In addition to the role of RNA modifications in brain tumor progression, we will also discuss potential opportunities to target these processes to improve the dismal prognosis for brain tumors.RNA modifications are emerging as critical regulators in cancer biology, thanks to their ability to influence gene expression and the predominant protein isoforms expressed during cell proliferation, migration, and other pro-oncogenic properties. The reversibility and dynamic nature of post-transcriptional RNA modifications allow cells to quickly adapt to microenvironmental changes. Recent literature has revealed that the deregulation of RNA modifications can promote a plethora of developmental diseases, including tumorigenesis. In this review, we will focus on four key post-transcriptional RNA modifications which have been identified as contributors to the pathogenesis of brain tumors: m Of the approximately 25,000 people diagnosed annually with primary malignant brain tumors in the USA, 80% are gliomas, one of the most lethal types of cancer , 131. Am6-methyladenosine (m6A), were previously limited by the inability to distinguish between certain nucleotides during reverse transcription [1-methyladenosine (m1A) and 5-methylcytosine (m5C), which are not found only in mRNA: m5C can also be found in transfer RNA (tRNA) and m1A in tRNAs, ribosomal RNAs (rRNAs) and long non-coding RNAs (lncRNAs) [6A, alternative polyadenylation (APA), alternative splicing, and adenosine to inosine (A-to-I) modifications. RNA modifications are a highly conserved mechanism utilized by eukaryotes throughout phylogeny to enhance biologic complexity [Before messenger RNA (mRNA) translation and protein synthesis can occur, nascent mRNA transcripts require processing and nucleic acid modifications. These include splicing out of introns, the non-coding sections of the transcripts, as well as methylation of certain bases. RNA modifications modulate most steps of gene expression, from indirectly controlling DNA transcription, by regulating expression of mRNAs encoding transcription factors, to directly affecting mRNA translation , 144. Whcription , 96. Howcription , 65, 69.lncRNAs) , 96. WitlncRNAs) . These mmplexity . RNA modmplexity . This memplexity . In contmplexity . This almplexity , 100. Whmplexity , 84, a cN6-methyladenosine (m6A) makes up the majority of the internal mRNA modifications in eukaryotes, and has emerged as a critical regulator in many aspects of RNA biology, including pre-mRNA splicing, polyadenylation, localization, and mRNA translation [6A is catalyzed by methyltransferases, demethylases, and binding proteins, otherwise known as “writers,” “erasers” and “readers”, respectively [6A generally occurs within long exons, around stop codons, and in 3′ untranslated regions (3′-UTRs) [6A writers are also located in intronic RNA regions [6A methylation may occur co-transcriptionally, before or during splicing. In addition, mRNA splicing factor precursors co-localize with m6A methyltransferases in nuclear speckles, suggesting the involvement of intronic m6A residues in alternative splicing [Of the known RNA modifications, the methylation of adenosine at the nitrogen-6 position to create nslation , 27, 95.ectively . M6A gen3′-UTRs) , 141. Ho regions , indicatsplicing , 69.6A can be added or removed facilitates rapid changes in gene expression, as m6A modifications are reported to promote mRNA decay through binding of specific degradative protein complexes [6A mRNA methylation, the multi-subunit writer complex comprises a catalytic subunit, known as methyltransferase-like 3 (METTL3), a second augmenting methyltransferase subunit (METTL14) utilized in substrate recognition, as well as the Wilms’ tumor 1-associating protein (WTAP) [6A modifications by promoting the recruitment of the METTL3-METTL14 complex to target mRNAs, in addition to inducing the translocation of the complex to nuclear speckles [WTAP mutations are extremely rare in cancer, occurring in only 0.5% of gliomas [The relative ease by which momplexes , 140. Thomplexes . To catan (WTAP) , 82, 127speckles , 69. WTAspeckles . WTAP mu gliomas . Instead gliomas . Moreove gliomas . While f6A readers” selectively bind to mRNAs modified with m6A. The specific type of reader protein regulates different functions: binding of YTH domain containing family protein (YTHDC1) to m6A induces mRNA splicing by recruiting splicing factor SRSF3 [6A [YTHDF1 and YTHDF2 mutations only occur in 0.9 and 0.5% of glioma cases respectively [6A readers in glioma progression [Proteins often referred to as “mor SRSF3 , whereasor SRSF3 , 50, 128or SRSF3 . To faciRSF3 [6A , 136. Whgression , 112. Whgression . Using pgression . While tgression . Althouggression , 105. It6A methylation is catalyzed by demethylases known as fat mass and obesity-associated protein (FTO), and ALKBH5 [6A modifications [ALKBH5 and no mutations have been reported in FTO [6A primary antibodies and performed microarray analysis. This approach identified ALKBH5 mRNAs targets, such as the proto-oncogene FOXM1. Demethylation of m6A residues in the 3′-UTR of the FOXM1 pre-mRNA results in increased transcript stability and enhanced FOXM1 protein expression [The reversal of md ALKBH5 , 146, boications , 146. Ind in FTO . Howeverd in FTO , is linkd in FTO , 143. Topression . Demethypression .METTL3 locus arise in ~ 1% of gliomas [6A levels are elevated in glioma compared to normal brain [ADAM19 and KLF4 were upregulated by METTL3 silencing and tumor suppressors such as CDKN2A and BRCA2 were downregulated. However, more recent work suggests a different scenario. Visvanathan et al., utilizing methylated RNA immunoprecipitation-seq (MeRIP-seq) on glioma cell line MGG8, followed by gene set enrichment (GSEA) and Gene Ontology (GO) analyses, proposed that METTL3 silencing may in fact disrupt tumorigenic pathways that facilitate glioma progression, such as NOTCH, c-Myc and NFκB [METTL3 significantly decreased proliferation of GBM cell lines U251 and U87MG in cell viability assays [According to the TCGA, genetic amplifications at the  gliomas . In addial brain , 125, thal brain . As a poand NFκB , 125. Thy assays . In vivoy assays . While iy assays , 108. Noy assays .6A methylation regulators are also emerging as critical components of GBM tumorigenesis [6A modifications are differentially expressed between different glioma grades. Specifically, there is positive correlation between WHO grade and expression of WTAP, YT521-B homology (YTH) domain containing family (YTHDF) and AlkB homolog 5 (ALKBH5), whereas there is a negative correlation between FTO and WHO grade [6A deposition around the start codon of serine and arginine rich splicing factor (SRSF) mRNAs. The methylation around the start codon then prevents NMD of SRSF mRNAs, thus resulting in increased alternative splicing and isoform switching in glioma [Additional migenesis , 126. ChHO grade . These rHO grade . A possin glioma .APA is a mechanism that allows a single gene to encode multiple mRNAs, and represents a critical post-transcriptional regulator of gene expression . For theThe process of polyadenylation is facilitated by a multimeric protein complex comprised of four primary subunits: the cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CSTF), mammalian cleavage factor I (CFIm) and cleavage factor II (CFIIm) , 40, 120One of the subunits of the CFIm complex, CFIm25, is believed to directly facilitate the recognition of certain PAS sequences, especially those rich in UGUA sequences . Depleti6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that acts to convert methylguanine back to guanine by removing the methyl or akyl group from the O6 position of guanine, without causing breaks in the DNA. Promoter methylation of the MGMT gene in GBM, present in over 40% of cases, results in improved survival in patients treated with TMZ in addition to radiotherapy [Ootherapy . APA hasotherapy . This lootherapy . Importaotherapy , 60. ThiAs mentioned, following transcription, processing of the pre-mRNA transcript precedes downstream translation and protein synthesis. In addition to the modifications discussed above, splicing results in the formation of multiple mRNA and protein isoforms from one gene Fig. . AlternaThe ubiquitous presence of alternative splicing within cells underscores its relevance for the pathogenesis of tumors, including GBM. Glioma, as described for many other tumors, display a high degree of intertumor heterogeneity , 124. AmMKNK1 and MKNK2 genes are rare in cancer, being present in 0.5 and 2.4% of cases respectively in glioma [PTCH1 while activating oncogenic genes such as GLI2 and CCND2 [The MAPK interacting kinase (Mnks) family of proteins include MNK1 and MNK2 and are the kinases responsible for phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) on Ser-209 . While gn glioma , both prn glioma . Previoun glioma , 42. A rn glioma . Unfortun glioma . On the n glioma . Typicaln glioma , 66. Then glioma , 133. Asn glioma . Finallyn glioma . This rend CCND2 . While nseed sequence (the miRNA recognition site in RNAs), affecting target specificity of the mature miRNA [A-to-I modifications comprise the irreversible, hydrolytic deamination of adenosine nucleoside bases by adenosine deaminases acting on dsRNA (ADARs), converting the adenosine to inosine in RNAs , 98, 106re miRNA , 57, 137re miRNA .Fig. 3TADARB1 gene occur only in 0.6% of cases [seed sequence [While found in all human tissues, A-to-I editing is most prevalent in the brain, and aberrant editing is linked to the development of various brain pathologies including amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, epilepsy and glioma , 93, 107of cases , ADAR2 asequence . In the sequence , 58, 74.GBM is known to utilize glutamate to promote proliferation and migration , a proce, it is unlikely that ADAR3 can directly edit pre-mRNA transcripts. However, as ADAR3 is known to bind to dsRNA despite not being able to perform A-to-I modifications, it has been hypothesized that ADAR3 could act as a direct physical block, preventing ADAR2 from mRNA binding and subsequent editing [In other studies, the activity of ADAR2 was deemed essential to prevent glioma proliferation and growth through the editing of the CDC14B pre-mRNA transcript involved in the Skp2/p21/p27 pathway . As ADAR editing . An alte editing . SequestThe studies outlined in this review highlight the importance of RNA modifications in brain tumor progression, specifically in glioma. The flexibility conferred by post-transcriptional control adds another dimension by which gene expression can be regulated beyond what is directly coded from DNA. The addition of multiple modifications on the same transcript could thus increase the complexity of multiple regulatory networks. This plasticity is particularly relevant for cancer cells to adapt to unexpected microenvironmental changes. However, as showcased throughout this review, deregulation of the RNA modification machinery and altered gene expression are associated with many of the hallmarks of cancer, such as apoptosis evasion and uncontrolled proliferation. Given the significant contribution to brain tumor malignancy, greater emphasis on clarifying the role of RNA regulation and modifications in glioma progression is needed. Targeting the regulatory enzymes controlling the post-transcriptional modifications discussed in this paper warrants further detailed investigation, as this remains an unexplored strategy that could ultimately improve the prognosis of brain tumor patients.


