P < 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest.Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA , whereas EV-D was notably enriched in human RNA . As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV,  In recent years, emerging methods based on liquid biopsy have shown great potential for disease diagnosis, therapeutic guidance, and recurrence monitoring ,2. AmongAmong EV-focused liquid biopsies, saliva offers unique advantages due to its noninvasiveness, ease of collection, cost-effectiveness, and safety . MoreoveCFS), EV-depleted saliva (EV-D), EXO, and MV from ten healthy volunteers. In this way, we identified common and unique characteristics of the RNA profiles across different saliva fractions, which provides a better understanding of specific RNA profiles associated with each saliva fraction. Thus, our work would offer valuable guidelines for researchers in preparing and selecting the most suitable saliva fraction for their biomarker studies, based on specific RNA of interest. This knowledge would contribute to enhancing the accuracy and effectiveness of saliva-based biomarker investigations.Despite the advantages of saliva EV as potential disease biomarkers, advancement in this field is hampered by major technical obstacles such as the lack of standardized protocol for RNA profiling, and guidance on saliva sample processing for biomarker analysis. To address these issues, we established an improved protocol for efficient isolation of saliva RNA and further performed small RNA-seq profiling to characterize multiple saliva fractions including cell-free saliva  were determined by RT-qPCR. As shown in 2FC of 1.4  from ten healthy volunteers. First, we determined the relative distribution of human and microbiome RNA from these individuals . Human RSpirochaetes, Actinobacteria, Fusobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. Interestingly, the microbiome composition was considerably variable among individuals as well as across saliva fractions. Specifically, CFS and MV had higher percentages of Bacteroidetes compared with EV-D and EXO, representing 36.3% and 43.2% of total microbiome reads, respectively. In contrast, EV-D and EXO had higher percentages of Firmicutes , whereas Proteobacteria was similarly expressed across all four saliva fractions (28.4% on average).Given the abundance of microbiome reads in our sequencing data, we further compared the taxonomic characteristics of microbiome in specific saliva fractions. As shown in r = 0.96–0.98 among the ten individuals), whereas the lowest correlation was found between EV-D and MV (r = 0.91–0.98). Next, we analysed specific microbiome phyla that were differentially expressed across these fractions. Bacteroidetes was identified as the most prevalent phylum in MV as compared to EV-D , long non-coding RNA (lncRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and other non-coding RNA (ncRNA) . CFS hadNext, we performed differential expression analysis by comparing the RNA profiles of all fractions across individual subjects . Comparer values of 0.88 on average.As previously described, miRNAs were significantly enriched in EXO. We further investigated the miRNA profiles across the four saliva fractions. First, we performed pairwise correlative analysis of miRNA expression profiles, with correlation coefficients between fractions or individuals ranging from 0.75 to 0.98 . Among aNext, we evaluated unique and overlapping miRNAs in the four fractions among 200 most abundantly expressed miRNAs . We obseCompared with the EV fractions, CFS and EV-D showed significantly higher levels of tRNA expression . InteresWe further categorized tRFs based on their matched amino acid. Glutamate tRFs were predominant in EXO and MV, representing 58.4% and 54.4% on average, respectively ; glycineNext, we categorized tRFs based on fragmentation patterns. We found that 5’-half tRF was the most abundant type across all four fractions, followed by 5’-tRF . EXO andAs shown in Bacteroidetes were the most abundant phylum in CFS and MV, whereas Firmicutes were most abundant in EV-D and EXO. Bacteroidetes and Firmicutes were the main phyla presented in human saliva [Bacteroidetes were negatively correlated with disease activity in ulcerative colitis, and Firmicutes levels in saliva reflected the gut condition [Firmicutes/Bacteroidetes ratio could serve as an indicator of obesity development [In this study, we have developed an improved small RNA-seq protocol for profiling different saliva fractions. Compared with other fractions, MV was highly enriched in microbiome RNA, which could be explained by the pelleting of bacterial cells during MV isolation . Interesn saliva . It has ondition ,16. In aelopment . Thus, tAs for human RNA composition, we found that rRNA was the most abundant RNA species across all saliva fractions, especially in EXO. Many studies reported the enrichment of rRNAs in EXO, which is consistent with our findings ,19. In oThe miRNA expression profiles in EXO have been intensely studied in recent years and many studies indicate that exosomal miRNAs could be used as biomarkers for human diseases. Compared with other saliva fractions, miRNAs were most abundant in EXO. The miRNA expression profiles of EXO and MV are highly correlated. Although EXO and MV differ in many aspects, they both feature EV-specific characteristics, such as membrane-bound vesicles, cargo selection for packaging, and cargo transport across cells . For theIn contrast to miRNAs, snoRNAs were significantly enriched in CFS and EV-D as compared with the EV fractions. snoRNAs are typically found in the nucleoli and are mainly responsible for rRNA posttranscriptional modification and maturation . InteresSimilar to snoRNAs, tRNA fragments (tRFs) were also found enriched in CFS and EV-D. The main role of full-length tRNAs is to act as key adaptor molecules for peptide elongation during translation. We found these tRNA reads were all derived from tRFs in our small RNA-seq libraries. tRFs are generated by specific cleavage of primary and mature tRNAs. Among all tRFs, two major subgroups including 5’- half tRFs and 5’-tRFs were most abundant in saliva fractions. Increasing evidence indicated that tRFs play important functions in human disease development ,33. ThusPrevious studies revealed that yRNAs and yRFs play important roles in disease development . yRNAs aWhile we provided a comprehensive analysis of distinct small RNA profiles in four saliva fractions, there are some limitations in this study. Firstly, we did not compare differential ultracentrifugation with other commercial EV isolation methods. To date, differential ultracentrifugation is widely considered as the gold standard for EV isolation, and it would be important to compare its performance with alternative methods. Although our primary focus was on RNA isolation, future studies should consider evaluating and comparing various EV isolation methods. Secondly, we only focused on relative changes in small RNA profiles across different saliva fractions, without performing absolute RNA quantitation. To this end, incorporating spike-in controls in future studies would enable more precise comparisons for absolute quantitation across different saliva fractions. Additionally, we did not analyse the mRNA species in this study. Our focus was on small RNA species by small RNA-seq analysis. Thus, data on mRNA from our sequencing results were limited; total RNA-seq in future studies would be helpful to delineate the expression profiles of mRNA or other long RNA species, thereby providing a complete picture of all RNA species in saliva.In summary, we have established an improved small RNA profiling protocol for saliva study. Based on our findings, saliva, especially the MV fraction, may serve as a useful resource for oral microbiome study; EXO or EV-D, are more suited for profiling human RNAs; CFS can be an optimal resource for balanced study of both human RNAs and microbiomes. This study presents unique characteristics of various circulating RNAs in saliva, which provides a guideline on selecting specific saliva fractions for targeted biomarker discovery.All the participants provided written informed consent to participate in the study. This study was approved by the Institutional Review Board of the University of Illinois Chicago.g for 15 min at 4°C to remove cells and debris. Cell-free saliva supernatant was then used for EXO and MV isolation or direct extraction of total RNA.Saliva was collected from ten healthy volunteers who were asked to refrain from drinking, eating, or chewing anything for at least 30 min before sample collection. For each volunteer, about 5 mL of unstimulated saliva was collected, and then mixed with 1 mL of RNAlater (Thermo Fisher). Two sample processing methods were assessed for initial treatment of saliva. Specifically, an aliquot of saliva was treated with either RNAlater or RNase Inhibitor (Thermo Fisher), and then centrifuged at 2600 × g for 1 hr at 4°C to pellet MVs. The MV-free supernatant was then transferred to a new tube and ultracentrifuged at 100,000 × g for 1 hr at 4°C to pellet EXOs. In this way, 1 mL of EV-depleted saliva (EV-D) was collected along with palleted MVs and EXOs for further RNA isolation. The size and concentration of four saliva fractions were measured by nanoparticles tracking analysis (NTA) with Nanosight NS300 .EVs were isolated by differential ultracentrifugation . In brieg at 4°C. The upper aqueous phase was transferred into a new tube and another 140 µL of chloroform was added, followed by incubation and centrifugation as mentioned above. Then, 1.25× volume of 100% ethanol and 1 µg of glycogen were added to the upper aqueous phase in the second repetition. The mixture was passed through MinElute spin column by centrifugation at 8,000 × g for 15 s. The column was then washed with 700 µL Buffer RWT, 500 µL Buffer RPE, and 500 µL 80% ethanol sequentially by centrifugation at 8,000 × g for 15 s and finally eluted with 10 μL of RNase-free water at 12,000 × g for 1 min.RNA from the four saliva fractions described above  was extracted using Trizol LS (Invitrogen), miRVana (Ambion), MagMAX (Applied Biosystems), Nucleospin (Takara), and miRNeasy Micro (Qiagen), respectively, following the manufacturers’ instructions. The modified miRNeasy method was performed according to the manufacturer’s protocol with specific modifications including glycogen co-precipitant and a second round of chloroform extraction. Briefly, 700 µL of the QIAzol Lysis reagent was added to 140 µL of sample and the mixture was vortexed and incubated at RT for 5 min. Then, 140 µL of chloroform was added to each sample and vortexed for 15 s. The mixture was incubated at RT for 3 min, followed by centrifugation for 15 min at 12,000 × Total RNA was quantified using NanoDrop spectrophotometer . RNA purity was assessed by checking the absorbance at 260 nm, 280 nm and 230 nm. To access the RNA integrity, the RNA Integrity Number (RIN) was determined using a 2200 Tapestation Instrument with RNA ScreenTapes  .To compare the efficiency of RNA isolation by different methods, RT-qPCR was used to quantify the yield of ten representative miRNAs, including miR-30d, miR-191-5p, miR-27a, miR-26a-5p, miR-21-5p, miR-24-3p, let-7a-5p, miR-92-3p, miR-148a, and miR-320a. The primers used are listed in Supplementary Table S3. Detailed procedures were carried out as described in our previous study . In brieSmall RNA-seq library preparation was performed using the NEBNext Small RNA-seq Library Preparation Kit with some modifications as previously described . Briefly2-transformed reads were used to calculate the correlation coefficients across all saliva fractions. Statistical analyses of differentially expressed miRNAs were performed using paired t-test. A p-value ≤0.01 with a log2 fold change (FC) ≥1 or ≤-1 was considered statistically significant.Raw FASTQ RNA-seq data were preprocessed, including trimming of adapter sequences and filtering of low-quality reads, as detailed previously . SubsequClick here for additional data file.


