing [6A CLIP data to determine true positives and negatives. The researchers then developed an individual Random Forest Classifier for each of the 4 most abundantly modified A-mers, 5-mers with an A in the central position, in their training set, reporting approximately 80% accuracy. EpiNano uses a significantly different approach, with the developers creating 4 distinct “curlcake” plasmids containing all 1,024 k-mers containing a modified A, while minimizing any potential RNA secondary structure [6A modifications in external samples. Building upon the approach used by EpiNano, Nanom6A uses the same curlcake data and expanded their number of base-call features to include match, mismatch, insertion, and deletion events [6A also changes to an Extreme Gradient Boosting (XGBoost) ensemble algorithm and was validated using meRIP-Seq and m6A-seq data from human and Populus trichocarpa (Black Cottonwood) samples. M6Anet employs an entirely different approach, by using a Multiple Instance Learning neural network and considering squiggle features for not only the target A-mer but also flanking k-mers from various human cell line samples [One of the first tools capable of utilizing nanopore signal for mquencing  , which are unlikely to occur naturally. As the premise of determining m6A modifications using nanopore sequencing is based upon characteristic differences between A and m6A, using additionally modified A-mers as the reference to determine the presence of m6A in a different context is an obvious source of error and uncertainty. Additionally, each of these methods depends on additional downstream analyses, including base-calling, alignment, and base-correction to perform modification prediction. The reliance on these types of data significantly reduces both the ease of use and specificity of these tools. More specifically, while m6ANano demonstrated some ability for transferred learning, with similar performance on both human and Populus trichocarpa samples, it is unclear if the other models are capable of such feats, and what the limitations of these tools are for other genomes, especially those with significant differences in both composition (AT richness) and levels of RNA modification like P. falciparum and other parasites.Despite the improvements in scope and sophistication of the employed methods, existing tools still have significant shortcomings. Some methods like NanomOther tools ,177,178 6A has proven its significance depending on its location along the transcript, the unique recognition by RBPs, and crosstalk from other intrinsic features such as secondary structure which has justified the continuation of investigation. Highly AT-rich genomes, such as P. falciparum, have adapted interesting methods of posttranscriptional regulation involving these m6A mediated events that the molecular parasitology field has not yet fully uncovered, and many questions are left to be currently answered. Emerging studies identified several core enzymes that are involved in RNA epigenetic modifications, much as methylation and pseudouridylation in parasitic protists (6A reader domain in apicomplexan parasites. A new study in the Trypanosoma parasite uniquely showed that m6A positively regulates mRNA stability. Additionally, the recent study on tRNA epitranscriptome unearthed the importance of these modifications in codon bias and protein translation in parasites, which could be a newer paradigm for gene regulation in these infectious agents. Although some research has only scratched the surface and others have provided definitive proof of the importance of this work through unique methods, the exciting fact is the field is heading to understand more about epitranscriptomic regulation in human parasitic species important in translational regulation of parasite-specific proteins. On the therapeutic side, specific details about m6A mediated 3′ end processing in all species that express the YTH containing polyadenylation ortholog should be exposed to provide clues for targeting “plant-like” polyadenylation factors in parasites. Splicing variants exist in most eukaryotic species and diligent work has begun to address alternative splicing associated with m6A in mammals, however, groundwork needs to be done in protozoans to create a similar foundation of knowledge.The dynamic role mprotists . Recent 6A to control mRNA fate will be important for our understanding how m6A contributes to cellular function in diverse cell types. Notably, studying the crosstalk within the mRNA dynamics has provided the need for the long-read methods and direct RNA-sequencing techniques that are important for the field to advance. However, the development of new, quantitative and qualitative methods for m6A detection has enabled new opportunities for researchers to study m6A in diverse organisms and to better understand how m6A abundance varies across individual sites and RNAs [P. falciparum’s life cycle has created difficulties in determining precise expression when utilizing