Accurate differential diagnosis between glioblastoma and brain metastasis is important. We aimed to differentiate these tumors by evaluation of the perienhancing area. Thirty patients with glioblastoma and solitary brain metastasis were included. The diameters of perienhancing and enhancing areas were measured, and the percentage of enhancing area was calculated. We measured Apparent diffusion coefficient (ADC) of perienhancing and enhancing areas. Intratumoral necrotic areas were measured. 3) was higher than in metastasis (0 mm3)  with a cutoff area of 11.8 mm2. The enhancing area of glioblastoma was 56.61% and metastasis was 42.55% (p = 0.08). The ADC values of the perienhancing part of GBM was 0.7 and metastasis was 0.79 (p = 0.052). The ADC value of the enhancing part of the GBM was 0.82 and metastasis was 0.8 . The intratumoral necrotic area of glioblastoma (152.25 mmThe ADC values of the perienhancing area were lower in glioblastoma with a near-significant p-value. Other perienhancing parameters demonstrated no significant difference between both tumors. The intratumoral necrotic area of glioblastoma is larger than metastasis. Glioblastoma and brain metastasis are two of the most common brain tumors in adults. Accurate imaging diagnosis is important because of the differing treatments for these conditions . Glioblastoma and solitary brain metastasis can sometimes appear similar on conventional MRI, which can make the definite diagnosis difficult. Both glioblastoma and brain metastasis demonstrate heterogeneous signal in conventional MRI with various types of enhancement surrounded by a perienhancing edematous area . Glioblastoma and metastasis differ in degrees of perilesional edema. Few previous studies have shown significant differences between the ratio of peritumoral edema and enhancing tumor . Perilesional edema was greater in brain metastasis than in glioblastoma. Diffusion-weighted image (DWI) and apparent diffusion coefficient (ADC) values are known to help differentiate between tumor types because they represent the movement of water molecules in vivo . Several studies have yielded inconclusive results when trying to make a differential diagnosis between glioblastoma and brain metastasis, especially when relying on values measured at the enhanced part of the tumor . Even though preliminary results are inconclusive, we believe DWI is a potential tool for differential diagnosis between these two tumors. In pathology, glioblastoma has combined peritumoral infiltration and edema but brain metastasis has pure perilesional vasogenic edema . Accordingly, ADC values of the perilesional area should be different between these two groups: glioblastoma should have lower ADC values than metastasis. In our study, we evaluated two main aspects of the perihancing area. We measured the maximal diameter of the area and also the ADC values relative to the enhancing area, to differentiate between glioblastoma and solitary metastasis. PatientsThirty consecutive patients with histopathologically proven solitary brain metastasis (n=15) or glioblastoma (n=15) who underwent pre-treatment MRI of the brain using DWI, between 1 January 2011 and 1 December 2016, were retrospectively reviewed. One patient had two lesions pathologically proven to be glioblastoma: the total number of tumors included was therefore 31. The study was approved by the local Institutional Review Board of ethical issues with a waiver of informed consent.Imaging techniques3 for both scanners. The diffusion gradients were applied sequentially in three orthogonal directions to generate 2 sets of axial DW images. The ADC maps were automatically generated from the datasets of DW images using the operating console  and ADC values were calculated. After administration of gadolinium-based contrast agent, multiplanar T1WI was performed. In all sequences, the field of view was 22-24 cm. and the section thickness was 5 mm. Seven patients were imaged using a 1.5T MR scanner  and twenty-one patients were imaged with a 3T MR scanner . Routine MR pulse sequences for the 1.5T scanner include: sagittal and axial T1-weighted image spin echo [T1WI SE] , axial T2-weighted image turbospin echo [T2WI turboSE] , axial fluid-attenuated inversion recovery [FLAIR] , and coronal T2GRE . Routine MR pulse sequences for the 3T scanner include: sagittal T1WI 3D TFE , axial T2WI TSE , axial FLAIR , and coronal T2GRE . Imaging parameters of DWI were as follows: 1819-8000/85-93 [TR/TE] with diffusion sensitivities b=0 and b=1,000 s/mmImage analysis All MRIs were analyzed by a senior neuroradiologist with 20 years’ experience in neuroimaging. We defined perienhancing areas as the non-enhancing hypersignal T2WI areas surrounding the enhancing tumor. At the perienhancing area, the maximal diameter in axial T2WI was measured, and the minimum ADC areas were visually inspected and five regions of interest (ROIs) were drawn in different places. At the enhancing area, the maximal diameter was measured in axial view on post-contrast T1WI and five ROIs for ADC values were measured  and 2. T2). We defined these areas as the non-enhancing hypersignal T2WI area within the enhancing tumor if they showed increased diffusion on DWI and ADC maps . The area was calculated from free-hand ROI drawings on the ADC map . A receiver operating characteristic (ROC) analysis was used to determine the cutoff value, sensitivity and specificity of the intratumoral necrotic area as a tool for differentiation between glioblastoma and brain metastasis. A p-value of < 0.05 indicated a statistically significant difference.Of the thirty patients included, sixteen were males (53.3%) and fourteen were females (46.7%). Their ages ranged from 7 to 71 years . Both glioblastoma and metastasis groups had fifteen patients, eight males and seven females. The mean age of metastasis patients was 58 (±10) years and the mean age of glioblastoma patients was 38 (±20) years. The primary tumors producing solitary metastasis were lung cancer , head and neck cancer , breast cancer , neuroendocrine tumor , renal angiomyelolipoma , and cholangiocarcinoma .-3mm2/s,) of perienhancing areas of glioblastomas and metastasis were 0.7 and 0.79 . The median of ADC values  of enhancing areas of glioblastomas and metastasis were 0.82 and 0.8 . The median of ADC ratios, calculated by dividing the perienhancing ADC value by the enhancing area ADC value, of glioblastomas and metastasis were 0.87 and 0.95 . The median of intratumoral necrotic areas (mm2) of glioblastomas and metastasis were 152.25 and 0 .2, sensitivity (87.5%) and specificity (66.67%) were obtained (A receiver operating characteristic (ROC) curve was used to analysis the intratumoral necrotic area for differentiation of glioblastoma and metastasis. The area under the ROC curve was 0.80 . When the cutoff value for the necrotic area was set as 11.8 mmobtained .Differentiation between glioblastoma and brain metastasis is significant for the planning of both diagnostic workup and treatment. Accurate pre-treatment diagnosis will be a benefit to both the patients and the clinicians. Heterogeneous cell structures were found in the solid tumoral parts of both glioblastoma and metastasis tissues in histology. However, at the peritumoral area, a glioblastoma typically has peritumoral infiltration, but a brain metastasis does not . Several studies have shown no significant difference in peritumoral ADC values between glioblastoma and metastases . Few studies have shown lower peritumoral ADC values in glioblastoma as compared to metastasis . We found median ADC values in the non-enhancing area at the edge of the tumor to be lower in metastases than in glioblastoma, with a near-significant p-value . This finding may help to distinguish between GBM and metastases and supports the hypothesis that a glioblastoma involves peritumoral infiltration but a brain metastasis does not .An enhancing mass with extensive perilesional brain edema is a classic characteristic of brain metastasis. However, the perilesional edema in metastasis can vary from minimal to extensive . A previous study found a statistically significant difference in the ratio of enhancing tumor to perilesional edema , whereas we found no such difference.3 (sensitivity of 87.5% and specificity of 66.67%). Intratumoral necrosis is believed to occur because of insufficient blood supply within a rapidly growing malignant tumor. However, for glioblastoma, microvascular hyperplasia is found around the necrotic area, which may represent an prothrombotic or vaso-occlusive state within the tumor  This may cause more intratumoral necrosis in glioblastoma as compared to brain metastasis, as reflected in the significant difference in our study. The intratumoral necrotic area in glioblastoma was higher than in metastases  with the cutoff point of 11.8 mmThis study had a few limitations. First, the study was retrospective in nature. Second, two different MR scanners (1.5T and 3T) were used, with slightly different technique, which may reduce comparability of the ADC values. Third, the sample size was small with a considerable age difference between the glioblastoma and metastasis group. Further prospective study with larger populations and uniform MR technique are required to extend the results.2.In conclusion, the ADC values of the perienhancing area were lower in glioblastoma than in solitary brain metastases with a near-significant p-value. Other perienhancing parameters, including ADC ratio and percentage of enhancement, demonstrated no significant differences between both tumors. The intratumoral necrotic area of glioblastoma is larger than metastasis with the cutoff value of 11.8 mm


Gliomas are the most common and lethal type of intracranial tumors. With the current treatment regime, the median survival of patients with grade IV glioma (glioblastoma/GBM) remains at 14–16 months. RNA editing modifies the function and regulation of transcripts. The development of glial tumors may be caused by altered RNA editing events.n = 1,083).In this study, we uncover the global RNA editome landscape of glioma patients from RNA-seq data of control, lower grade glioma (LGG) and GBM samples  to be edited  in 73% (editing ratio 0.8) of control samples compared to LGG  and GBM  samples. GABRA3 transcript level was found to be downregulated in glioma compared to control in a grade-specific manner with GBMs having the lowest level of the transcript. Further, GABRA3 transcripts were observed to be higher in edited compared to unedited glioma samples. The transcript and protein levels of exogenously expressed gene were found to be higher for edited compared to unedited GABRA3 in glioma cells. Further, exogenously expressed edited GABRA3 inhibited migration and invasion of glioma cells efficiently but not the unedited GABRA3.Collectively, our study discovered a reduction in RNA editing during glioma development. We further demonstrate that elevated RNA editing maintains a high level of GABRA3 RNA and protein in normal glial cells which provides a less migratory environment for the normal functioning of the brain. In contrast, the reduction in GABRA3 protein levels, due to lower stability of unedited RNA, results in the loss of function which confers an aggressive phenotype to GBM tumor. Thirty percent of all central nervous system tumors and eighty percent of malignant brain tumors are composed of glial cells and are called gliomas . AstrocyRNA editing is a molecular process by which RNA sequences are altered post-transcriptionally through base conversion or insertion/deletion. RNA editing increases proteomic diversity in cancer. In mammals, especially in humans, the most common type of editing changes include A-to-I and C-to-U base modifications . A-to-I In the present study, we propose to understand the role of RNA editing in glioma development. Until the last 6 years, various groups have identified RNA editing events by protein sequencing or cDNA sequencing of individual genes. Reduced A-to-I editing was observed in several human tumor types, including brain tumors . Also, iThe importance of RNA editing in glioma pathogenesis was reported in an earlier study where authors studied the role of ADAR2 in GBM growth . In a mophs000178). RNA sequencing data of 