N6-methyladenosine (m6A) and C5-methylcytosine (m5C) have been most broadly studied in different RNA viruses. Various studies, however, reported varying results with regard to number and extent of the modification. Here we investigated the m5C methylome of SARS-CoV-2, and we reexamined reported m5C sites in HIV and MLV. Using a rigorous bisulfite-sequencing protocol and stringent data analysis, we found no evidence for the presence of m5C in these viruses. The data emphasize the necessity for optimizing experimental conditions and bioinformatic data analysis.The addition of chemical groups to cellular RNA to modulate RNA fate and/or function is summarized under the term epitranscriptomic modification. More than 170 different modifications have been identified on cellular RNA, such as tRNA, rRNA and, to a lesser extent, on other RNA types. Recently, epitranscriptomic modification of viral RNA has received considerable attention as a possible additional mechanism regulating virus infection and replication.  The SARS-CoV-2 pandemic has created an immense surge of virus-related research activity. Large efforts have been directed toward the elucidation of the structure of the SARS-CoV-2 genome as well as the RNA and protein products made from it. Nine dominant subgenomic (sg) sense RNAs are transcribed from the ∼30 kb long positive-strand genomic RNA, all of which share the same 3′UTR and have a poly(A) tail that can vary in length . Most buN6-methyladenosine (m6A) appearing as the most prevalent type (5C) among others have been reported  of human coronavirus HuCoV-229E RNA reported numerous putative m5C sites distributed along sgRNAs  and murine leukemia virus  protein that was reported to contain eight methylated Cs (5C in this region (Asp were readily detected with our protocol (Treatment of isolated RNA with bisulfite leads to the deamination of cytosine (C) to uracil (U) at controlled pH and temperature conditions, while C that is methylated at the carbon 5 atom is largely protected from this reaction . Sequencructures . Moreoveructures . Thus, tlated Cs  for anallated Cs A. Total s region B,C. In cprotocol C.5C pattern at a transcriptome-wide level and with higher coverage compared to the PCR protocol, we subjected isolated poly(A) RNA from infected Vero E6-TMPRSS2-ACE2 cells to bisulfite treatment, library preparation and deep sequencing. Agilent Bioanalyzer results of the poly(A)RNA revealed strong enrichment of the abundant viral sgRNAs demonstrating robust infection of the cells . To control for the efficiency of C-to-U conversion, unmethylated ERCC standard RNA was spiked into the samples before bisulfite treatment and deep sequencing. For a rigorous analysis of the sequencing data, we applied a modified version of the meRanTK software (version 1.3.0). Thereby, the meRanCall module of the original analysis workflow . The 3′end in particular was covered by a large number of reads reflecting the transcription of sgRNAs from this part of the genome  protocol  produced viral RNAs, only in our study full replication of the virus occurred with production of infectious viral progeny and secondary infection. However, we also examined the gag-pol region in a different HIV isolate (D117/II) and received the same negative result .HIV and MLV are two other RNA viruses for which cytosine methylation was reported recently. In the case of HIV, mprotocol  or a bisprotocol . While tus study . The res HIV RNA B. These 5C peaks, only five high confidence sites were identified using bisulfite-sequencing . To better understand the reason for the discrepancies, we reanalyzed the original bisulfite-sequencing data sets (5C sites in the host transcriptomes of MLV and HIV infected cells (present in two replicates), respectively, including the well-characterized methylation sites in mitochondrial 16S rRNA , no m5C was detected in the reported viral RNA regions. These results support our experimental analyses in the respective viruses using PCR-bisulfite sequencing  and/or m5C readers (ALYREF) perturb virus replication  are inherently error prone.In this study, we have revisited the question of whether RNA viruses, such as SARS-CoV-2, HIV or MLV are subject to cytosine methylation. Using a rigorous bisulfite-sequencing protocol and stringent data analysis, we found no evidence for epitranscriptomic modification of viral RNA by cytosine methylation in any of the examined viruses. Our findings emphasize the necessity for the implementation of stringent experimental standards when studying m7 cells were seeded in a 175 cm² cell culture flask 1 d prior to infection. The cells were infected with a SARS-CoV-2 D614G isolate  with an MOI of 0.001 and harvested after 48 h. PM1 cells  were cultured in RPMI-1640 Medium (Sigma) supplemented with 5% fetal calf serum and 2% L-Glutamine (Gibco). 2 × 106 PM1 cells/well were seeded in a six-well plate and directly infected with replication competent HIV NL4-3 or HIV-1 D117/II isolate. Cells were harvested 48 h post infection. Mus dunni cells were cultured in RPMI-1640 Medium (Sigma) supplemented with 10% fetal calf serum and 2% L-Glutamine (Gibco). 1 × 106 cells/well were seeded 1 d prior to infection in a six-well plate. The cells were infected with an MOI of 0.1 Friend Murine Leukemia Virus (FMLV) and harvested 48 h post infection.African green monkey Vero E6 cells stably overexpressing TMPRSS2 and ACE2 (Vero E6-TMPRSS2-ACE2) have been generated as described previously  and wereFor transcriptome-wide analysis of SARS-CoV-2 methylation, poly(A) RNA was prepared from infected Vero E6-TMPRSS2-ACE2 cells using the NEBNext mRNA Magnetic Isolation Module (NEB), followed by two additional rounds of poly(A)RNA enrichment by Dynabeads (Ambion) according to the manufacturer's protocol. Bisulfite treatment was performed as described previously  using thAsp (positive control) were amplified and sequenced in parallel. Primer sequences are listed in Supplemental Table S2.Total RNA was extracted from virus-infected cells using TRIzol (Sigma-Aldrich) and digested with DNase I (NEB) for 20 min at 37°C followed by purification using phenol chloroform extraction and isopropanol precipitation. An amount of 1.2–1.5 µg total RNA was used for bisulfite treatment exactly as described above. cDNA synthesis was performed by the GoScript Reverse Transcription System (Promega) with the addition of 1 µg custom made random hexamers lacking the C base followed by PCR amplification using bisulfite compatible primers of the following genomic RNA regions: SARS-CoV-2 regions 28893–29051 and 29114–29274, HIV regions 2056–2200 and 9055–9256, MLV regions 303–468 and 6906–7040. Amplicons were cloned into pGEM-T vector (Promega) and plasmid DNA from individual colonies was analyzed by Sanger sequencing. To determine deamination efficiency, spike-in ERCC sequence 136 (negative control) and human tRNAChlorocebus sabaeus genome (Ensembl v106) extended with the SARS-CoV2 genome  and ERCC sequences using meRanGs from our previously published meRanTK software collection  to include a C-cutoff parameter, a signal-to-noise cutoff filter and a gene-specific methylation rate calculation. The following parameter settings were used: allowed duplicates <20 (-md 20), coverage >20 reads (-mcov 20), C-cutoff of 3 (-C_cutoff 3), signal-to-noise rate threshold 0.9 (-snr 0.9), methylation rate >0.2 (-mr 0.2). To determine statistical significance  of the m5C calls, we used the gene-specific conversion rate (-gcr). In case the m5C position was not within gene regions defined in the GTF annotation files from Ensembl, we used the conversion rate determined by the unmethylated ERCC sequences.Raw BS-seq reads were first cleaned from sequencing adapter contaminants using Trimgalore and aligned to the llection . For m5CFor reanalysis of the published BS-seq data, MLV (GSE139606) and HIV (GSE157193) data sets were downloaded from the Gene Expression Omnibus (GEO) database and processed exactly as described above.BS-seq data have been deposited with the Short Read Archive (SRA) database under BioProject accession number PRJN908293.Supplemental material is available for this article.