short-read sequencing instruments. Direct RNA sequencing allowing long-read capabilities has opened the door for deciphering transcript levels from characteristic repeat sequences and splicing variants during its transcriptome-wide data mining potential.There are significant mechanistic details that remain to be discovered in the field of epitranscriptomics. Investigations into the factors that determine which readers are recruited to mand RNAs . The dep6A-associated proteins in P. falciparum in the context of life cycle inside of human RBCs that provides greater insight into timeliness of mRNA processing events. Exploration of stage-specific, global transcript levels correlating to reader protein abundance can help indicate individual reader roles relating to the transcripts’ fate. Further guidance and understanding of how the epitranscriptome is utilized can allow the molecular parasitology field to expand on spatiotemporal requirements to gain insight on metabolomics, immune evasion, transmission, RBC invasion, cell cycle regulation, etc., ultimately to understanding pathogenesis in a more dynamic, RNA-inclusive manner.As seen in


Although a plethora of single proteins, ribonucleoproteins, complexed RNAs, and short RNA sequences have emerged as nodal contributors to the splicing cascade, the role of RNA secondary structures in warranting splicing fidelity has been underestimated. Recent studies have leveraged the establishment of novel high-throughput methodologies and bioinformatic tools to shed light on an additional layer of splicing regulation in the context of RNA structural elements. This short review focuses on the most recent available data on splicing mechanism regulation on the basis of RNA secondary structure, emphasizing the importance of the complex RNA G-quadruplex structures (rG4s), and other specific RNA motifs identified as splicing silencers or enhancers. Moreover, it intends to provide knowledge on newly established techniques that allow the identification of RNA structural elements and highlight the potential to develop new RNA-oriented therapeutic strategies against cancer.Alternative splicing represents an essential process that occurs widely in eukaryotes. In humans, most genes undergo alternative splicing to ensure transcriptome and proteome diversity reflecting their functional complexity. Over the last decade, aberrantly spliced transcripts due to mutations in  Therefore, U1 snRNP complex recognizes an AGGURAGU sequence which corresponds to the 5′ splice site (splicing occurs after AG), while U2AF binds to the 3′ splice site that contains a polypyrimidine tract rich in uracils followed by an AG dinucleotide after which 3′ splicing takes place. An additional branchpoint sequence is located 18–40 nts upstream of the 3′ splice site where an adenine serves as the essential nucleophile that starts splicing after U2 snRNP anchoring (cis-splicing elements have been identified throughout exons that can promote or inhibit the splicing of specific genes via interactions with serine/arginine-rich (SR) proteins or heterogeneous nuclear ribonucleoproteins (hnRNPs), respectively  and E/ISSs (exon/intron splicing silencers), appear to be responsible for inverting their silencer or enhancer actions or generating new splicing element functions - and small molecule-based strategies that could potentially repair specific pre-mRNA aberrant splicing and treat the underlying pathologies with a focus on aggressive cancer.+, further stabilize the formation of rG4s by fitting the space between two G-quartets (RNA G-quadruplexes (rG4s) are secondary RNA structures formed by guanine-rich sequences organized in stacks of G-quartets promoted by Hoogsteen hydrogen bonds between guanines . G4 struquartets . rG4s arquartets .in cellulo (in vivo mapping of rG4s contemplates the recruitment of specific RNA binding proteins (RBPs) that recognize, bind, and unwind RNA G4 structures , suggesting that rG4s plausibly control genome decoding by influencing the transcriptome . In addiAlthough there are hints in the literature suggesting the involvement of rG4s and other RNA structures in alternative splicing, a unanimous functional mechanism remains controversial . In moreWhile rG4s within intronic regions appear to modulate splicing by promoting exons inclusion , a G4 elProteome-wide pull-down experiments coupled with mass spectrometry have identified several hnRNPs and splicing factors that bind G4 structures in pre-mRNAs and alter the splicing regulation presets . AdditioThe importance of RNA structural elements in shaping the alternatively spliced transcriptome in tumorigenesis was highlighted recently . MoreoveEpithelial-to-mesenchymal transition (EMT) is an essential process in cancer that cultivates stem-like properties in cancer cells, dictating phenotypic plasticity during tumorigenesis and metastasis. It has been reported that an rG4 in intron 8 of CD44, a marker for cancer cell stemness, regulates the inclusion of the upstream variable exon 