45 glioma cell lines were also downloaded from CCLE through CGHub (dbGaP accession number phs000178). Samples downloaded from TCGA and CCLE study were in BAM format, we converted BAM format to fastq format and used for further analysis. We also downloaded 174 LGG and 100 GBM RNA sequencing samples from Chinese Genome Glioma Atlas (CGGA) via SRA (SRP027383)  (n = 63) were downloaded from SRA (We downloaded (downloaded in March 2014) 529 lower-grade glioma and 172 GBM RNA and exome sequencing samples from TCGA through the Cancer Genomics Hub  . RNA seqfrom SRA . Data dohttp://broadinstitute.github.io/picard/) followed by re-alignment and base re-calibration. GATK unifiedgenotyper tool (http://evs.gs.washington.edu/EVS). For each read first six bases were discarded so as to remove the mismatches which were caused due to random-hexamer priming. Following steps were used to filter out the spurious changes: (a) Editing events were removed which showed changes within the 4 bps of known splice junction. (b) Editing events present in homopolymer runs of ≥5 were removed. (c) Editing events present in different locations with high similarity found in BLAT  Benjamini/Hochberg FDR correction value is less than 0.05, (b) absolute difference between average RNA editing of tumor samples vs. control brain samples is greater than 0.2 and (c) absolute difference in percentage of samples edited, tumor vs. control brain, is more than 10% then that RNA editing event was considered as significantly differential RNA editing event.We compared RNA editing ratios between control brain samples and tumor samples to find out differentially edited events. We calculated average editing ratios for both the conditions. Editing difference was calculated by subtracting average value of control brain samples from average value of tumor samples. Significant testing was carried out by using Mann–Whitney Significant differentially edited events (glioma vs. control brain sample) were used for gene ontology (GO) analysis using DAVID bioinformatics resources 6.7  . MissensGlioma cell lines were obtained from ECACC, Salisbury, UK. All cells were cultured in Dulbecco’s Modified Eagles’ Medium (DMEM) containing 10% Fetal Bovine Serum (FBS). The cells were grown at 37 °C in 5% CO2.Antibodies used include anti-GABRA3 , DDK , Actin . GABRA3 overexpression construct: (NM_000808) Human cDNA ORF Clone with catalog number RC206286.Escherichia coli DH5α and plated on Kanamycin LB agar plate. Mutant colonies were picked and plasmid was isolated. The mutation status was verified by Sanger sequencing.pCMV-Entry-GABRA3 plasmid was subjected to SDM using QuikChange Multi Site Directed Mutagenesis Kit . A total of 100 ng of plasmid was taken and mixed with buffer, dNTPs, mutation primer and Pfu polymerase enzyme mix. During PCR reaction, extension was performed at 65 °C for 14 mins (2 mins/kb of plasmid length). The whole mix was then incubated with DpnI enzyme at 37 °C for 1 h. A total of 2 μl of the reaction mix was transformed in 6 cells were plated in 35 mm dishes. A total of seven μg of each of VC or GABRA3 plasmids were transfected using Lipofectamine 2000™ in OptiMEM™ medium. After 6 h of transfection, the medium was changed to DMEM containing 10% FBS. Cells were plated for experiment or harvested after 24 h of transfection for RNA or protein isolation.A total of 0.5 × 10t-test was used for calculating statistical significance between two groups. *, ** and *** denote p value of <0.5, <0.1 and <0.001 respectively.For every experimental condition, 50,000 cells were plated in triplicates in the upper-chamber (Boyden chamber from BD Biosciences) in serum-free medium. In the lower chamber, DMEM containing 10% FBS was added. The cells were allowed to migrate for 6–8 h. Next, cells were fixed in 100% chilled methanol and stained using 0.2% crystal violet. The experiment was carried out in two biological replicates and the quantification is shown for one experiment. Unpaired t-test was used for calculating statistical significance between two groups. *, ** and *** denote p value of <0.5, <0.1 and <0.001 respectively.Activation of the matrigel chambers  were performed by incomplete DMEM for half an hour. For each experimental condition, 75,000 cells were plated in the upper chamber in serum-free medium. A total of 10% FBS was added in the lower chamber. The cells were allowed to invade for 20–22 h after which cells were fixed and stained using 0.2% crystal violet stain. The experiment was carried out in two biological replicates and the quantification is shown for one experiment. Unpaired ™ and RNA was isolated immediately following phenol-chloroform extraction method. The RNA was quantified using nanodrop method and quality assessment was performed by running 1 µg of RNA on agarose-formaldehyde gel. A total of 2 µg good quality RNA was used per reaction for cDNA conversion. Applied Biosystems™ High Capacity cDNA Reverse Transcription kit (Part no. 4368813) was used. The cDNA strand synthesis was carried out in Biorad S1000™ Thermal Cycler. Thermo Scientific’s DyNAmo reagent was used for real time qPCR. Applied Biosystems™ 7900HT Fast Real-Time PCR system was used to measure RNA levels. GAPDH was used as internal control. The experiment was carried out in two biological replicates and the quantification is shown for one experiment.Cells were harvested using TrizolThe primer sequences used in the current study:CMV promoter FP: GCTCGTTTAGTGAACCGTCAGGABRA3 5′ UTR FP: CAGTCACACCACAGCGTCTGABRA3 Common RP: GGTTCCAGGGAGAATATTAATCAGGGAPDH FP: GTCTCCTCTGACTTCAACAGCGGAPDH RP: ACCACCCTGTTGCTGTAGCCAAFAST™ protease inhibitor cocktail was used to lyse the cells. After 1 h, the cell debris were pelleted at 14,000 rpm and 4 °C for 30 min. The protein concentration of each sample was determined by Bradford assay. A total of 50 µg of protein was used for SDS-PAGE at 12% concentration of the resolving gel and run at 80 V for 3 h. The proteins were then transferred onto PVDF membrane, blocked using 5% skimmed milk and subsequently probed using primary and secondary antibodies. The experiment was carried out in two biological replicates and the quantification is shown for one experiment.Cells were trypsinized and pelleted down at 4 °C. RIPA lysis buffer containing 1 mM NaF, 1 mM PMSF and 1X SIGMAThe advent of Next Generation Sequencing (NGS) has facilitated identification of novel RNA editing events. The availability of RNA sequencing as well as, matched whole exome sequencing (WES) data provide us with the opportunity to study RNA editing events that are present in the RNA but do not occur in the DNA. Hence, we aimed to unravel RNA editing events in glioma pathogenesis through analysis of RNA-seq and WES data. For this purpose, data of glioma tumor tissue samples from The Cancer Genome Atlas (TCGA) was analyzed. Further, RNA-seq samples from Chinese Glioma Genome Atlas (CGGA), glioma derived cell lines from Cancer Cell Line Encyclopedia (CCLE) and control brain samples from Sequence Read Archive (SRA) were analyzed to find out potential RNA editing events.A brief schematic of the overall workflow has been given in We found higher frequency of RNA editing events in Alu repeat region (94.81%) compared to non-Alu repeat (2.24%) and non-repeat (2.95%) regions in control brain samples . We obsen = 734,436) in control brains samples as compared to glioma samples in different data sets such as TCGA LGG , TCGA GBM , CGGA LGG , CGGA GBM  and CCLE glioma cell lines   –S7. We c140,952) . We foun140,952)  and S8B.140,952)  and S8D.140,952)  and CGGA140,952)  datasets140,952) . From thWe compared RNA editing ratio of control brain samples with LGG samples of TCGA cohort to find out differential RNA editing events in LGGs. We found increased level of editing ratio in 176 editing events in TCGA LGGs as compared to control brain samples and 4,842 editing events had a reduced level of editing ratio in TCGA LGGs as compared to control brain samples . SimilarNext we checked the concordance of RNA editing events in TCGA and CGGA data sets. We found 85.54% of RNA editing events (both upregulated and downregulated) in CGGA LGGs were present in TCGA LGGs with downregulated editing events showing a higher overlap  and 5B. In depth analysis of the genes that underwent RNA editing identified several interesting facts. Two genes, MLLT6 and MPP2, that were most edited in control brain compared to GBM have been implicated in leukemogenesis and cell adhesion/cytoskeleton regulatory functions . We alsoR = 0.51; p value < 0.0001; It is evident that the majority of the aforementioned missense edited genes, that came up in our pathway enrichment analysis, play roles in neuronal signaling. These genes were found to be highly edited in normal brain and editing ratio and percentage of edited samples decreased significantly in glioma patients. We performed further investigation on GABRA3 because we found it to be highly edited  in normal brain (73%) with an average editing ratio of 0.8 while the editing percent and ratio reduced significantly in GBM samples (5.2% and 0.5 respectively). Indeed, GABRA3 editing reduced significantly in all datasets—TCGA LGG, CGGA LGG, TCGA GBM and CGGA GBM compared to control brain . MoreoveTo understand the effect of loss of editing in glioma cells, we exogenously overexpressed edited and unedited forms of GABRA3  in GABRANext generation sequencing techniques have helped to identify novel RNA editing events in many studies , 2013. IA-to-I changes are the most common type of RNA editing events in mammals, especially in humans . A-to-I Our study revealed RNA editing levels to be higher in control brain samples and it is significantly reduced in glioma samples which correlate with the RNA expression levels of the ADAR enzymes ADARB1 and ADARB2. Indeed, it was shown in a previous report that ADAR enzymes are downregulated in glioma and overexpression of the gene leads to tumor suppressive effects . HoweverThe editing events of GRIA2 and GABRA3 as unearthed by this study have been previously reported to be essential for the functioning of normal mammalian brain . Moreoven = 2), the results are confirmed by similar regulation in the larger TCGA and CGGA datasets.From literature, we see that GABRA3 editing increases during brain development in rats with a simultaneous decrease in the expression . HoweverThis opposing regulation of GABRA3 by editing could be cell type-specific and development-specific. In fact, it is possible that the regulation in adult brain is different. In the healthy adult human brain, editing of GABRA3 is found in different regions of the brain irrespective of enrichment of neuronal or glial populations . MoreoveIn a recent study, it is shown that loss of editing in GABRA3 leads to invasive phenotype in breast cancer . In thisThus, the present study unfolds the entire RNA editome landscape of glioma tissues which will help scientists in understanding the importance of post-transcriptional sequence alterations in diseased conditions. Furthermore, the effect of loss of editing of GABRA3 during glioma progression highlights the importance of RNA editing for the maintenance of tissue homeostasis. The study will open up novel avenues of research and therapeutic interventions for glioma.10.7717/peerj.9755/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9755/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9755/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9755/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9755/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.9755/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9755/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.9755/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.9755/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.9755/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.9755/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.9755/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.9755/supp-13Supplemental Information 13Click here for additional data file.