Hundreds of different species of small RNAs can populate a bacterial cell. This small transcriptome contains important information for the adaptation of cellular physiology to environmental changes. Underlying cellular networks involving small RNAs are RNA–RNA and RNA-protein interactions, which are often intertwined. In addition, small RNAs can function as mRNAs. In general, small RNAs are referred to as noncoding because very few are known to contain translated open reading frames. In this article, we intend to highlight that the number of small RNAs that fall within the set of translated RNAs is bound to increase. In addition, we aim to emphasize that the dynamics of the small transcriptome involve different functional codes, not just the genetic code. Therefore, since the role of small RNAs is always code-driven, we believe that there is little reason to continue calling them small noncoding RNAs. Recently, the explosion of metatranscriptomic analysis of complex, often non-culturable, microbial populations sampled in diverse environments has revealed a universe of unassigned sequencing reads amounting to about 50% of total non-ribosomal RNAs. This large fraction of RNAs may consist of mRNAs coding for unknown proteins, RNA regulatory elements, or RNA viruses. It was predicted that a significant portion (about 20%–30%) of these orphan RNA sequences could be assigned to small RNA (sRNA) families , where sBy base-pairing, sRNAs can influence the translatability and/or stability of target mRNAs. sRNAs can also interplay with RNA-binding proteins (RBPs), modulating, e.g., by sequestration or acting as guides, their regulatory activity. Reciprocally, RBPs can influence sRNA stability and expression. The regulatory activity of sRNAs can also be challenged by other RNAs, the so-called RNA sponges, which allow the coordinated expression of different target genes. Therefore, small RNAs have several modes of action for regulating gene expression . BecauseIn the past few years, protocols based on RNA-seq have revolutionized our approach to studying the transcriptome dynamics of prokaryotes . ReferriIn this perspective paper, we aim to highlight the complexity of small transcriptome functional codes that are emerging from recent approaches and the challenges of integrating their results to provide a more detailed description of the cellular networks in which sRNAs are involved.per se as global translation regulators by direct binding to mRNAs.In the last few years, high-resolution transcriptome mapping obtained with different techniques based on RNA-seq, both in several longstanding and emerging model bacteria, has revealed the presence of many species of sRNAs  other thEscherichia coli and Salmonella enterica, as well as in a broad range of bacterial species, suggest that the prevalent mode of sRNA regulatory action is through base-pairing, either negatively or positively regulating the translation and/or stability of multiple trans-encoded mRNAs (E. coli (Many studies in the well-characterized ed mRNAs . Althouged mRNAs . Therefoed mRNAs , there ied mRNAs . The keyed mRNAs  that hased mRNAs . The pro(E. coli  and sugg(E. coli . The Hfq(E. coli . The pos(E. coli . This ma(E. coli .Taken together, all these aspects suggest the presence of a multifaceted “RNA interacting code” that governs the Hfq hub and makes it very versatile and capable of multiple intertwined switches. The functional interaction of a significant portion of the small transcriptome with Hfq is a good example of the need to better understand the mechanisms behind the regulatory roles of sRNAs.E. coli have begun to unravel this complexity. By considering only the Hfq function of RNA matchmaker, Two recent pieces of work in With the combination of these pieces of information, we can envisage an extraordinary multi-gate device  where: (In this view, for an sRNA molecule to regulate through Hfq-assisted base-pairing, it must win the competition for the proximal face and be timely enough to find a target mRNA molecule bound to the distal face. Therefore, the competition among sRNAs for the Hfq proximal face can be an important gate of the base-pairing mechanism. In addition, a Class II RNA could further interfere with the base-pairing mechanism by reducing the likelihood of a target mRNA being bound to the distal face.Furthermore, the mRNA displacement activity of Class II sRNAs may be an important player in modulating Hfq-mediated regulation of mRNA translation and stability. In an mRNA-specific manner, we can speculate that Class II sRNAs may be able to increase mRNA stability either directly, by challenging the recruitment of RNase E, or indirectly by alleviating the translational repression operated by Hfq, resulting in the protection of the mRNA from RNase E by translating ribosomes. Conversely, Class II sRNAs may also reduce the stability of certain mRNAs if Hfq binding plays the role of protecting them from degradation. It might be supposed that Class I sRNAs are excluded from this type of regulation. However, by competing with a Class II sRNA for the Hfq proximal face, Class I sRNAs may also regulate the translation and stability of non-target mRNAs.In summary, according to this view of the Hfq hub, the sRNA-mediated regulation of mRNAs can be conveyed from the proximal to the distal face of Hfq with a mixed code of RNA–RNA and RNA-protein interactions. The decoy function of Class II sRNAs at the distal face would appear to be a key element in the modulation of Hfq mRNA binding activity and thus in the determination of gene regulation effects.E. coli, the activity of CsrA is mainly modulated by the sRNAs CsrB and CsrC, while Pseudomonas aeruginosa has two paralogs, RsmA and RsmN, and the four sRNAs RsmV, RsmW, RsmY, and RsmZ that function as antagonists. Thus, if the Hfq hub can be envisioned as multi-gated, the CsrA/RsmA RBPs support only one mechanism of competition, that between sRNAs and mRNAs for binding to the protein, which could be likened to what occurs at the distal face of Hfq. The antagonist sRNAs mentioned appear to be exclusively dedicated to the regulation of CrcA/RsmA orthologs by protein titration, and no base-pairing activity with mRNAs is known. However, sRNAs were discovered to perform both functions. In E. coli, McaS was characterized as an Hfq-dependent sRNA base-pairing to some targets (E. coli GadY, Spot 42, GcvB, and MicL, which are Hfq-dependent base-pairing sRNAs, were found to bind CrsA with high affinity (The small transcriptome also interacts with other RBPs, such as those in the CsrA/RsmA family  of small targets . McaS al targets . More reaffinity . Like Mcaffinity . Interesaffinity . This suaffinity .The above examples represent the set of rules, or codes, that govern the function of sRNAs as regulators of gene expression through base-pairing and protein-binding. Besides, the genetic code can also be used. Indeed, about ten sRNAs were shown to be dual-function, i.e., both regulators and mRNAs . The smaE. coli as an example (Taking a recent ribosome profiling approach with stalled initiation complexes in  example , many neE. coli, but it may be similar in other species, it is noteworthy that one-third of the small proteins are estimated to be localized in the cytoplasmic membrane (E. coli small membrane protein YohP and its smRNA (Furthermore, at least in membrane . Althougmembrane , recent membrane . This cots smRNA . Future Never more than in the last few years have we come to realize that the small transcriptome performs its function of regulation of gene expression through a combination of RNA–RNA and RNA-protein interactions. Moreover, some sRNAs can also be smRNAs. Recent global approaches to RNA biology  allow usE. coli (lacZ led to the conclusion that Spot 42 did not function as an mRNA (To address these questions, a major future challenge will be to compare the different functional datasets to discover their degree of overlap . From thE. coli , is veryE. coli . However an mRNA . In 2022 an mRNA . The sug an mRNA . Besides an mRNA . Therefo an mRNA , or how In summary, in the small transcriptome resides an important share of code-driven information necessary for the adaptation of cellular physiology to environmental changes. Traditionally, sRNAs are generally considered to be noncoding, since only a few are known to contain translated ORFs. In this Perspective, we have tried to show that the number of sRNAs belonging to the translatome is likely to increase. In addition, the dynamics of the small transcriptome involve other functional codes and not only the genetic code. Therefore, we suggest a multi-code-driven functionality of sRNAs that would render the adjective noncoding misleading, as it only refers to “no coding for a protein.” Consequently, we believe that there is little reason to continue to call them noncoding sRNAs. Here, we have been focusing on sRNAs. However, given the generalized presence of multiple functional codes in RNA, sometimes coexisting on the same molecule, we like to think that in the future it will be avoided the use of the adjective noncoding even for RNAs such as rRNAs, tRNAs, miRNAs, and siRNAs, etc., which are very often belittled as such.SF, TB, and GB collaborated to conceptualize and write the manuscript. All authors contributed to the article and approved the submitted version.The research on sRNAs in the laboratory led by GB has been supported in recent years by the “Fondazione per la Ricerca sulla Fibrosi Cistica,” Verona, Italy, grant FFC#10/2020, with the contribution of Delegazione FFC di Firenze and Delegazione FFC di Prato, and grant FFC#14/2021 with the contributions of Delegazione FFC Ricerca Brindisi Torre, Delegazione FFC Ricerca di Prato, and Emanuela Cricri e Amici della Ricerca. The authors acknowledge support from the University of Milan through the APC initiative.