8. Expression of this CD44 variant maintains the epithelial phenotype , indicatin silico folding software an SSE  in the pre-mRNAs of PLEC which encodes plectin, a cytoskeletal protein essential for tissue integrity, and ERRFI1, a negative regulator of EGFR signaling. This SSE is located near cassette exons, and due to its high composition of GCs, it folds into a stable stem bearing a small bulge around the fifth nt on the top of the stem. The stability of the SSE secondary structure is crucial to interact directly and in a primary sequence-independent manner with SNRPA1, a component of U2 snRNP that is responsible for the branchpoint recognition site in the initial steps of pre-mRNA splicing. Intriguingly, it has been reported that SNRPA1, beyond its nodal role as the core of U2 complex, acts as a regulator of PLEC and ERRFI1 alternative splicing by interacting with SSEs. Notably, this specific interaction promotes the invasion and migration of breast cancer cells resulting in metastatic lung colonization, while SNRPA1 upregulation is associated with a poor prognosis of the disease , such as pyridostatin analogues that stabilize telomeric dG4s or the recently developed G4-specific fluorescent probe (SiR-PyPDS) used for live imaging analysis of individual G4 structures in cells. Additional evidence suggests that universal G4 probes, such as G4-specific antibodies BG4, can detect fully folded and stable dG4 and rG4 structures equally. In addition, a recent study showcases a next-generation quadruplex ligand (N-TASQ) to probe rG4 structures in untreated living cells (Within the last decade, the increased need for mapping G4 or analogous structures within the human transcriptome in order to understand their functional relevance in RNA processing and turnover has led to the development of various detection tools and analysis methods. Apart from genome- and transcriptome-wide studies focusing on the reconstitution of G4 structure ng cells .in cellulo. Therefore, downstream applications have often been used to verify secondary structural determinants and functionality within predicted motifs. Most of those methodologies used for decades are low throughput, such as circular dichroism spectroscopy, and are employed to recapitulate predicted secondary structures within transcripts by reconstituting their folding in vitro.On the other hand, several algorithms or web-based platforms can detect G4 structures within a given DNA or RNA sequence. Initial versions of such tools, such as the QGRS mapper , can prea priory folding prediction (In recent years the widespread usage of highly sensitive RNA-seq methodologies led to revisiting existing low throughput RNA analyses. That resulted in the development of combinatory approaches that leverage the classical RNA structural detection to be applicable in complex RNA sequence mixtures without requiring ediction . These nediction . In paraediction .As discussed in this review, along with transcriptional, post-transcriptional regulation is at the core of genome decoding. Such events are primarily involved in many diseases, including cancer, calling for the development of novel and efficient RNA-targeted therapies. In this context, ASO-based approaches have come to the limelight to repair aberrantly spliced transcripts and treat various disorders . ASOs arcis-acting elements within the exclusive exon 10 of PKM2, aiming to force the switch to the PKM1 isoform. One identified exonic splicing enhancer recognized by the SRSF3 splicing activator seems critical for producing the PKM2 splice variant. A second-generation ASO designed to anneal to the enhancer sequence disrupts the interaction with the SRSF3 and diminishes PKM2 production, leading to apoptosis induction in glioblastoma cells (in vivo. Notably, the increased PKM1 isoform production led to concomitant metabolic rewiring in cancer cells and reduced tumor formation in xenograft mouse models (As mentioned above, a hallmark of cancer metabolism is an isoform switch of PKM1 to PKM2 caused by a unique, mutually exclusive alternative splicing event . Hence, ma cells . Furtherma cells . Neverthe models .Altogether, ASOs are considered novel and promising therapeutic approaches toward personalized medicine, and various ASO-based treatments are currently in the preclinical stage, including aggressive cancer . HoweverThe abundancy of RNA structures within the transcriptome indicates the existence of a structural code that defines an additional layer of accuracy and precision in the modulation of gene expression. Moreover, distinguishable characteristics of this code, including the plasticity of RNA structures and their versatile recognition by RNA-binding proteins, allow for distinctive regulatory responses both at the transcriptional and translational levels. As highlighted in this review, increasing evidence implies that such RNA structural code influences regulatory systems to facilitate metastatic signaling integration, which is critical for cancer progression . Consequ