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition. The causative agent of the COVID-19 disease, the SARS-CoV-2 virus, has an unusually large genome that encodes for many proteins. Among them are four structural proteins  important for RNA packing and virion assembly. Molecular understanding how new SARS-CoV-2 virions arise could direct new antiviral strategies urgently needed to combat the current pandemic. In our study, we describe how the N protein binds single- and double-stranded RNA, a key process for virion assembly. Our structural insights identified a large charged RNA binding groove on the surface of the N-terminal domain of the N protein that might play an important role in the higher-order supercoil structure formation in the context of the multiple copies of the dimeric full length nucleocapsid phosphoprotein. Coronaviridae family glucose (for NMR experiments) in 37°C and 220 rpm until OD600 reached 0.6. Then the expression was induced by adding 0.5 mM IPTG and the culture was further incubated shaking (220 rpm) for 16 h at 18°C. The cells were centrifuged  and the pellet was lysed by sonication in lysis buffer  and the lysate was cleared by centrifugation . His-tagged protein was purified from the supernatant by affinity chromatography on a Nickel-NTA (Machery-Nagel) according to the manufacturer's instructions, 6×His tag was cut off by TEV protease . Protein sample was then passed through Nickel-NTA to remove the 6×His tag and uncut protein. The protein for NMR experiments was further purified by size-exclusion chromatography on a Superdex 75 HiLoad 26/60 column  in buffer containing 20 mM Na2HPO4, 50 mM NaCl, 0.01% NaN3, pH 5.5. Purity of the protein was checked using SDS-PAGE. Protein was concentrated to 1.19 mM and used for NMR. For further NMR measuring of binding RNA, protein was diluted to 300 μM and flash frozen in liquid nitrogen and stored at -80°C.DNA with coding sequence for SARS-CoV-2 N-NTD (residues 44–180) was obtained as an artificial gene (Thermo Scientific), cloned to pHIS-Parallel2 and expressed as a fusion protein with 6×His tag followed by cleavage site for TEV protease on its N-terminus. To examine the RNA binding mode of the N-NTD we used ssRNA 7mer (5'-CUAAACG-3') and 10mer (5'-UCUCUAAACG-3') and dsRNA that was prepared by annealing of 7mer 5'-CACUGAC-3' and 5'-GUCAGUG-3' (Sigma) at the final concentration 200 μM of each oligonucleotide and water supplemented with 50 mM NaCl. The mixture was incubated at 60°C for 15 min and then cooled slowly at 26°C. For ssRNA titration, the ssRNA was added to 40 μM protein in molar ratios 1:0.25, 1:0.5, 1:0.75, 1:1, 1:2 and 1:4. For dsRNA titration, the annealed RNA was added to 50 μM protein in molar ratios 1:0.3125, 1:0.625, 1:1 and 1:2.For fluorescence anisotropy assays, the protocol for expression and purification of SARS-CoV-2 N-NTD wild type and all mutants was the same as for the protein used for NMR, except the expression medium was LB medium and size-exclusion chromatography buffer was 10 mM Tris pH 8.0, 150 mM NaCl, 3 mM β-mercaptoethanol. Pure proteins were concentrated to ~2 mM.15N/13C/1H) cryoprobe. The sample volume was either 0.16 or 0.35 mL, in SEC buffer, 5% D2O/90-95% H2O. A series of double- and triple-resonance spectra [1H-1H distance restraints were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.NMR spectra were acquired at 25°C on an 850 MHz Bruker Avance spectrometer, equipped with a triple-resonance ( spectra  were rec spectra  implemen spectra  for initwww.nesg.org). The statistics for the resulting structure are summarized in Structural calculation was carried out in CYANA  using NO15N/1H HSQC spectra of the protein in the free state was calculated as the geometrical distance in ppm to the peak in the 2D 15N/1H HSQC spectra acquired under different conditions using the formula: To follow changes in the chemical shifts of a protein upon RNA binding, we calculated chemical shift perturbations (CSPs). The CSP of each assigned resonance in the 2D l widths .ε groups were significantly perturbed for residues 88, 89, 92 and 177 but remained at their original positions for 68, 93, 95. We combined this information in generating the inter-molecular distance restraints used in YASARA docking calculation that consisted of an initial energy minimization followed by a 100 ns of molecular dynamics using the default md_fast.mrc macro. The 3.9 Å upper distance limits were set between K65 NZ–U1 P, K61 NZ–U2 P, R88 NH1 –U5 P, R89 NH1 –U4 P, R92 NH1 –A6 P, K102 NZ–A7 P, R107 NH1 –A8 P, T166 CG2 –G10 C5, and R177 NH1 –C9 P and R177 NH2 –C10 P.The structure of the N-NTD in complex with the 7mer RNA duplex was calculated using HADDOCK . The RNAThe binding of wild type and all mutants of the SARS-CoV-2 N-NTD (residues 44–180) to RNA was measured using fluorescence anisotropy . BrieflyThe protein sequences of N-NTD sequence from selected closely and distantly related to SARS-CoV-2 was retrieved from available PDB structures  and NCBI sequence  databases, respectively. The multiple sequence alignment (MSA) of amino acid sequences was created using MAFFT v7 server . The final MSA of N-NTD figures with enhanced graphics showing color coded sequence similarities and secondary structural elements above the sequence derived from PDB ID:6YI3 was created using an online server Easy Sequencing in PostScript (ESpript) v3.0 (espript.ibcp.fr).pymol.org).All structural figures were prepared using PyMOL v2.3.5 . The SARS-CoV-2 N-NTD in complex with the 7mer dsRNA under PDB accession code 7ACS and the complex with 10mer ssRNA under PDB accession code 7ACT.S1 Fig(A) Structural superimposition of SARS-CoV-2 N-NTD NMR structure PDB ID: 6YI3 (green), shown along with the backbone ribbon representation of 39 lowest energy NMR structure ensemble (light gray) aligned with 6M3M (purple), 6VYO (cyan) and 6WKP (yellow), illustrating the highly flexible basic finger subdomain and termini. (B) Superimposed Cα trace of currently available four SARS-CoV-2 N-NTD structures are represented as sausage model (ENDscript 2.0), where the radius is proportional to the deviation of r.m.s. between Cα pairs per residue between structures and white color shows the termini that is only present in the NMR structure. (C) Structural superimposition of SARS-CoV-2 N-NTD NMR structure (PDB ID: 6YI3) colored in green with SARS-CoV (1SSK—pink), MERS-CoV (4UD1—lilac), HCoV-OC43 (4J3K - orange), HCoV-NL63 (5N4K - purple), IBV (2GEC—blue), and MHV (3HD4—brown) with its respective electrostatic surfaces calculated for comparison. (D) Multiple sequence alignment of SARS-CoV-2 with other related coronaviral N-NTD with available structures and (E) Superimposed Cα trace of SARS-CoV-2 N-NTD NMR structure along with available coronaviral structures are represented as a sausage model, where the radius is proportional to the deviation of r.m.s. between Cα pairs per residue between structures and coloring based on sequence conservation (high-red to low-white).(TIF)Click here for additional data file.S2 Fig1H-15N-HSQC spectral superimposition of free (dark blue) and RNA bound, which revealed specific chemical shift changes indicating the molecular interaction with RNA-10mer (light blue) and dsRNA (red), each labeled cross peak corresponds to the backbone or side-chain chemical shift of individual amino acid.(TIF)Click here for additional data file.S3 FigCPS are shown as color coded intensity gradient for both complexes, In addition, residues that were used for docking of the 10mer ssRNA molecule are highlighted (A) using YASARA and 7mer dsRNA (B) using HADDOCK .(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Long non-coding RNAs (lncRNAs) have a size of more than 200 bp and are known to regulate a host of crucial cellular processes like proliferation, differentiation and apoptosis by regulating gene expression. While small noncoding RNAs (ncRNAs) such as miRNAs, siRNAs, Piwi-interacting RNAs have been extensively studied in male germ cell development, the role of lncRNAs in spermatogenesis remains largely unknown.In this article, we have reviewed the biology and role of lncRNAs in spermatogenesis along with the tools available for data analysis.Till date, three microarray and four RNA-seq studies have been undertaken to identify lncRNAs in mouse testes or germ cells. These studies were done on pre-natal, post-natal, adult testis, and different germ cells to identify lncRNAs regulating spermatogenesis. In case of humans, five RNA-seq studies on different germ cell populations, including two on sperm, were undertaken. We compared three studies on human germ cells to identify common lncRNAs and found 15 lncRNAs  to show consistent differential expression across these studies. Some of the targets of these lncRNAs included CENPB, FAM98B, GOLGA6 family, RPGR, TPM2, GNB5, KCNQ10T1, TAZ, LIN28A, CDKN2B, CDKN2A, CDKN1A, CDKN1B, CDKN1C, EZH2, SUZ12, VEGFA genes. A lone study on human male infertility identified 9879 differentially expressed lncRNAs with three  of them showing specific and high expression in immotile sperm in comparison to normal motile sperm. A few lncRNAs  have been functionally validated for their roles in spermatogenesis. Apart from rodents and humans, studies on sheep and bull have also identified lncRNAs potentially important for spermatogenesis. A number of these non-coding RNAs are strong candidates for further research on their roles in spermatogenesis. According to the central dogma, DNA is transcribed into mRNA, which is translated into protein, and the latter perform most of the functional, structural, and regulatory roles in various cellular processes. Nevertheless, it was found that protein coding genes in both lower and higher organisms are similar and are not enough to account for the complexity of higher organisms, hence challenging the protein centric view. With the advent of high-throughput technology, it was found that the majority of the genome in humans is transcribed and out numbers the current gene annotations. Moreover, a majority of these transcripts do not get translated into proteins. These transcripts which are not translated into proteins are known as the noncoding RNAs (ncRNAs) , 2. EarlFAL1 [GAS5 [Long non-coding RNAs have been reported to regulate a variety of cellular processes such as differentiation, proliferation and apoptosis. The differential expression of lncRNAs in human diseases highlights their roles in various biological processes. It has been reported that three lncRNAs: Peril, Evf2 (Dlx6os1) and linc-Brn1b are important for neural development , 11. SevFAL1 , GAS5 [2L1 [GAS5 , HOTAIR L1 [GAS5 , HOTTIP L1 [GAS5 , PCGEM1 L1 [GAS5 , PCA3 [2L1 [GAS5 . The divTill date many potential lncRNAs have been reported in spermatogenesis and male infertility, however, very few of them have been validated and functionally characterized. A comparison of the findings across these studies can lead to the identification of lncRNAs important for spermatogenesis which could also be exploited as markers of male infertility. Therefore, in this article, we have reviewed the long noncoding RNAs (lncRNAs), their genomic locations, databases for lncRNA prediction and analysis, recent discoveries made with respect to spermatogenesis and male fertility, and discuss important lncRNAs known to play a functional role in spermatogenesis and male fertility.LncRNAs (long non-coding RNAs) are defined as the non-protein-coding RNAs, which are at least 200 nucleotides in length , 29. LikLncRNAs can be classified on the basis of their genomic location i.e. depending upon where from these RNAs are transcribed. These can be grouped into five broad classes: stand-alone lncRNAs, natural antisense transcripts, pseudogenes, long intronic ncRNAs, promoter-associated transcripts, and enhancer transcripts Fig. .Fig. 2GC. elegans, yeast, Arabidopsis thaliana [Stand-alone lncRNAs  are transcribed from a distinct genomic loci and do not overlap with protein-coding genes –33. Theythaliana –37. Xistthaliana , HOTAIR thaliana , H19 [40thaliana  are someNatural antisense transcripts are transcribed from the opposite sense strand , 42. GenPseudogenes are ‘relics’ of genes, which are non-functional and do not encode for any protein . They arArabidopsis [Arabidopsis thaliana, COLDAIR, an intron present in the FLC (flowering repressor locus) gene, is responsible for plant vernalization [It has been known that introns harbour small ncRNAs, such as snoRNAs and miRNAs. Several transcriptome