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of this meta‐analysis was to summarize the available results of immunotherapy predictors for small cell lung cancer (SCLC) and to provide evidence‐based information for their potential predictive value of efficacy.We searched PubMed, EMBASE, Web of Science, The Cochrane Library, and ClinicalTrials . The hazard ratios (HR) and its 95% confidence intervals (CIs) and tumor response rate of the included studies were extracted.p = 0.54), with high heterogeneity ; disease control rate [DCR] , with high heterogeneity ; overall survival (OS) ; and progression‐free survival (PFS) , with high heterogeneity . TMB‐High (TMB‐H): OS ; PFS . Lactate dehydrogenase (LDH) >upper limit of normal (ULN): OS . Asian patients: OS ; White/Non‐Asian patients: OS . Liver metastasis patients: OS ; PFS . Central nervous system (CNS) metastasis patients: OS ; PFS .Eleven studies were eventually included and the pooled results showed that programmed cell death ligand 1 (PD‐L1) positive: objective response rate (ORR)  in SCLC patients. LDH, baseline liver metastasis and CNS metastasis may be used as markers/influencing factors for predicting the efficacy of ICIs in SCLC patients. Non‐Asian SCLC patients had better efficacy with ICIs in our results. Immune checkpoint inhibitors (ICIs) are essentially humanized monoclonal antibodies that target programmed cell death protein 1 (PD‐1), programmed cell death ligand 1 (PD‐L1), or cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4) (CD152) to activate T cells and promote the immune system to recognize and attack cancer cells.Many potential biomarkers/influencing factors related to the efficacy of ICIs have been proposed, with PD‐L1 and tumor mutation burden (TMB) being the most frequently studied. PD‐L1 is a ligand for the programmed cell death 1 (PD‐1) receptor.22.1This meta‐analysis was performed following the PRISMA guidelines according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses Protocols (PRISMA‐P) statement. In addition, this meta‐analysis was registered on the PROSPERO database (PROSPERO ID: CRD42022291538). Ethics approval is not required, as this study involved published studies.Two reviewers (LLL and YCF) independently searched the following databases from January 1, 1975 to November 1, 2021: PubMed, EMBASE, Web of Science, and The Cochrane Library. Unpublished materials searched through the following databases from January 1, 1975 to November 1, 2021: ClinicalTrials. The search strategies in the electronic databases are listed in Table 2.2Two reviewers (LLL and YCF) independently selected articles. Disagreements or uncertainties were resolved by the investigators (XHT and CZ).2.2.1Patients with pathologically confirmed SCLC;Prospective research;ICI therapy was applied;At least one biomarker of PD‐L1, TMB, LDH, liver metastasis, central nervous system (CNS) metastasis.Studies published in English.2.2.2Cell experiments, animal research, reviews, summary, case reports, commentaries, letters;Studies with insufficient data;Studies with less than 10 patients;Keep articles with the most recent available data from multiple publications of the same study.2.3Two reviewers (LLL and YCF) independently extracted data using a predesigned data extraction form. The results were compared by the investigators (XHT and CZ), and resolved any disagreements or uncertainties. Extracted details include study characteristics , enrollment period, interventions (types of treatment), and results. We collected hazard ratios (HR) and 95% confidence intervals (CIs) to assess OS and PFS; and response rates or number of people per item to assess objective response rate (ORR) and disease control rate (DCR).2.4Overall survival (OS)Progression‐free survival (PFS)Objective response rate (ORR)Complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD).(4) DCR2.5Two reviewers (LLL and XHT) independently assessed the risk of bias for all candidate studies. Disagreements were determined by the investigators (JBH and CYF). Randomized controlled trials (RCTs) were evaluated with Cochrane Collaboration's tools, and single‐arm or multi‐arm nonrandomized trials used the methodological index for nonrandomized studies (MINORS).We evaluated RCTs through the following items and each item was classified as high, low, or unclear: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other bias. In the MINORS, there are 12 items: 1–8 items: A clearly stated aim, inclusion of consecutive patients, prospective collection of data, endpoints appropriate to the study aim, unbiased assessment of endpoint, follow‐up period appropriate to the major endpoint, loss to follow‐up no more than 5%, prospective calculation of the sample size. 9–12 items: A gold standard intervention in the control group, contemporary groups, baseline equivalence of groups, and adequate statistical analyses. The first 8 items were used to evaluate single‐arm studies and all 12 items were used to evaluate comparative studies. Each item was scored as 0 (not reported), 1 (reported but inadequate), or 2 (reported and adequate).2.6p > 0.10, I2 < 50%) and the random effects model was used when heterogeneity was significant . The heterogeneities were explored by using subgroup and sensitivity analyses. Begg's and Egger's tests were used to determine publication bias.Statistical analyses were performed using Review manager 5.3 software and Stata 14.0. HR and 95% CI were pooled to evaluate the OS or PFS. Dichotomous variables were expressed as relative risk (RR) to evaluate ORR or DCR. Heterogeneity between studies was assessed by the Cochran's Q test. The fixed effects model was used when heterogeneity was acceptable  were included.Quality assessment tools were used with Cochrane collaboration's tools and MINORS, respectively. Since the CheckMate 032 study3.33.3.1p = 0.54), with high heterogeneity , a random‐effect model was used , with high heterogeneity , a random‐effect model was used . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in patients with PD‐L1 negative compared with control treatment . With low heterogeneity , a fixed‐effect model was used . With high heterogeneity , a random‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was not significant in patients with PD‐L1 negative compared with control treatment . With high heterogeneity , a random‐effect model was used . With acceptable heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in patients with low TMB (TMB‐L) compared with control treatment . With low heterogeneity , a fixed‐effect model was used . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in patients with TMB‐L compared with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Two studies explored the association between TMB and PFS. The pooled results indicated that the clinical benefit of ICIs was significant in patients with TMB‐H compared with control treatment . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in patients with LDH ≤ ULN compared with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Three studies explored the association between LDH and OS. The pooled results indicated that the clinical benefit of ICIs was not significant in patients with LDH > ULN compared with control treatment . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in White/Non‐Asian patients compared with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Four studies explored the association between race and OS. The pooled results indicated that the clinical benefit of ICIs was not significant in Asian patients compared with control treatment . With acceptable heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in non‐liver metastasis patients compared with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Five studies explored the association between liver metastasis and OS. The pooled results indicated that the clinical benefit of ICIs was not significant in liver metastasis patients compared with control treatment . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in non‐liver metastasis patients compared with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Two studies explored the association between liver metastasis and PFS. The pooled results indicated that the clinical benefit of ICIs was not significant in liver metastasis patients compared with control treatment . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in non‐CNS metastasis patients compared with those with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Four studies explored the association between CNS metastasis and OS. The pooled results indicated that the clinical benefit of ICIs was not significant in CNS metastasis patients compared with those with control treatment . With low heterogeneity , a fixed‐effect model was used. The pooled results indicated that the clinical benefit of ICIs was significant in non‐CNS metastasis patients compared with those with control treatment . With low heterogeneity , a fixed‐effect model was used in Figure Two studies explored the association between CNS metastasis and PFS. The pooled results indicated that the clinical benefit of ICIs was not significant in CNS metastasis patients compared with control treatment , disease control: 7 (25%), and disease progression: 21 (75%). We discussed and changed the total number of IC0 (n = 29) to 28, and this change did not affect the heterogeneity of the DCR results in this study. There were two papers that included the KEYNOTE‐158 study,Before discussing the results of PD‐L1, we would like to describe the parts of the included studies that need to be highlighted. First, only two of the included studies had extractable DCR data and five studies had extractable ORR data. Three results of Anish Thomas et al.Although study has shown that positive expression of PD‐L1 is associated with better OS in SCLC patients,In terms of immune response mechanisms, MTB‐H should be associated with better efficacy. TMB‐H indicates the presence of more neoantigens in the tumor microenvironment, promoting an inflammatory response that leads to epigenetic alterations.LDH has been proposed as a prognostic efficacy predictive marker for a variety of malignancies, such as B‐cell non‐Hodgkin lymphoma,The pooled results of baseline racial data showed that non‐Asian or white SCLC patients benefited more from ICIs relative to controls than those of Asian ethnicity. Similar results were reported in patients with high TMB tumors treated with ICIs and in patients with SCLC treated with ICIs.The pooled results of this study showed that SCLC patients without liver or CNS metastases at baseline had a longer OS and PFS benefit after treatment with ICIs or ICIs+ chemotherapy compared to those with controls. The occurrence of metastasis is undoubtedly related to poor prognosis. The study has shown that the metastasis of some malignant tumors is related to histological subtypes, liver metastasis, and CNS metastasis are more potential in SCLC.The occurrence of brain metastases is considered to be associated with progression, and higher T stage is considered an independent risk factor for the occurrence of CNS metastasis in SCLC.Limitations of this study: 1. Large heterogeneity was observed in some of the pooled results. The reasons may be small sample size, single center, and some studies with open labels. 2. There are some unavailable data, such as clinical benefit information for smoking patients. Smoking has been a risk factor for SCLC, but the data we extracted could not be combined; as the subgroups in the CASPIAN study5Based on the results of this meta‐analysis, the available research results do not support the recommendation of PD‐L1‐positive and TMB‐H as predictors for the use of ICIs in SCLC patients. LDH, baseline liver metastasis, and CNS metastasis may be used as biomarkers/influencing factors for predicting the efficacy of ICIs in SCLC patients. Non‐Asian SCLC patients achieved better clinical benefit with ICIs. More multicenter, large‐sample, well‐designed studies are needed to confirm these results.Lin‐Lu Li: Conceptualization ; data curation ; formal analysis ; methodology ; software ; validation ; writing – original draft . Cheng‐Feng Yu: Data curation ; formal analysis ; writing – original draft ; writing – review and editing . Hong‐Ting Xie: Data curation ; writing – original draft ; writing – review and editing . Zheng Chen: Data curation ; writing – original draft . Bo‐Hui Jia: Formal analysis ; software ; writing – original draft . Fei‐Yu Xie: Software ; writing – original draft . Ya‐Fang Cai: Data curation ; writing – original draft . Peng Xue: Writing – review and editing . Shijie Zhu: Conceptualization ; funding acquisition (lead); methodology ; project administration (lead); supervision (lead); validation (lead); writing – review and editing .The authors declare that they have no conflicts of interest regarding the publication of this paper.Table S1–S23Click here for additional data file.