studies have found that many long transcripts are encoded by the introns of annotated genes , 51. Tilbidopsis , 53, 54.lization .Promoter-associated long RNAs (PALPs) and enhancer-associated RNAs (eRNAs) are lncRNAs, which are transcribed from promoters and enhancers by RNA Pol II . Both prGene regulation is a complex process, which takes place at transcriptional, post-transcriptional and translational levels Fig. . Long noLncRNAs are involved in epigenetic gene regulation and genomic imprinting, such as X-inactive specific transcript (XIST), AIR, H19 . In femaA.There are several non-coding RNAs, which regulate gene expression by binding to the target mRNA through sequence complementarity. There are certain lncRNAs which work as competitive endogenous RNAs (ceRNAs), also called miRNA sponges because they contain binding sequences for miRNAs . These cB.MicroRNAs can also regulate the expression of lncRNAs . For exaC.Long non-coding RNAs can interrupt in miRNA function by competing for its target site on mRNA. A very good example of this is the binding of lncRNA BACE1-AS (β-site amyloid precursor protein cleaving enzyme 1- antisense transcript) to its sense partner, where miR-485-5p binds, thus impairing the function of miR-485-5p in Alzheimer’s disease .After transcription, long non-coding RNAs can regulate the expression of miRNAs by working as a miRNA sponge. It can also interfere with the function of miRNA by competing for its target site on mRNA. Moreover, miRNA can also regulate the expression of lncRNAs by binding to its 3’UTR region. This type of regulation has been reported in human, mouse and rat –71.A.ThThe process of alternative splicing takes place in both animals and plants to generate different mRNA isoforms . It was A.LncRNAs play important roles in regulating the translation process as well. LncRNAs regulating target genes at the translational level have been reported in human and mouse . LncRNA-B.The translation of Uchl1  is facilitated by the lncRNA generated from its antisense strand AS-Uchl1, which recruits polysomes with the help of a repetitive domain SINEB2 to promote a cap- independent translation .LncRNAs can also modulate the post-translation modifications (PTM) of a protein by masking the PTM sites, which has been reported in both human and mouse , 79. LncHigh throughput studies, such as, microarray and deep sequencing, have identified a huge number of non-coding RNAs . Around All the databases provide information about lncRNAs identified in humans. Databases which provide information of mouse lncRNAs are DIANA-LncBase, CHIPBase, lncRNAdb, Noncode v3.0, and the Functional lncRNA Database. LncRNAdb and Noncode v3.0 databases provide information on lncRNAs expressed in different species, from yeast to plants. There are certain databases that provide information about the cell or tissue specific lncRNAs; for example, lncRNAdb, CHIPBase, Noncode v3.0, DIANA-LncBase, and lncRNome. Only lncRNAdb and Noncode v3.0 provide information about the cellular localization of the lncRNAs. There are certain databases which provide information about lncRNAs and their association with diseases: Noncode v3.0, lncRNAdb, DIANA-lncBase, lncRNADisease, and lncRNome. Few databases provide information about the association of lncRNAs with other non-coding RNAs. For instance, databases like DIANA-LncBase, LNCipedia, the Functional lncRNA Database, and lncRNome give information about the association between miRNAs and lncRNAs.Microarrays with probes specific to lncRNAs detection became available about a decade back. These include Arraystar Mouse Stringent and Affymetrix Genechip 2.0 ST series and LncRNA microarray, which are based on updated lncRNA genomic annotation resources such as NONCODE, ENCODE, RefSeq, UCSC Genes, NRED, and fRNAdb. These platforms allow direct measurement of the levels of known lncRNA candidates. Only three microarray studies have investigated lncRNAs in relation to spermatogenesis –101  conducted another microarray study to identify lncRNAs expression at different developmental stages: embryonic day (E) 12.5, E 15.5, postnatal day 7 (PND 7), 14, 21, and adult . They foIn a recent study, microarray analysis of transcripts from mouse brain, kidney, heart, liver and testis was performed  Table 22. It wasBecause of the falling cost of the Next Generation Sequencing (NGS) technology, RNA seq has become a popular approach and is commonly used for transcriptome analysis. Its advantage over microarray technique is its sensitivity, coverage, resolution and the capability to identify new transcripts. Thousands of lncRNAs have been identified in human and mouse transcriptome sequencing projects . RNA seqAt least three studies have investigated lncRNAs in human germ cells Table . Zhu et LncRNA profiling of human sperm is very limited Table . SendlerMost of the RNA seq studies have been undertaken on mouse testis or sperm Table . Laiho eZhang et al. (2017) undertook strand-specific RNA sequencing that revealed a large repertoire of lncRNAs in mouse mature sperm . They idSelvaraju et al. 2017) undertook sequencing to identify the spermatozoal transcripts in fresh bull semen   conducted RNA sequencing to identify lncRNAs and their role in the testes development of high-grain (HG) diet fed sheep . A totalIn another study, Wang et al. 2019) undertook an integrated analysis of lncRNAs and mRNAs in high and low motility Holstein bull sperm, in order to identify mRNAs regulated by lncRNAs and their impact on sperm motility  4). BelowNarcolepsy candidate-region 1 gene (NLC1-C) is an lncRNA expressing in the cytoplasm of spermatogonia and early spermatocytes. It was found that the over-expression of NLC1-C promoted cell growth whereas its loss inhibited cell growth and promoted apoptosis . MicroarMeiotic recombination hot spot locus (Mrhl) RNA is a 2.4 monoexonic residing in the nucleus. Mrhl was first identified by Nishant et al. (2004) while working on a 17.2 kb fragment of a novel meiotic recombination hot spot in mouse chromosome 8 . Later, HongrES2 is a 1588 bp lncRNA, which is co-transcribed by chromosomes 5 and 9 in rats. Its expression is seen in the cauda region of epididymis . The expTestis specific X-linked (Tsx) is an lncRNA, which is located within the X-inactivation center. It is known to be highly expressed in the pachytene spermatocytes and not in spermatogonia or round spermatids, suggesting its role in regulating the meiotic division. An increased number of abnormal pachytene spermatocytes undergoing apoptosis were seen in Tsx knockout mice, suggesting its role in the progression of meiosis in mice .Sohlh1 and prevents early meiosis of spermatogonia by repressing Stra8 [Similarly, Dmrt1-related gene (Drm) is an exclusively expressing lncRNA in testis. It was discovered by Zhang et al. (2010) in an attempt to clone the Dmrt1 gene when they found another RNA isoform of different size and sequence on the 3’half . Later, ng Stra8 , 129. ThChan et al. (2006) used two different techniques, SAGE and whole genome tiling microarray, in three different germ cell stages: type A spermatogonia, pachytene spermatocytes, and round spermatids to identify the protein coding genes . Later, LncRNA- testicular cell adhesion molecule 1 (lncRNA-Tcam1) is a 2.4 kb nucleotide long nuclear lncRNA. It is exclusively expressed in mouse male germ cells and is assumed to play a crucial role in meiosis. In a recent report, it was found that lncRNA-Tcam1 regulates immune related genes in mouse germ cells . The ind−/− males as compared to the wild-type mice (oligospermia), they also found morphological defects in the head and mid-piece region of sperm (teratospermia). Thus, these findings revealed an essential role of the Tug1 locus in male fertility. Further investigations showed that Tug1 locus harbours three distinct regulatory activities: cis-acting repressive DNA function, trans- acting Tug1 lncRNA and peptide (TUG1-BOAT) having role in maintaining mitochondrial membrane potential [Tug1 locus resides on chromosome 11 in murine and is among the most conserved lncRNAs between mouse and humans . The expotential .Prss/Tessp gene cluster on chromosome 9 [Prss/Tessp gene. The Prss/Tessp gene cluster specifically activates in the primary spermatocytes at the late pachytene stage and has been found to be critical for meiotic progression of the germ cells. The molecular mechanism by which tesra enhances the activity of Prss/Tessp gene promoter still needs to be explored [Tesra is a 4435 nucloetide long testis-specific lncRNA, which is present at the mosome 9 . The expexplored . Howeverexplored .Gfra1) gene. LncRNA033862 was found to be highly dysregulated in response to GDNF (a ligand of GFRA1) in mouse SSCs [This lncRNA resides on chromosome 19 and is transcribed from the antisense strand of the GDNF family receptor alpha  performed a microarray study to investigate the expression profile of lncRNA in different germ cell populations  . They foIn a microarray study, the expression of Gm2044 was found to be highest in the pachytene spermatocytes of mouse testis . FurtherOnce thought to be junk, non-genic regions of DNA encode several non-coding RNAs, which play important roles in gene regulation. The advent of next generation sequencing has accelerated the discovery of lncRNA along with other types of non-coding RNAs. Investigations on lncRNAs expression in the embryonic and post-natal testis identified about 1000 lncRNAs that show change in expression with the onset and progression of spermatogenesis. At least one study investigated lncRNAs in different germ cells in adult testis and identified about 1500 such RNAs showing change in expression during spermatogenesis. Specific investigations on particular lncRNAs identified their impact on sperm count , sperm motility , sperm capacitation (mil-hongrES2) and meiosis . There are certain lncRNAs involved in maintaining the balance between stemness of spermatogonia and their differentiation. For example, Spga-lncRNA, AK015322, LncRNA033862 are involved in maintaining the stemness of spermatogonia while Mrhl lncRNA binds to the Sox8 promoter to regulate spermatogonial differentiation in mouse. Comparison across three studies on human germ cells identified 15  common lncRNAs that can be great candidates for further analysis for their role in spermatogenesis and their differential expression in infertility.A number of databases and analysis tools have now become available to facilitate the data analysis and comparison. Transcriptome sequencing offers the identification of novel RNAs along with known RNAs; therefore, sequencing studies should provide new candidate RNAs for further investigation in spermatogenesis and male infertility. Several lncRNAs have been identified which play roles in different developmental stages and adult mouse testes. Hong et al. (2018) identified testis-specific lncRNAs in mouse, of which 26 most abundant are strong candidates for further investigations. Bao et al. (2013) reported 28 candidate lncRNAs  which were differentially expressed across different developmental stages in mouse.Although many potential lncRNAs have been discovered till date with the help of advanced technologies and public lncRNA annotation resources, yet the functional annotation of lncRNAs in spermatogenesis is in infancy and has tremendous possibilities. Long noncoding RNAs involved in spermatogenesis can be identified by using public lncRNA annotation, but their specific localization and biological function in testis need experimental evidence. A number of studies have undertaken RNA sequencing in testis, germ cells and sperm , 138–140New techniques have been developed to identify the function of lncRNAs through their interaction with other molecular species. These techniques involve the identification of interacting partners like RNA, protein and/or DNA and isolation of a component of interest (RNA/protein). For identification of RNAs linked to proteins, RIP (RNA immunoprecipitation) and HITS/PAR-CLIP (High-Throughput Sequencing of RNA/PhotoActivatable-Ribonucleoside-Cross Linking and Immunoprecipitation) techniques can be used. Other techniques like RNA pull down and/or ChIRP/CHART would be useful to identify proteins and DNA sequences associated with a particular lncRNA.


SOX2. Recently, we identified the RNA-binding motif protein 24a (Rbm24a) which binds to and regulates sox2 in zebrafish and mice. Here we show that morpholino knockdown of rbm24a leads to microphthalmia and visual impairment. By utilizing sequential injections, we demonstrate that addition of exogenous sox2 RNA to rbm24a-deplete embryos is sufficient to suppress morphological and visual defects. This research demonstrates a critical role for understanding the post-transcriptional regulation of genes needed for development.Congenital eye defects represent a large class of disorders affecting roughly 21 million children worldwide. Microphthalmia and anophthalmia are relatively common congenital defects, with approximately 20% of human cases caused by mutations in  Congenital eye defects represent a large class of disorders affecting roughly 21 million children worldwide ,4,5. (SOX2) account for approximately 20% of human anophthalmia cases [SOX2 is crucial. While the transcriptional regulation of SOX2 has been explored, only recently has the post-transcriptional aspect of its regulation been appreciated [Mutations in SRY (sex determining region Y)-box2 ia cases . As a tria cases ,8,9. Thureciated .Sox2 [SOX2′s known role in microphthalmia and anophthalmia in human patients, knockdown and knockout Rbm24-deficient mice and zebrafish often displayed eye defects including microphthalmia and/or anophthalmia [rbm24a knockdown, mutation, and overexpression are also associated with cardiac defects. Previous inquiries found Rbm24 mutant mice to be embryonic lethal, dying between E7.5 and E14.5. The embryonic lethality observed is thought to be cardiomyopathy due to numerous cardiovascular malformations, which included ventricular septum defects, reduced trabeculation and compaction, dilated atria and ventricle chambers, thinner atrioventricular endocardial cushions, sarcomere disarray, and fibrosis [rbm24a depletion. Previously, we identified the RNA-binding protein, RBM24, as a post-transcriptional regulator of Sox2 ,12. Conshthalmia . In addifibrosis ,16. Mostsox2, to perform more in-depth analyses as to the relationship between Rbm24a and sox2. To do so, we analyzed the expression pattern of rbm24a and found, consistent with previous studies in zebrafish, Xenopus, mice, and chick, its expression in the somites, heart, and lens in a spatiotemporal manner [rbm24a in zebrafish development, we performed morpholino knockdown and CRISPR mutagenesis which phenocopied previously reported morphants and mutants [rbm24a morpholino and exogenous sox2 RNA to determine the extent to which sox2 can suppress the rbm24a-depleted microphthalmia. We found phenotypic suppression of the rbm24a-induced microphthalmia. We also performed a visual assay which tests for light/dark detection on these embryos and found partial visually functional rescue by sox2 RNA. This work highlights the study of post-transcriptional modification and their target RNAs during eye development. It is challenging to study global factors such as Rbm24a due to their wide-ranging impacts on a multitude of target RNAs. We chose to focus on a single proposed RNA target, l manner ,21,22,23 mutants ,20,23. WZebrafish are maintained in standard conditions under the approval of the University of Iowa Institutional Animal Care and Use Committee . Embryos are collected from natural spawning and raised between 28 and 30 °C. No more than 50 embryos are kept per 100 mm plate. Embryo plates are cleaned of dead daily and water changes are made as needed.rbm24a morpholino , splice-blocking rbm24a morpholino, or AltR CRISPR construct (3 nL). The morpholinos were ordered from Gene Tools. rbm24a AUG MO sequence: 5′-GCATCCTCACGAAACGCTCAAGTGC-3′. rbm24a SB MO sequence: 5′-TTGATATAATCCTCACCTGGCTGCA-3′. The AltR crRNA was ordered from IDT [rbm24a AltR crRNA sequence: 5′-GGACUUUCCAGUCUGUCUGUGUUUUAGAGCUAUGCU-3′. RNA for rbm24a (100–400 pg), sox2 (200–300 pg), and EGFP (200–300 pg) was generated from cDNA that was cloned into the pCR8/GW/TOPO vector  or pCS2 +  before linearized templates were transcribed using the SP6 Ambion mMessage mMachine kit . rbm24a and sox2 RNAs have N-terminal Myc tags. sox2 RNA lacks a 3′ UTR. Microinjection needles were measured via a capillary tube to ensure dosages fell in the aforementioned ranges. Sequential injections were performed utilizing the same needle between morpholino-injected embryos (morpholino-only and morpholino with RNA) and RNA-injected embryos (RNA-only and morpholino with RNA) to ensure consistent dosage between experimental groups. An app was used to calculate the amount of construct injected . When noted, sequential injections were performed on a single day per set and involved the usage of the same clutch of eggs, needle, and dosage for all embryos injected.Embryos at the 1–2 cell stage were injected with either a translation-blocking rbm24a-Forward-5′-ATGCATACCACGCAAAAGGAC-3′, rbm24a-Reverse-5′-CAGTCTGTCTGTCGGTAATCA-3′.Uninjected and injected embryos underwent gDNA extraction at 2–4 dpf. Briefly, 20 µL of 50 mM NaOH per embryo was added before embryos were heated at 95 °C for 15 min. Samples were cooled and neutralized with 1 µL of 1 M Tris pH 8.0 per 10 µL of NaOH. The resulting gDNA underwent PCR amplification before being sequenced and insertion/deletion (indels) detection was performed utilizing Synthego ICE . rbm24a-The automated vision startle response, VIZN, was performed on 5 or 6 days post-fertilization (dpf) larvae as previously described . PhenotyData in tables were analyzed statistically with Fisher’s exact test implement in R . VIZN asrbm24a is expressed in the developing heart, somites, and lens [rbm24a mutant. Similar to other organisms, including mice and humans, the zebrafish Rbm24a contains two domains: the RNA recognition motif (RRM) and an Alanine-rich region. The RRM domain spans exons 1 and 2 and is the domain responsible for binding to target RNAs. This domain is highly conserved, with 96.2% identity shared between zebrafish and humans. The Alanine-rich region is in exon 4 and has an unknown function in Rbm24a   . rbm24a knockdown phenotypes. When compared to uninjected embryos, morphant embryos injected with a low dose (1–1.3 ng) displayed only microphthalmia, while embryos injected at a higher dose (1.7–2 ng) phenocopied the CRISPR-injected F0 embryos with both microphthalmia and cardiac edema   . The overbm24a knockdown phenotype. We utilized EGFP RNA as a control for RNA injection. EGFP is not naturally found in zebrafish and should have no function. Additionally, we can check for successful injection by screening embryos for EGFP fluorescence (rbm24a morpholino (1.7–2 ng) and embryos injected with both rbm24a morpholino and EGFP RNA (200–300 pg) showed similar phenotypes, including many with microphthalmia and microphthalmia with cardiac edema (p = 0.1050), while the number and nature of the affected embryos in both the rbm24a knockdown and rbm24a knockdown with EGFP RNA are also very similar (p = 0.5884)  . Taken tsox2 as a target of Rbm24a in both zebrafish and mouse models [rbm24a led to decreased levels of sox2. We hypothesize that supplying exogenous sox2 RNA would supplement the reduced endogenous sox2 RNA levels. We previously identified the binding site in the 3′ UTR of Sox2 for RBM24 via mouse cell culture studies [sox2 RNA construct which lacked the 3′ UTR to prevent Rbm24a binding (referred to as sox2 RNA). To test the functional role of sox2 as a target of Rbm24a, we performed sequential injection of rbm24a morpholino followed by sox2 RNA. Knockdown of rbm24a (1.7–2 ng) generated embryos with microphthalmia or microphthalmia with cardiac edema when compared against the uninjected control  were often normal in morphology  significantly fewer times (approximately two out of five times) than the uninjected control  than the uninjected control. Sequential injection of rbm24a morpholino and sox2 RNA larvae responded significantly more times (about three out of five) than rbm24a morpholino-injected larvae. However, the rbm24a morpholino and sox2 RNA larvae were also statistically significantly different from the uninjected control larvae. Larvae injected with only sox2 RNA responded similarly (about four out of five times) to the uninjected control  between embryos. It is possible that the transfer of these biomolecules could have played a role in suppressing the microphthalmia phenotype [Recently, a zebrafish TALEN mutant for  defects . In the cultures . From thnockdown A–C’. Ourhenotype . Follow-sox2 overexpression embryos, perhaps stemming from the ubiquitous overexpression of sox2. Ubiquitously expressed exogenous sox2 may be able to substitute for cardiac members of the SOX family including Sox6 which has been shown to be a cardiomyocyte regulator in murine cells, Sox9 which is involved in heart valve development in mice, and Sox17 which has been shown to be essential for specification of cardiac mesoderm in mouse cell cultures [rbm24a/sox2 interactions with specificity to the eye.We occasionally observed cardiovascular defects in the cultures ,30,31. Irbm24a, leads to microphthalmia at low-dose knockdown and microphthalmia with cardiac edema at increased knockdown doses. Additionally, we demonstrated that the microphthalmia induced by rbm24a functionally impacted the visual capabilities of the larvae, as knockdown fish performed worse in a visual behavior study. Previously, we identified sox2 as a target of Rbm24a [sox2 RNA is able to phenotypically and partially functionally suppress the rbm24a knockdown larvae. This shows functional validation of a suspected target RNA by an RNA-binding protein. This work broadly demonstrates the key role RNA-binding proteins and post-transcriptional modification plays during development. It also highlights that genes not associated with disease, such as rbm24a, can impact a well-studied disease-associated gene (sox2), making it critical to better understand post-transcriptional regulation and the potential impacts of RNA-binding proteins and their associated target RNAs.In this study, we demonstrated that knockdown and mutation of the RNA-binding motif protein 24a gene, f Rbm24a . When se