To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5′–3′ and 3′–5′ RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3′–5′ RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3′–5′ RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer. Early discoveries concerning splicing of RNA molecules guided the curiosity of researchers towards identifying RNA ligases and ligation mechanisms that are responsible for the maturation of RNA species, such as mRNAs and tRNAs, that contain introns. Although some RNA ligation processes are performed by self-catalysing RNAs, other processes are dependent on protein enzymes. There are only two identified proteins that catalyse RNA ligation in human cells, namely RtcB , which catalyses 3′–5′ RNA ligation and C12orf29, which catalyses 5′–3′ RNA ligation and was identified very recently [XBP1 mRNA after removal of a 26-nucleotide intron [XBP1 mRNA begins with endoribonuclease activity of IRE1α (hereafter named IRE1), an endoplasmic reticulum (ER) transmembrane protein that is activated as part of the unfolded protein response [XBP1s (‘s’ for spliced) mRNA encodes a transcription factor that enhances transcription of itself, as well as several genes involved in the unfolded protein response and lipid synthesis [RtcB enzymes are responsible for 3′–5′ RNA ligation in archaea, bacteria, algae and animals . The subresponse . XBP1s  Fig. . The secHuman RtcB was first identified in human HeLa cell extracts, which possess a 3′–5′ RNA ligase activity between 2′,3′-cyclic phosphate and 5′-OH termini . This RNbona fide substrate as suggested by RtcB’s reduced efficiency with 3′-phosphate ends and the observation of an RNA 3′-terminal phosphate cyclase activity in HeLa cell extracts that converts the 3′-phosphate into 2′,3′-cyclic phosphate [Although both 2′–3′ cyclic phosphate and 3′-phosphate ends can undergo ligation by RtcB, it seems that the former is the hosphate , 16. In hosphate . The prehosphate . Convershosphate .Pyrococcus horikoshii and 73% with the nematode Caenorhabditis elegans [Comparative genomic and structural studies show that RtcB proteins are evolutionarily conserved. For example, human RtcB shares 51% amino acid identity with RtcB from the archaeon  elegans , 20. Mor elegans , 21.Pyrococcus horikoshii, Pyrobaculum aerophilum and the bacteria Escherichia coli have been solved. These help shed light on the mechanism of action of RtcB, identifying key amino acids in the catalytic site responsible for coordination of the metal ions to achieve RNA ligation H can override this protective mechanism leading to inactivation of RtcB [2O2 that could inactivate RtcB. Since it is so tightly controlled, a regulatory function for the redox sensitivity of RtcB has been suggested, but not yet shown. For example, it could play a role as a molecular switch during oxidative stress coupled to ER stress.There is some evidence that RtcB activity is sensitive to inactivation by oxidative stress. Murine cells with oxidative stress exhibit an accumulation of tRNA exon halves . Later, rikoshii . More rerikoshii . PYROXD1 of RtcB . It is sThe first identified function for RtcB was in ligation of select tRNAs that require removal of an intron for maturation. In fact, in humans, 28 of 416 tRNAs contain introns, including all 13 tRNAs that transfer tyrosine to ribosomes . Thus, RIn metazoans, RtcB accomplishes tRNA ligation as part of a multi-subunit ligase complex called the tRNA ligase complex. The human tRNA ligase complex was identified through activity-guided purification of tRNA ligase activity from HeLa cell extracts, revealing a complex containing RtcB and DDX1, RTRAF, FAM98B and Ashwin . FurtherInterestingly, RtcB and some of the other subunits of tRNA ligase complex have been reported to also be part of other protein complexes, suggesting a dynamic relationship between the components. For example, RtcB, DDX1 and RTRAF are associated with kinesin 5 as part of an RNA-transporting complex in mouse neurons . AdditioXBP1 mRNA maturation was demonstrated subsequent to reports of its role in tRNA ligation [XBP1 mRNA, resulting in the removal of a 26-nucleotide intron. The 5′ and 3′ mRNA fragments produced are re-ligated by RtcB [Identification of RtcB as the ligase responsible for ligation , 5, 20.  by RtcB , 5, 20.  by RtcB –11.XBP1 mRNA fragments. In fact, XBP1 splicing, including cleavage and rejoining of the cleaved fragments can be reconstituted in vitro with just recombinant IRE1 (which performs the double cleavage of XBP1 mRNA at two stem loops) and RtcB, although only about 50% of the cleaved XBP1 exons were rejoined [It is not clear which, if any, of the components of the tRNA ligase complex are required for XBP1 splicing apart from RtcB itself. While a complex containing RtcB, RTRAF, DDX1 and FAM98B can be found in both the nucleus and the cytosol , this doMuch of what we understand about RtcB function and regulation in human cells is inferred from observations in other organisms including prokaryotes. RtcB has a major impact on translation in cells due to its roles in tRNA and XBP1s maturation. It can directly affect translation at the ribosomal level by regulating the levels of tRNAs that require splicing. It also exerts an indirect effect on protein expression through production of XBP1s, which maintains ER homeostasis due to the expression of its target genes. Moreover, RtcB may also target other unidentified RNA substrates, linking RtcB to other functions of which we are currently unaware. Here, we summarise what is known about the consequences of RtcB activity in various biological systems, from bacteria to human cells and finish by examining the potential of RtcB as a therapeutic target in human cancer.E. coli [E. coli suggests involvement in re-ligation of damaged rRNA. RtcB deletion leads to diminished recovery from ribosomal damage after exposure to antibiotic-induced stress conditions [E. coli upon stress release, due to its ability to re-ligate rRNA in ribosomes modified by endoribonuclease MazF as part of the stress response [The functional importance of RtcB during genotoxic and ribosomal stresses was uncovered through studies on the bacterial operon encoding the orthologs RtcA and RtcB . RibotoxE. coli . MoreoveE. coli . Furthernditions . RNA-seqresponse . In addiresponse . Furtherresponse , 51.Salmonella enterica Serovar Typhimurium may be due to RtcB DNA-capping activity [E. coli, RtcB can produce a DNA3′pp5′G at DNA broken ends and this RtcB-induced DNA cap could then protect the 3′end of E. coli DNA from exonucleases [Myxococcus xanthus RtcB paralogues, which demonstrate variable activities and/or efficiency levels for RNA/DNA ligation and capping [Other studies support a role for RtcB in ribosomal stress and DNA repair beyond its classical role in RNA ligation. It has been suggested that the survival advantage conferred by RtcB under conditions of DNA damage in activity . In E. cucleases . These o capping . FurtherGenetic manipulation of RtcB  in animal models represents an opportunity to decipher the role of RtcB activity in multicellular organisms. There are only a few studies in this area but they have revealed roles for RtcB in physiology, neurology and immunology.C. elegans, RtcB KO causes growth deficits and shorter life span, which can be rescued by expression of pre-spliced tRNA [C. elegans also causes defects in vulval development and sterility due to failure in oocyte maturation, which are independent of both XBP1 and tRNA maturation  to a neurodegenerative disease [C. elegans due to α-synuclein overexpression and or treatment with the Parkinson mimetic 6-hydroxydopamine could be rescued with co-expression of RtcB [C. elegans [Drosophila and cultured mouse neurons [Several studies demonstrate a role for RtcB in neuronal systems and neurodegeneration. In a  disease . Downreg of RtcB . RtcB-me elegans . This ef elegans . Thus, R elegans . Thus, t elegans . These o neurons . In thesIn the immune system, XBP1s is well characterised as essential for differentiation of B lymphocytes into plasmocytes and subsequent antibody production , 65. AccSeveral lines of evidence support a potential role for RtcB in human cancer, and thus it may have value as a therapeutic target. RtcB has been shown to mediate resistance to antioestrogen treatment in MCF7 breast cancer cells . Mass spTwo independent studies identified RtcB as a target of miR-34a in cancer. A miRNA microarray analysis found miR-34a to be the most downregulated miRNA in breast cancer cell lines compared to healthy mammary epithelial cell lines ; and an Taken together, these studies show that the role of RtcB impacts various fields such as neurology, immunology and cancer. However, further studies are needed to elucidate many of these aspects.Since the discovery of RtcB as the enzyme responsible for RNA ligase activity in human cells, several groups tried to resolve the mechanisms governing its activity and its various functions. In this review, we summarised the findings obtained from three broad strategies to decipher RtcB functions: analysis of its crystal structure, biochemical analysis of its regulation and what is known about its functions in diverse organisms from bacteria to humans. However, there are still several key questions that remain unanswered.bona fide subunit in the complex, investigation into Archease’s ability to increase RtcB RNA ligation activity by converting it into a multiple-reaction enzyme is a worthwhile pursuit, albeit technically difficult due to the transient nature of its interaction with the tRNA ligase complex.While RtcB activity itself is now well known, thanks to the large number of studies describing its ligase activity, information about the specific functions of each of the tRNA ligase complex subunits is relatively patchy. Regarding DDX1, while its roles as a helicase and as a guanylylation factor of RtcB have been recognised, its importance for the proper positioning of RNA fragments in the catalytic site of RtcB is mostly speculative and moreover, its knockdown does not completely abrogate RNA ligation. Similarly, RTRAF knockdown causes a reduction of RNA ligation in HeLa cells, an effect that was attributed to a concomitant loss of DDX1 without excluding a potential functional role of this protein in the complex . While sXBP1 mRNA splicing, some studies involving defective RtcB excluded an involvement of both tRNA and XBP1 ligation in the induced phenotype [E. Coli RtcB has been used to identify cleaved mRNA fragments with 5′-OH ends in yeast, revealing multiple mRNA targets as well as HAC1, the yeast homologue of XBP1, demonstrating that such strategy could be used to detect these aforementioned unidentified RtcB targets [Interestingly, although RtcB has been mostly studied in the context of tRNA maturation and henotype , 62. Thuhenotype , 52, 53. targets .Finally, observations of the role of RtcB in multicellular organisms indicate a role in neurobiology, immunology and cancer, three areas linked to dysregulation of ER homeostasis –76. AlthHowever, targeting RtcB ligase activity as a treatment for cancer may present significant challenges for several reasons. First, the binding of GMP to the catalytic core of RtcB might make the ligase activity of RtcB virtually undruggable. A similar challenge has been encountered in the long-standing difficulties in targeting RAS in cancer . However