Riboswitches and toehold switches are considered to have potential for implementation in various fields, i.e., biosensing, metabolic engineering, and molecular diagnostics. The specific binding, programmability, and manipulability of these RNA-based molecules enable their intensive deployments in molecular detection as biosensors for regulating gene expressions, tracking metabolites, or detecting RNA sequences of pathogenic microorganisms. In this review, we will focus on the development of riboswitches and toehold switches in biosensing and molecular diagnostics. This review introduces the operating principles and the notable design features of riboswitches as well as toehold switches. Moreover, we will describe the advances and future directions of riboswitches and toehold switches in biosensing and molecular diagnostics. In addition to the regulatory applications, the RNA-based regulatory system, specifically riboswitches and toehold switches, has attracted scientific interest to develop systems for molecular detection applications as biosensing and molecular diagnostics ,2. A ribToehold switches belong to riboregulators which are regulatory molecules that control gene expression in trans through the base pairing with target RNA sequences . Since tThe complicated evolution of infectious pathogens and increasing demand for more efficient, accurate, and affordable detection methods are the main driving force for the recent innovation. The tendency of development moves from the benchtop experimental platform with intensive equipment, qualified professionals, and lengthy waiting time to the portable platform, with POC devices providing simple operation with fast and accurate results ,20,21. MBiosensing and molecular diagnostics are emerging fields with market shares that have dramatically increased over the last few decades. Both fields have huge markets with a forecast for increasing growth over the coming years. According to the industry analysis report, the biosensor global market size was valued at 19.6 billion USD in 2019 . The EurAs the recent progress of the applications in biosensing and molecular diagnostics is remarkable, it is time for reviewing the development so far and the latest advancements. In this review, we focus the discussion on riboswitches and toehold switches in the field of biosensing and molecular diagnostics. We firstly provide a general look at the operating principles and the design strategies of the riboswitch, as well as the toehold switch. Then, we describe the advancements and future directions of these tools in molecular detection.Biosensors are analytical devices used for the detection of an analyte by utilizing the biological entity with a physicochemical detector ,30. The Riboswitches, both natural and synthetic, have so far been considered as a potential approach to overcome the limitations of protein-based biosensors. Riboswitches are quickly synthesized in vitro, flexible in engineering (both aptamers and expression platforms), and can provide a fast response to recognise elements due to the avoidance of complicated protein–protein interactions, even before considering their high specificity and sensitivity ,8,10. A The operating principles of the riboswitch as a biosensor have been described in various reviews ,10. In gFor the transcriptional process, regulation is determined by the completeness of RNA polymerization controlled via a terminator or anti-terminator structure in the riboswitch. In this case, the mechanism is mainly dependent on the interplay between the transcription process and structural conformation of the RNA ,7,9. In Synthetic riboswitches with engineered signaling modules have been developed to increase the diversity of the riboswitch response to different types of metabolites ,7,8,38. However, there are some issues to be considered when riboswitches are used for biosensing design, including the possibility of gene activation by the integration of engineered signaling modules with response domains , the strConventional riboregulators prevent the initiation of translation through sequestration of the RBS sequence. The cis-repressor locating on the 5′ end of target mRNA complementarily binds to its RBS sequence to prevent the translation occurring. In the presence of the trans-activator RNA, the cis-repressor binds, and the RBS sequence is released to allow the translation to occur . HoweverGreen et al. proposed a solution by developing the toehold switch . The toeThe toehold switch possesses the characteristics that make it a potential tool for employment in molecular diagnostics. In comparison with the conventional riboswitch, the toehold switch sequesters the region around the start codon and leaves the RBS and start codon unpaired, which allows the possibility to generate a large number of potential toehold and trigger sequences without any constraints C.In the following paragraphs, we briefly review general design approaches for the use of the riboswitch and toehold switch in applications of biosensing and molecular diagnostics.Owing to their intrinsic ability to directly regulate and interfere with both translation and transcription processes by forming specific structural alternatives, the use of riboswitches has gained significant interest in synthetic biology and metabolic engineering in recent years ,43,48,49Many applications exploit the ligand-binding property of riboswitches for the construction of regulatory systems  and artiThe engineered aptamer domain can be devised from well-studied natural riboswitches, where its characteristics, such as structure, dissociation constant, and mode of action, are well-documented. Moreover, different computational assisted methods for the generation of the initial RNA sequence pool provide greater complexity and uniformity to the aptamers . The iniDepending on applications, riboswitch designs can differ vastly. For translational regulation-based riboswitches, design is rather straight forward. The changes in the RNA secondary structures are reversible throughout the lifetime of RNAs depending on their corresponding ligand-binding states. Therefore, it is primarily controlled through equilibrium thermodynamics. The most stable secondary structure of an RNA can be approximately determined by the minimum energy model, or Turner energy model, which solves the combinatorial optimization problem of all additive contributions of strong, local interactions such as base pairing, base stacking, and looped regions . The parFor transcriptional regulation-based riboswitches, the expression is controlled irreversibly through RNA polymerization interference using a terminator/anti-terminator structure in nascent transcript. Due to the presence of the interplay between structure formation of RNA folding, and rate of transcription, it is proposed that transcriptional regulation-based riboswitches are to a large extent controlled by cotranslational kinetic effects . To desiThe functionality of a riboswitch relies on its ability to form structural alternatives. In a riboswitch context, where folding traps and competing secondary structures are present, it is challenging to predict the functionality and performance of the switch. As riboswitch functionality is strongly affected by thermodynamic interactions, depending on the surrounding structures and sequences, gene expression can vary. To tackle this issue, various tools have been employed to predict plausible RNA folding structures  ,68,69,70Thermodynamic modeling allows us to impose and weigh different criteria for a specific design. However, the current computational assisted thermodynamic modeling has limitations . TherefoToehold switches address fundamental limitations of earlier riboregulator designs, namely low dynamic range, orthogonality, and programmability. By incorporating linear–linear interactions instead of loop–loop and loop–linear interactions, toehold switches are more kinetically and thermodynamically favorable compared to traditional riboregulators . A high A typical design of the synthetic diagnostic system can be divided into three modules: a sensing module, a processing module, and a reporting module A 31]. I. I31]. IAfter defining the desired toehold switch secondary structure, conserved sequences, and interaction domain sizes, the trigger RNA sequence pool which later serves as the templates for toehold domain design is generated and validated with the assistance of computational algorithms, The Nucleic Acid Package (NUPACK) program, a software suitable for the design and analysis of nucleic acid systems, has been employed for the design of many toehold switch-based devices ,77. The RBS-linker upon RNA refolding. However, these modifications also pose a risk of signal leakage [Some modifications could be made to increase the dynamic range of the RNA sensor, such as increasing the loop size of the toehold switch, reduction in hairpin stem length by 1 bp, and removal of some GC bps at the bottom of the stem to decrease the stability of the stem. A reduction in the length of the stem-loop region can also increase the translation rate of the RNA sensor as the stem-loop region can detrimentally affect the ∆G leakage . Further leakage .Escherichia coli  riboswitch controls the termination of Rho-dependent transcription in E. coli , the S-aE. coli , the cycifficile , and theifficile .C. difficile which no longer bind to FMN, instead they bind to the FMN precursor and degradation products [Discoveries showed the dynamic nature of the riboswitch in the regulation of gene expression through its activities or complex interactions with other cellular factors. Genetic-based and computational-based approaches have been deployed to characterize the existence of various classes of natural riboswitches so far ,94,95. Wproducts . These f12 constructed by employing the entire natural riboswitch with different types of output signals was employed for quantifying its concentration and studying the synthesis as well as the import mechanism of this coenzyme B12 in E. coli. The biosensor exhibited an impressive specificity and high sensitivity to the target metabolites [12 with simple yet high-throughput assays and demonstration of the utilization of riboswitch-based biosensors for tracking the metabolite transport [l-lysine-producing E. coli was deployed to illustrate the efficiency of the synthetic RNA system in accelerating the evolution of overproducing strains. The amount of high-productivity strains accounted for up to 75% of total population after four enrichment cycles. This system was also successfully examined for their widespread applicability in the case study of l-tryptophan [Efforts were made to manipulate natural riboswitches. The biosensor of the coenzyme Babolites . Based oransport . A high-yptophan . Even thyptophan .Saccharomyces cerevisiae [S. cerevisiae. The connection of product concentration with GFP fluorescent through riboswitch-based biosensors allows a high-throughput screen of enzyme activity. The combination of FACS and flow cytometry in the screening system resulted in a 33-fold increase of enzyme activity and 22-fold of product selectivity after seven rounds of in vivo screening without any false positive screen [It has been demonstrated that riboswitch-based biosensors are not only deployable in bacteria but are also capable of being utilized in higher organisms, such as the theophylline biosensor in revisiae , TPP andrevisiae ,13,83. Te screen . This sye screen .crcB of Bacillus cereus integrated in the cell-free expression platform for detection fluoride, a contaminant in groundwater, with high efficiency at levels above 2 ppm, the Environmental Protection Agency’s regulatory standard. The system illustrated good detection results both in lab-scale and field conditions with a prolonged storage time using lyophilization [Intensive efforts have been given to develop riboswitch-based sensors to control various mammalian cellular processes . Based olization .S-adenosylmethionine (SAM), adenosine 5′-diphosphate (ADP), TPP, S-adenosyl-L-homocysteine (SAH), cyclic di-GMP, and cyclic di-AMP in bacteria [Although these riboswitches have been employed for various applications, limitations remained due to the gene-based reporter output signal, which can be affected by expression conditions or the need for oxygen in chromophore maturation . Many efbacteria ,105,106.bacteria ,107. Thebacteria . Improvebacteria ,107. Thebacteria . Synthetbacteria ,107.The structural features