RNAs that are able to prevent degradation by the 5’–3’ exoribonuclease Xrn1 have emerged as crucial structures during infection by an increasing number of RNA viruses. Several plant viruses employ the so-called coremin motif, an Xrn1-resistant RNA that is usually located in 3’ untranslated regions. Investigation of its structural and sequence requirements has led to its identification in plant virus families beyond those in which the coremin motif was initially discovered. In this study, we identified coremin-like motifs that deviate from the original in the number of nucleotides present in the loop region of the 5’ proximal hairpin. They are present in a number of viral families that previously did not have an Xrn1-resistant RNA identified yet, including the double-stranded RNA virus families Hypoviridae and Chrysoviridae. Through systematic mutational analysis, we demonstrated that a coremin motif carrying a 6-nucleotide loop in the 5’ proximal hairpin generally requires a YGNNAD consensus for stalling Xrn1, similar to the previously determined YGAD consensus required for Xrn1 resistance of the original coremin motif. Furthermore, we determined the minimal requirements for the 3’ proximal hairpin. Since some putative coremin motifs were found in intergenic regions or coding sequences, we demonstrated their capacity for inhibiting translation through an in vitro ribosomal scanning inhibition assay. Consequently, this study provides a further expansion on the number of viral families with known Xrn1-resistant elements, while adding a novel, potentially regulatory function for this structure. A relatively novel type of viral RNA structure has been discovered in the 3’ untranslated regions of an expanding number of flaviviruses and plant viruses10. These Xrn1-resistant RNAs (xrRNAs) have the capacity of resisting the highly processive RNA degradation of 5’ to 3’ exoribonuclease Xrn111. Through this resistance, genomic RNA further downstream is protected, resulting in the accumulation of subgenomic RNA (sgRNA) species13. A distinct type of xrRNA termed ‘coremin’ (xrRNAC) was discovered in Beet necrotic yellow vein virus (BNYVV)15. This conserved RNA motif was determined to be necessary for accumulation of the sgRNA ncRNA3, through the action of stalling degradation by Xrn1 from the 5’ side. In BNYVV, this ability of xrRNAC to stall Xrn1 influences viral RNA silencing suppression mechanisms, and is essential for the virus to achieve long-distance movement through the plant16.RNA viruses make use of a large variety of structures that may influence, or even hijack their host’s cellular mechanisms, ranging from transcription to translation and antiviral immune responses18, and the Tombusviridae and Solemoviridae (xrRNALT)20, no structure of xrRNAC has been solved yet. Initial analysis of xrRNAC motifs within Beny- and Cucumoviruses revealed a conserved sequence of a 4-bp hairpin (hp1) carrying a 4-nt loop (lp1), followed by a spacer of 8–10 nucleotides21. Subsequent work focusing on the BNYVV RNA3 xrRNA (xrRNABNYVV) has elucidated more specifically its structural and sequence-specific requirements for stalling Xrn110. It was determined that a second hairpin (hp2) which holds only structural conservation has to follow the conserved hp1 and spacer. Furthermore, Xrn1 resistance was retained only when lp1 followed the consensus YGAD, showing that some variation is possible within xrRNAC that was not reflected in the sequences found in nature so far. These parameters lead to the discovery of novel xrRNA sequences within a variety of plant virus families10. With more xrRNAs being identified in an expanding number of viruses, we wondered to what extent certain variations of this motif are distributed throughout positive-sense, single-stranded RNA viruses, or even beyond. In this study, we show that xrRNAC-like motifs are present in untranslated regions and also intergenic regions (IGRs) of the genomes of several distantly-related viruses. Several of these novel putative xrRNA sites represent the first case of such sequences in double-stranded RNA (dsRNA) viruses. Notably, these candidate xrRNAs carried five or more nucleotides as lp1, diverging from the canonical four found in xrRNABNYVV. Through in vitro degradation assays within either the xrRNABNYVV, or the original genomic context, we show how such lp1 identities influence the Xrn1 resistance of xrRNAC structures. These experiments were coupled with an investigation into the importance of xrRNAC hp2 in order to find the minimal size and stability required for retaining Xrn1 resistance.Contrary to the large structures that hold an analogous function within the FlaviviridaeLT species in IGRs may lead to them ending up in the 5’ UTR of sgRNAs, either due to synthesis from subgenomic promoters, transcription from prematurely terminated negative-strand RNA, or from incomplete degradation by Xrn122. Consequently, the Tombusviridae that house such IGR xrRNALTs may make use of their 3’ cap-independent translation enhancer elements in order to produce translationally active sgRNAs that are protected by xrRNAs25. Furthermore, the increasingly widespread distribution of xrRNA species and the short sequence required for its function in the case of xrRNAC, highlights how such structures may play a regulatory role in any RNA, coding or non-coding. These factors prompted us to investigate to what extent these structures are able to stall scanning ribosomes, and how this compares to stalling of Xrn1. We show how xrRNAC indeed stalls scanning ribosomes, and that this ability is lost when mutations known to prevent stalling of Xrn1 are introduced. However, a true correlation between Xrn1 resistance and ribosomal stalling could not be demonstrated, since several xrRNAC variants that have been shown to stall Xrn1, actually lost some ribosomal stalling ability. Overall, this study helps to further map the consensus of a functional xrRNAC, which should support the identification of novel instances of such sequences. The ability of these sequences to stall scanning ribosomes augments our understanding of the stalling mechanism by xrRNAC and provides an explanation for the genomic location in which these structures may be found.Recent studies have emphasized how the presence of xrRNA26 were carried out using variants of the original coremin motif10 GUCCGAAGACGUUAAACUAC. In these variants the 2nd and 3rd base pairs from the top were individually replaced by other Watson–Crick base pairs, or the loop size was extended by inserting N, NN, NNN, or NNNN at various positions in the YGAD consensus. These variants were used to screen several families of RNA viruses, adjusting the scoring parameters for mismatches and gaps. Hits were manually checked for strand polarity, location in the genome and presence of a putative hp2.BLAST searches10, using forward and reverse oligos that were purchased from SigmaAldrich in desalted form, with reverse complementary 3’ ends that allow compatibility in PCR reactions . This method yields products with a T7 promoter sequence (GTAATACGACTCACTATA), followed by a 12 nt leader sequence and the construct of interest, as depicted in Fig. Templates for production of RNA to be tested for Xrn1 resistance were designed and produced as described in Dilweg et al.10. After the addition of an equal volume of denaturing loading buffer , RNA was denatured for 5 min at 75 °C. These samples were run on 8 M urea 14% polyacrylamide gels in TBE buffer, equilibrated at 60–65 °C. In the case of MRV JP-B and PicaV-C constructs , as described earlierRenilla luciferase reporter vector pMRL27 was digested with HindIII and MfeI in order to insert pairs of complementary oligonucleotides that introduced KspAI and Van91I restriction sites between the T7 promoter sequence and the start codon of the Renilla luciferase ORF. Digestion of the resulting plasmid with KspAI and Van91I allowed insertion of pairs of complementary oligonucleotides that housed the xrRNABNYVV-derived constructs of interest. Subsequent linearization on the XhoI-site downstream of the Renilla luciferase ORF, and purification with the PureYield™ Plasmid Miniprep System (Promega), yielded products suitable for run-off transcription as described above, though at minimum 1 h incubation time was applied in order to allow for production of the larger transcript. Reaction mixtures were treated with 1 unit RQ1 RNase-free DNase for 20 min at 37 °C, after which 20 μL Milli-Q water was added. Free nucleotides were removed by filtration through illustra™ MicroSpinTM G-25 columns , and RNA concentration of the flowthrough was determined by measuring absorbance at 260 nm and checking by agarose gel electrophoresis. Transcript concentration was diluted to 25 ng/μL with Milli-Q water.The For each measurement, a premix was prepared containing per sample 5 μL nuclease-treated rabbit reticulocyte lysate (Promega), 0.5 μL of 1 mM amino acid mixture without methionine, 0.5 μL of 1 mM amino acid mixture without lysine and 2 μL of 30-fold diluted Renilla-Glo™ Luciferase Assay Substrate . Of this mixture, 8 μL was transferred to a 96-wells reaction plate per construct, in triplicate. To each well, 2 μL of 25 ng/μL RNA was added and mixed well, with intervals of 10 s. Luminescence was measured continuously for at minimum 100 min on a GloMax® Microplate Reader, with appropriate intervals between each well. For each time-point, means were calculated and normalized against the maximum mean luminescence reached by the sp.mut construct  or untranslated region (UTR)) and the presence of a second hairpin following directly downstream of the conserved spacer sequence . Of note is Pea enation mosaic virus-1 (PEMV-1), which carries a putative xrRNAC in its IGR for the isolate ID, or in a 3’UTR in isolate LK.These hits provide for several viral families a first indication for the presence of an xrRNA within their genome. Notably, the double-stranded RNA (dsRNA) virus families of Hypoviridae and Chrysoviridae carry several candidate xrRNA sequences, within the 3’ UTR of Rosellinia necatrix hypovirus 2 (RnHV2), in a satellite-like RNA derived from Botrytis cinerea hypovirus 1 (BcHV1), and in the 3’ UTRs of all four Chrysothrix chrysovirus 1 (CcCV1) genomic RNAs. For the hits found within Tombusviridae and Solemoviridae, this represents the first time that two different types of xrRNA are found within a viral family, as both families house xrRNAC -like motifs function as actual xrRNAs, we tested Xrn1 resistance for some of the hits within their original contexts, using the exact hp1, spacer and hp2 sequence as they occur in the genome . Considering this knowledge, we were curious to see whether it would be possible to rescue the non-functional CCGAA by testing a sequence that would follow YGNAD. This was not the case however, as CGAAA did not result in any Xrn1-resistant RNA  was determined through comparison with a construct carrying a substitution of the spacer C and U with two A’s (sp.mut), a version that has previously been shown to be unable to stall Xrn110. It appears that the production of luciferase occurs much more rapidly within the sp.mut construct, leading to roughly a five-fold increase in maximum relative luminescence (MRL) after about an hour of translation compared to the wildtype construct , UGAAG (lp1b) and ACGAA (lp1c), all show a strong loss of ribosomal stalling  reached an MRL that was twice that of the WT construct, although still around 0.4 times the MRL of sp.mut, indicating that heptaloops in an xrRNAC may partially retain the ability to stall ribosomal scanning.The Xrn1-digestion assays using xrRNAing Fig. , even woSince many of these motifs were found in plant viruses, we investigated the main constructs also in a wheat germ extract. This led to basically the same results  CDSs, highlight the importance of mapping the interplay of translation regulation and Xrn1-mediated decay that viruses employ.In this study, we show how xrRNAnce Fig. . Like xrC sequences allow for a relatively stable hp2 , which are known to be crucial for stalling Xrn1. Furthermore, the constructs testing the Xrn1-resistant hexaloop variants CGAAAA, CGAAAU and CGAAAG, indicated ribosomal stalling on par with, or better than the wildtype. In contrast, the non-Xrn1 resistant CGAAAC lp1 appears to lose this ability at least partially, reaching an MRL comparable to the spacer mutant. As such, these assays provide a potential for additional information on the structural integrity of xrRNAC variants, where Xrn1-digestion assays do not account for structures ‘more’ resistant than wildtype. Furthermore, the pentaloop constructs, which are all unable to resist Xrn1, appear to slow down ribosomes even less than the spacer mutant, indicating an even stronger loss of thermodynamic stability and/or tertiary structure than caused by mutations in the spacer.Following the discovery that xrRNABNYVV may influence not only thermodynamic stability of the construct, but also the ratio of functionally versus non- functionally folded structures. A recent study highlighted the importance of this folding process for Zika xrRNA, showing how misfolded intermediates without the intricate structure necessary for stalling Xrn1 may ultimately form39. Furthermore, Xrn1 and ribosomes do not process RNA in the same way. While Xrn1 is unable to progress through an xrRNA structure even after 20 h of incubation (Supplementary Fig. We must however take into account that certain substitutions within the xrRNAC motifs from BLAST-searches is unlikely to be exhaustive, and it is therefore likely that more viruses carrying such structures can be found. Since the intrafamilial conservation of these motifs was not explored thoroughly, it remains unclear to what extent these structures are abundant throughout. The expansion of lp1 sequences that may confer Xrn1 resistance in the context of xrRNABNYVV, and the minimal hairpin that is required, should aid further interrogation of viral genomes for these motifs. Knowing that they are able to stall scanning ribosomes, we may also look into the 5’ UTRs of viral families with internal translation initiation capacities. Conversely, how often xrRNAC motifs are positioned in the genome such that they mostly evade ribosomes, as opposed to a position where they may provide a more regulatory, ribosome-inhibiting function, is yet open to question.The manual assessment of novel xrRNASupplementary Figures.