and operation mechanism of toehold switches were aforementioned. In this section, we focus on the achievements in diagnostic applications so far and how the toehold switch can pave the way to a promising future.The Ebola epidemic, first occurring from 2013–2016 in West Africa and continuing from 2018–2019 in the Republic of Congo, posed a huge threat to human lives. Not far from where the pandemic broke out, in 2014, Pardee and his colleagues developed a portable biosensor that can sense the Ebola virus RNA rapidly in vitro by incorporating a cell-free system on paper based on a prior biosensor to detect a specific RNA in Ebola . The conA similar system was applied in molecular diagnostics one year later when the Zika virus raged through Brazil in early 2015. In this device, a toehold switch-based sensor was developed in the cell-free paper-based platform as before. Moreover, the combination of the isothermal amplification method called nucleic acid sequence-based amplification (NASBA) and the CRISPR/Cas9 system into a NASBA-CRISPR cleavage assay increases the specificity and sensitivity of the system. The selective ability of CRISPR to cleave allows the system to discriminate the RNA sequences between Zika with Dengue and two variants of Zika virus, the African and American Zika strains. Furthermore, the system can detect the Zika virus from the macaque plasma sampled at the concentration of 2.8 fM without the need for thermocyclers . The celFrom the foundation of previous accomplishments, the utility of toehold switch-based biosensors was extended. The synthetic genetic system was demonstrated to analyze up to 10 different species from the gut microbiome in humans via sensing the specific sequence of 16S rRNA in 3–5 h . MoreoveCurrently, the gold standard in molecular diagnostics is real-time reverse transcription-polymerase chain reaction (RT-PCR). With the specificity, sensitivity and high-throughput, the RT-PCR and its derived techniques have been intensively deployed for the detection of different pathogens, especially for virus detection. The RT-PCR has illustrated its efficiency in the diagnosis of various pathogenic viruses, i.e., Zika, Ebola and recently the novel coronavirus (SARS-CoV-2) ,110,111.Since the first development in 2014, with advances in molecular techniques, the sensitivity and specificity of the toehold switch-based detection system have been improved to be comparable with the “gold standard” RT-PCR detection method ,18,26,80The riboswitch and the toehold switch are at their initial stages of commercial applications. The intensive progress has led to their utilities in a variety of fields from metabolic engineering, synthetic biology to molecular diagnostics. However, both riboswitches and toehold switches still have not had a fully developed system that can be deployed in large-scale applications, which exposes a number of possibilities for further studies and developments in the future.The discovery of the riboswitch, a cis-regulatory element, in bacterial cells has led to the implementation of this bioregulator in various fields, especially in biosensing and molecular diagnostics. Several studies have aimed at developing approaches to engineer riboswitches to deploy them in novel applications. Since the use of natural riboswitches to detect specific metabolites in bacterial cells, the engineering of riboswitches has taken significant improvements, which has led to the creation of artificial riboswitches with engineered aptamers and expression platforms, including RNA-based fluorescent biosensors. Their functions have also evolved numerous applications from mutant strain screening to real-time metabolite imaging. Their spectrum of application has widened from prokaryotes to eukaryotes and from in vivo to in vitro. Moreover, unlike protein-based reporting techniques, such as Western blotting, immune-staining, or LC-MS, riboswitch-based reporters can detect metabolites, enzymes, or proteins in living cells without labor-intensive purification steps . Due to The emergence of the toehold switch is a remarkable milestone in the development of the riboregulator, which paved the way for broader and deeper implementations. With the binding ability for any RNA sequence, toehold switches are adaptable with various applications, which is evident through various studies. The compatibility of the toehold switch system with various detection platforms as fluorescent , colorim


IgH) translocations are found in a majority of pPCL cases, supporting a central relation to pathogenesis of pPCL. However, two independent IgH translocations are barely detected at the onset of pPCL, and their significance is yet to be elucidated. Here, we report a case of an aggressive pPCL with simultaneous IGH/MYC and IGH/CCND1 translocations. A 73-year-old man was referred to our hospital with back pain and diagnosed as having pPCL with more than 50% circulating plasma cells. Cytogenetic analysis revealed 47, Y, t  , t  , and +18. IGH/MYC and IGH/CCND1 translocations were confirmed by fluorescence in situ hybridization analysis. Bortezomib and dexamethasone treatment achieved rapid elimination of peripheral malignant plasma cells, and the patient maintained a partial response for 18 months. After biological relapse, he received salvage therapy with ixazomib, lenalidomide, and dexamethasone, followed by pomalidomide and dexamethasone, and exhibited stable disease for an additional 14 months. Although IGH/MYC translocation in association with dysregulation of antiapoptotic pathway leads to worse prognosis in lymphomas, the novel agent-based regimen showed good efficacy, suggesting that IGH/MYC plays a different role in the pathogenesis of MM. IGH/CCND1 and IGH/MYC translocations may have contributed to abrupt onset of pPCL in this case.Primary plasma cell leukemia (pPCL) is an aggressive variant of multiple myeloma (MM). Immunoglobulin heavy chain ( Plasma cell leukemia (PCL) is a rare and aggressive form of multiple myeloma (MM), and the incidence of PCL was reported to be less than 1% of MM cases . In the IgH) gene translocations involving CCND1, FGFR3 and MMEST, CCND3, MAF, and MAFB are important oncogenic pathways observed in about 40% of MM and in 87% of pPCL cases [CCND1 is the most prevalent partner of IgH translocation, detected in 20–71% of pPCL cases [IgH translocations are speculated to be mediated by errors in IgH class switch recombination during B cell maturation, they are considered to be an early oncogenic event [IgH translocations that target MYC are thought to be a very late and progressive event in the course of MM and rarely identified at the onset of MM, especially in pPCL [Recurrent immunoglobulin heavy chain , and free light chain (FLC) analysis demonstrated a slight increase in free λ monoclonal protein. Bone marrow examination revealed an infiltration of atypical plasma cells, comprising 55% of overall cellularity. Cytogenetic analysis revealed 47, Y, t  , t  , and +18 in 2 of the 20 cells studied. On immunohistochemistry, atypical plasma cells were strongly positive for cyclin D1 and partially weakly positive for MYC (IGH/MYC and IGH/CCND1 translocations by fluorescence in situ hybridization (FISH) analysis at the first relapse . Malignant plasma cells rapidly disappeared from peripheral blood after one cycle of Bd therapy. He achieved a partial response after three cycles of Bd therapy. His IgG level was stable at around 1200 mg/dL for 18 months; however, it increased to 3083 mg/dL after 15 cycles of Bd therapy. As second-line therapy, IRd therapy  and 20 mg of dexamethasone weekly) was administered. IRd therapy was effective, and his IgG level decreased to 1380 mg/dL after five cycles. He achieved a partial response again. Unfortunately, he fractured the right femoral neck after 12 cycles of IRd therapy. He also developed severe pneumonia; therefore, we discontinued IRd therapy. Thereafter, bone marrow examination was performed, which revealed increased atypical plasma cells, and additional aberrations, including der (11)t  and der (14)t , were found on cytogenetic analysis . Due to IGH/MYC and IGH/CCND1 translocations at onset that showed a good response to novel agent-based treatment.We report a case of pPCL harboring IgH translocations. In pPCL, IgH translocations were observed in 87%, and CCND1 was the most common translocation partner observed in 20–71% [IGH/CCND1 translocation in the pathogenesis of pPCL. Recently, as high BCL2/BCL-XL and MCL-1 expression ratios were reported in t  MM, IGH/CCND1 translocation may contribute to plasma cell survival by altering the antiapoptotic pathway [There are two main types of primary cytogenetic abnormalities in MM: hyperdiploidy and primary n 20–71% , supportMYC is dysregulated or overexpressed in most human cancers and contributes to cell activation, cell proliferation, and apoptosis [MYC is involved in the progression from MGUS to MM [MYC is dysregulated by genetic rearrangements, such as translocations and amplifications, and is also modulated by deregulation of upstream pathways such as IRF4, DIS3/LIN28B/let-7, or MAPK [poptosis . In MM, US to MM  and alsoUS to MM . MYC is  or MAPK .MYC is speculated to be a late and progressive event [IgH with MYC and CCND1 were reported in only six cases of newly diagnosed MM, and this is the second case in pPCL [Ig translocation with ve event  and has ve event . To our  in pPCL , 13–16. MYC and BCL2 is recognized as a distinct entity under the WHO 2016 and known to have very poor prognosis [BCL2 may counteract the proapoptotic function of MYC, resulting in rapid cell growth and resistance to therapy [MYC and CCND1 in pPCL is not known, especially when treated with novel agent-based regimens. Despite leukemic presentation, frailty, as well as MYC and CCND1 rearrangements, doublet and triplet regimens of novel agents had good efficacy in the patient and responded to treatment for more than 30 months after diagnosis. Recently, it was reported that IGH/MYC translocations in MM have extragenic IGH breakpoints, which are distinct from other B cell malignancies that usually have breakpoints in IGH switch regions [IGH/MYC-possessing MM.Double-hit lymphoma with rearrangements of rognosis . Concomi therapy . HoweverD genes  were ectopically expressed in almost all MM cases from an early stage, implying a unifying and initial oncogenic event in MM [CCND1 and MYC to the IgH gene enhancer may lead to simultaneous activation of initiating and progressive oncogenic pathways, resulting in abrupt onset of aggressive pPCL.Interestingly, a previous study observed that cyclin nt in MM . In thisIGH/MYC and IGH/CCND1 translocations over time during treatment, which suggested that MYC and CCND1 signaling play a role in disease progression. There were discrepancies between cytogenetic analysis and FISH analysis in the time period and number of additional translocations. These may reflect the high sensitivity of FISH analysis in detecting chromosomal translocations [We also observed the accumulation of ocations .IGH/MYC and IGH/CCND1 successfully treated with novel agent-based regimens.Here, we described a case of double-hit primary plasma cell leukemia with MYC and CCND1 signaling in the pathogenesis of pPCL for further understanding and identifying new therapeutic targets of pPCL.It is important to evaluate the