Parents play an important role in the sexual and reproductive health (SRH) of their adolescents. Parent–adolescent SRH information communication is cardinal and is expected to improve SRH outcomes of adolescents.The aim of this systematic review was to search for effective SRH information communication interventions in lower- and middle-income countries (LMICs) to inform the adaptation of parent–adolescent SRH information communication intervention.This is the first phase of an explanatory sequential mixed method study. The systematic review was carried out by employing Joanna Briggs Institute software for reviews. Search sources included Cochrane Reviews Library, EMBASE, CINAHL, PubMed, OVID, Scopus, Web of Science and Science Direct. A systematic search strategy was formulated, making use of the key terms: adolescent, teenager, youth, parent, mother, father, caregiver, reproductive, sexual, health, information, communication and intervention.Five articles met the inclusion criteria for full-text screening. The interventions included addressed sociodemographic covariates; parent–adolescent general communication; parental monitoring; parent–adolescent communication about sex-related topics; parent’s sexual communication skills; parent’s self-efficacy in sexual communication; parent’s responsiveness to sexual communication; communication frequency; quality of sex‑related communication and information-motivational-behavioural skills.Findings suggest that evidence-based SRH information communication interventions are effective in improving parent–adolescent SRH information communication to optimise safe SRH behaviour in LMICs.This systematic review identified effective SRH information communication interventions in LMICs, which can form the basis of further qualitative exploration for adaptation of a culturally sensitive intervention in Ghana. Adolescence is a period of transition between childhood and adulthood, which occurs between 10 and 19 years of age  experience poor SRH outcomes, which include sexually transmitted infections (STIs) including HIV/AIDS, unsafe abortions, and early and unwanted pregnancies  and the JBI manual guided the conduct of this systematic review. The review was prospectively registered in the PROSPERO database with registration number CRD42022297526.The search strategy aimed to locate both published and unpublished studies. The researcher developed a full search strategy for Cochrane Reviews Library, EMBASE, CINAHL, PubMed, OVID, Scopus, Web of Science and Science Direct. The search strategy, including all identified keywords and index terms, was adapted for each included database and/or information source. This included (‘Adolescents’ OR ‘Teenagers’ OR ‘Young Adults’) AND (‘Parents’ OR ‘Caregiver’ OR ‘Mother’ OR ‘Father’ OR ‘Guardian’) AND  AND (‘Information’ AND ‘Communication’) AND (‘Interventions’ OR ‘Strategies’ OR ‘Best Practices’). The reference list of all included sources of evidence was screened for additional studies. Only studies published in the English language, and only those published between January 2011 and December 2021, were included, as many studies were produced in that period.Following the search, all identified citations (1706) were collated and uploaded into Mendeley version 1.19.8, and duplicates were removed. Titles and abstracts were then screened by two independent reviewers, F.B.A. and D.K.K., a doctoral student and a Professor, for assessment against the inclusion criteria for the review. Potentially relevant studies were retrieved in full and their citation details were imported into the JBI System for the Unified Management, Assessment and Review of Information (JBI SUMARI). The full text of selected citations was assessed in detail against the inclusion criteria by F.B.A. and D.K.K. Reasons for the exclusion of articles in full text that did not meet the inclusion criteria were recorded. Any disagreements that arose between the reviewers at each stage of the selection process were resolved through discussion. The results of the search and the study inclusion process were reported in the PRISMA flow diagram  or both to an SRH information communication intervention, in LMICs. It considered both experimental and quasi-experimental study designs, including randomised controlled trials, in LMICs.Selected studies were critically appraised by F.B.A. and D.K.K. independently using the Joanna Briggs Institute standardised critical appraisal tools  in the pre-to-post increases in the intervention means compared with the control means. The number of new topics discussed as baseline was greater among intervention parents  than among the parents in the control group .Bogart et al. :602–608 F = 16.74; (p ≤ 0.01) albeit with a small effect size (d = 0.3), which shows that there is a non-overlap of 21.3% in the two distributions. At 6 months, the results showed a statistically significant difference between the experimental and control groups, which favoured the former F = 17.9; (p < 0.001) albeit, also with a small effect size (d = 0.3). At 1 year follow-up, the results still demonstrated a statistically significant difference that favoured the experimental group, with a small effect size (d = 0.4). The marginal increment in Cohen’s d may highlight that the longer the participants receive and practice the intervention, the more they are influenced by it. This may translate to improved outcomes.Seif et al. :1–13 assp = 0.001), and 1 month after the intervention, between the intervention (30.41 ± 10.07) and control (42.47 ± 9.62) groups (p < 0.001).Ziaei et al. :1–9 deteKatahoire et al. :91–104 et = 5.162; p < 0.001) (parents or caregivers) and 0.26  (students). The study assessed the attitudes towards SRH communication and found that there was a significant difference between the two groups on positive attitudes with effect sizes equal to −0.31  for parents or caregivers and 0.20  for students. In the intervention group, the reduction in negative attitudes towards sex-related communication was significantly greater than in the control group, with effect sizes equal to −0.17  parents or caregivers and −0.19  for students.The effect sizes were 0.36 . With regard to parents’ SRH communication skills and their responsiveness, those in the intervention group showed higher mean score changes at the first and second follow-ups than the baseline (p < 0.001 for each) but this did not remain statistically significant from the first follow-up to the second .Ahari et al. :1–8 evalThe effectiveness of these interventions could have been influenced by factors such as the components of the interventions and how the interventions were delivered.In summary, the study sought to identify effective interventions to improve parent–adolescent SRH information communication that optimise safe and healthy SRH behaviour in LMICs. The findings highlight that the identified interventions were effective in improving communication. This behavioural change was influenced by the improvement in SRH communication skills, which was evidenced by the improved quality and self-efficacy regarding SRH communication. Sexual and reproductive health communication skill was influenced by the SRH information communicated and participants’ motivation to communicate, expressed as attitudes in most of the studies. This has been presented as a conceptual framework to understand how behavioural change was influenced by the indicated factors .Regarding the information communicated to recipients of the various interventions, it was also observed that most of the topics were similar, with a few exceptional topics such as alcohol and drug use, sex exchange for gifts, dating and masturbation. It was also significant that Bogart et al. :602–608,The aim of this review was to identify effective interventions to improve parent–adolescent SRH information communication that optimise safe and healthy SRH behaviour in LMICs.Considering the differing nature of these SRH issues, it may be difficult to draw stronger conclusions regarding the overall effects of the interventions. Additionally, the small effect numbers reported across some studies may highlight a need for cautious interpretation of the review findings. These caveats notwithstanding, the review findings suggest that interventions focused on information communication may be able to improve parent–adolescent SRH information communication.Most of the studies involved both parents and adolescents in the communication process, and both parents and adolescents were trained. This is effective in promoting communication that can be started by any of the parties. An adolescent may have a need that must be communicated and that cannot wait for the parent to initiate the communication, and vice versa. Involving both parents and adolescents has proven to be effective in the studies identified in this systematic review and other studies . The researchers limited the age of adolescents to 13 to 16, which may have excluded some effective SRH interventions for younger or older adolescent populations.The review offers a comprehensive summary of effective parent–adolescent SRH information communication interventions in LMICs. It highlights the components of the identified interventions, thematic analysis of the intervention delivery characteristics, frequency, quality, attitudes towards communication on SRH and communication skills. The findings of the systematic review provide information that can guide the adaptation of a culturally sensitive parent–adolescent information communication intervention in the context of Ghana, which is a LMIC and can also inform stakeholders’ decisions to further invest in the adaptation of interventions. This can broaden the understanding of healthcare providers and policymakers about what works most effectively in improving SRH outcomes.


Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This   From novel vaccine platforms, illustrated by the SARS-CoV-2 mRNA vaccines, to gene therapy, RNA stands poised to play a central role in therapeutics of the future . ThroughWe and others have shown that these products arise from rebinding of released RNA to the enzyme in a mode that allows its 3′ end to pair with upstream sequences in the RNA and prime extension encoded by the RNA itself . Under hRebinding of the product RNA to the RNA polymerase should involve (at least) nonspecific, electrostatic interactions. Thus, one would expect that increasing ionic strength should reduce rebinding, and therefore reduced self-primed extension. We have observed that elongation complexes of T7 RNA polymerase are relatively tolerant to moderate salt concentrations , but iniAs illustrated in Figure In this work, we demonstrate that enhancement of promoter binding using partially single-stranded or nicked/gapped promoter constructs yields initiation complexes that are more tolerant to increasing ionic strength. Transcription under optimized salt concentrations allows efficient initiation, while inhibiting the product rebinding that leads to self-primed extension. We further describe a novel approach toward generating uniquely gapped promoter constructs in long double-stranded DNA templates. Together, these approaches allow RNA synthesis with a dramatic reduction in self-primed extension (higher purity of the encoded RNA). Since primed extension derives from correct length RNA, the improved purity is accompanied by an overall increase in yield of the desired RNA.Escherichia coli strain BL21 carrying the plasmid pBH161, purified and characterized as previously described (His-tagged T7 RNA polymerase was prepared from ® (Uracil-Specific Excision Reagent) II Enzyme (New England Biolabs) system to excise dU, as indicated in the figures, to introduce a gap in the nontemplate strand. USER® II excision reactions for oligonucleotide templates contained 10 μM duplex DNA, excision reactions for 104-nucleotide-long guide RNA templates contained 1.25 μM DNA and excision reactions for longer (mRNA-length) RNA templates contained 0.35 μM DNA. In each, the DNA was dissolved in NEB CutSmart® buffer with 20 U/ml of Thermolabile USER® II Enzyme system. After 1 h incubation at 37°C, the reactions were heat inactivated at 65°C for 10 min.DNA oligonucleotides used as transcription templates were purchased from Integrated DNA Technologies; sequences are shown in High-yield transcription reaction contained 1 μM of the indicated DNA, 1 μM T7 RNA polymerase, and 7.5 mM each of guanosine triphosphate (GTP), cytidine triphosphate (CTP), adenosine triphosphate (ATP) and uridine triphosphate (UTP). Reactions were carried out at 37°C for 4 h in a buffer containing 40 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM potassium glutamate, 0.25 mM ethylenediaminetetraacetic acid (EDTA) and 0.05% Tween 20 and were supplemented with 2000 U/ml of RNase Inhibitor, Murine (New England Biolabs) and 5 U/ml of inorganic pyrophosphatase .High-yield transcription reaction contained 0.125 μM of the indicated DNA, 0.125 μM T7 RNA polymerase, and 7.5 mM each of GTP, CTP, ATP and UTP. Reactions were carried out at 37°C for 4 h in a buffer containing 40 mM magnesium acetate, 30 mM HEPES, 25 mM potassium glutamate, 0.25 mM EDTA and 0.05% Tween 20 and were supplemented with 2000 U/ml of RNase Inhibitor, Murine (New England Biolabs) and 5 U/ml of inorganic pyrophosphatase .Transcription reactions contained 0.035 μM of the indicated DNA, 0.035 μM T7 RNA polymerase, 5 mM of each of GTP, CTP, ATP and UTP, and 4 mM of CleanCap AG (TriLink), where indicated. Reactions were carried out at 37°C for 1 h in a buffer containing 30 mM magnesium acetate, 30 mM HEPES, 25 mM potassium glutamate, 0.25 mM EDTA and 0.05% Tween 20 supplemented with 2000 U/ml of RNase Inhibitor, Murine (New England Biolabs) and 5 U/ml of inorganic pyrophosphatase (yeast) and were stopped by heat inactivation at 70°C for 5 min. The reactions were treated with TURBO™ DNase I (Invitrogen) to remove the template and then cleaned up with Monarch RNA Cleanup Kit (New England Biolabs).Immunoblot analysis was carried out by spotting purified mRNA on a positively charged Nylon membrane , blocking the membrane with 5% (w/v) nonfat dried milk in TBS-T buffer  and probing it with anti-dsRNA antibody  at 4°C overnight. Immunoblots were probed with IRDye™ 680 or 800 conjugated secondary antibodies  for 1 h and imaged with a Typhoon FLA 9500 imager to determine dsRNA contamination. The amount of RNA analyzed is indicated in the figures. Poly I:C was used as a dsRNA control for the immunoblots.Synthesized, purified mRNAs were transfected into human embryonic kidney cells (HEK 293) using the TransIT-mRNA Transfection Kit (Mirus Bio). The expression from the luciferase mRNA was analyzed by measuring the luciferase activity from the media measured 6 h post-transfection. The luciferase activity was measured with BioLux Cypridina Luciferase Assay Kit (New England Biolabs) using a Centro LB 960 luminometer (Berthold) in relative light units.32P] ATP (PerkinElmer) in the reaction mixture . All gel experiments were repeated at least twice and showed no significant differences. Quantifications of gel data from the Typhoon FLA 9500 laser imager used ImageJ v2.1.0/1.53c (https://imagej.nih.gov/ij/)  gel electrophoresis. Transcribed RNAs were labeled by including  construct of Figures The results shown in Figure ® II Enzyme system to create a gap at position −4. As expected, the transcription from this DNA yields the same salt tolerance and, critically, the same reduction in self-primed extension observed in Figure Finally, to reproduce the results of Figure ® II Enzyme gapping approach allows users who cannot assemble DNA from pieces to take advantage of this substantial improvement to in vitro transcription.The results from the above sets of experiments clearly confirm that for the fully double-stranded system, with its native binding affinity, increasing salt concentrations decreases initiation (and self-primed extension). The pss[−5], nicked and gapped constructs all increase promoter binding and so increase the salt tolerance of initiation, while at the same time allowing salt to inhibit self-primed extension. The novel Thermolabile USERin vitro transcription is routinely generated by the PCR. By including the promoter sequence in the PCR primer, one can include a dU base (as a part of the primer) to direct excision of the base at promoter position −4, as above. Accordingly, we prepared PCR primers in which the primer corresponding to the nontemplate strand of the promoter contains a dU base uniquely at position −4 of the promoter. Following PCR and standard product cleanup, we incubated the double-stranded DNA with the Thermolabile USER® II Enzyme.The approach characterized above should be readily extendable to long RNAs, including CRISPR guide RNA (sgRNA), messenger RNA or long noncoding RNAs. Double-stranded DNA of arbitrary length for As an initial demonstration, template encoding a 104-nucleotide guide RNA was PCR amplified with dU at position −4 of nontemplate, as shown in Figure ® II system and then transcribed under increasing salt concentrations, as before. The data in Figure The above assays intentionally used relatively short RNAs, as the analytical tools provide good to reasonable resolution at these lengths, but there is no reason that the synthesis of much longer RNAs will not benefit equally well. To confirm this expectation, a PCR amplified template with dU at position −4 and encoding a 1700-nucleotide mRNA was reacted with the USERAt these lengths, primed extension products are expected not to be resolved. Instead, the effect of the increasing salt on dsRNA synthesis was analyzed by carrying out an immunoblot with anti-dsRNA antibody (J2 clone) that specifically binds to dsRNA longer than ∼40 bp. Immunoblot data shown in Figure in vivo and these modifications can be added during or after transcription  protein contacts with the duplex promoter DNA upstream of and including position −5 drive binding and that some of this binding energy is used to drive (unfavorable) local melting of the promoter from position −4 to ∼+3 ,19,30–33Combining high-salt transcription with the more tightly binding pss[−5] promoter variant does yield a dramatic reduction in self-primed extensions, while maintaining high yields of the encoded RNA, as demonstrated in Figures beyond the melting region (pss[+2]) shows the high salt sensitivity of the fully double-stranded construct, as shown in Figures de novo initiation). Assembling such a nicked construct by combining the three DNA fragments yields the expected behavior, as illustrated in Figures Consistent with the mechanistic model, a construct that is double stranded in the melting region, but is single stranded ® II Enzyme system to generate a single nucleotide ‘gap’ at position −4, functionally substituting for the nick between positions −5 and −4. Paralleling the results in Figures Assembly of a nicked promoter DNA from fragments will work well for those transcribing up to ∼50–70-nucleotide RNAs but transcribing a 70-nucleotide RNA requires a DNA template strand of ∼90 nucleotides, and at these lengths, the yields and purity of chemically synthesized DNA are low. With an eye toward generating longer RNAs, we can replace the T at position −4 of the nontemplate strand with a dU and then target that position with the USEROf course, the potential utility of this new approach derives from the ability to generate DNA templates (incorporating a dU at position −4 of the promoter through the upstream PCR primer) encoding arbitrarily long RNAs. The results presented in Figure Moving to significantly longer RNAs, we next characterized the synthesis of a 1700-nucleotide mRNA. The Bioanalyzer results in Figure To further demonstrate the generalizability of the approach and to introduce another analytical assay, a luciferase mRNA (M3) was synthesized incorporating a 5′ cap and encoding a poly-A tail (1821 nucleotides in length). The results shown in Figure in vitro transcription, RNAs are also extended by a templated, self-priming mechanism by the single subunit RNA polymerases such as T7. While a priming stem containing As ‘paired’ with As is predicted to be unstable, addition of a single U to the 3′ end creates a priming structure that is more stable. Addition of a second and a third U would then generate good complementarity to prime extension. Alternatively, the poly-A tail may loop back (over a long distance in this case) to prime in the mixed nucleotide region of the RNA. Future studies will distinguish between these two mechanisms, but the utility of high-salt synthesis from gapped promoter constructs is unambiguously clear.The dramatic reduction of the dsRNA by-products in the poly-A tail containing mRNAs confirms the hypothesis that during The results presented here demonstrate the broad utility of synthesizing RNA under elevated salt concentrations using promoter DNA engineered to have increased binding affinity for the polymerase. Increased salt reduces rebinding of ‘good’ product RNA, preventing it from being converted into undesired dsRNA impurity, while the increased promoter binding allows good yields under the higher salt conditions.In this system, there is predictable interplay between salt sensitivity and the concentrations of the DNA and the RNA polymerase. End users will want to empirically determine the ideal concentration of added salt, to balance this and to balance trade-offs of yield versus purity. Higher concentrations of DNA and RNA polymerase (and longer reaction times) may also lead to increased concentrations of product RNA, necessitating higher salt to prevent/limit rebinding.While those synthesizing short RNAs  can readily adapt their protocols to high salt, longer RNAs can be synthesized from PCR-generated DNA, with a fairly simple and inexpensive adaptation in the PCR. For ‘worst case’ sequences, this can yield a much higher total yield of the target RNA. For all RNAs, the RNA should be of higher purity, with reduced or eliminated levels of contaminating RNAs derived from self-primed extension of the RNA.The data underlying this article will be shared on reasonable request to the corresponding author.gkad027_Supplemental_FileClick here for additional data file.