Rb1L/L/Trp53L/L mouse model, we identify the NCAMhiCD44lo/– subpopulation as the SCLC metastasizing cell (SMC), which is progressively transitioned from the non-metastasizing NCAMloCD44hi cell (non-SMC). Integrative chromatin accessibility and gene expression profiling studies reveal the important role of the SWI/SNF complex, and knockout of its central component, Brg1, significantly inhibits such phenotypic transition and metastasis. Mechanistically, TAZ is silenced by the SWI/SNF complex during SCLC malignant progression, and its knockdown promotes SMC transition and metastasis. Importantly, ectopic TAZ expression reversely drives SMC-to-non-SMC transition and alleviates metastasis. Single-cell RNA-sequencing analyses identify SMC as the dominant subpopulation in human SCLC metastasis, and immunostaining data show a positive correlation between TAZ and patient prognosis. These data uncover high SCLC plasticity and identify TAZ as the key molecular switch in orchestrating SCLC phenotypic transition and metastasis.Small-cell lung cancer (SCLC) is a recalcitrant cancer characterized by high metastasis. However, the exact cell type contributing to metastasis remains elusive. Using a  Small-cell lung cancer (SCLC) is characterized by very poor prognosis, with ∼15% global lung cancer incidence and a five-year survival lower than 7% [Rb1 and Trp53 [Rb1L/L/Trp53L/L (RP) model typically expresses neuroendocrine markers including neuronal cell adhesion molecule (NCAM) and achaete-scute complex homolog 1 (ASCL1), and frequently metastasizes into distant organs [P130, an Rb-related gene, or Pten in the RP model, significantly accelerates malignant progression and SCLC metastasis [Previous studies have shown that ∼90% of human SCLC harbors concurrent inactivating mutations or deletions of nd Trp53 . Homozygtastasis ,5. Moreotastasis –8.hiCD44loEpCAMhi subpopulation from the RP model is identified as harboring a strong capability to form tumors in allograft assay [SCLCs are characterized by high heterogeneity –15. It ift assay . Moreoveft assay . The NE ft assay . It has ft assay . TherefoThe switch/sucrose-non-fermentable (mSWI/SNF) complexes, including canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF) and non-canonical BAF (ncBAF), are essential for chromatin remodeling ,18. All The Hippo pathway is initially defined as an important pathway during organ size control, and functions mainly via the synergetic interaction between the transcription factor TEAD1-4 and transcriptional co-activator YAP/TAZ (WWTR1) . The onchiCD44lo/– cells as the major subpopulation responsible for SCLC metastasis. Moreover, this subpopulation is progressively transitioned from the non-metastatic NCAMloCD44hi cells via the SWI/SNF-complex-mediated TAZ silencing. Our data highlight the important link between epigenetically regulated TAZ and SCLC plasticity and metastasis.We here identify NCAMRP tumors using NE marker NCAM and mesenchymal marker CD44. In primary RP tumors, we indeed observed the heterogeneous expression pattern of these two markers intra-tumorally and inter-tumorally  staining in pression , increasern Fig. A. These hiCD44lo/– and NCAMloCD44hi subpopulations from primary RP tumors . We found that the NCAMhiCD44lo/– cells grew in culture as oncospheres with a suspension growth pattern . In contrast, the metastasis analyses revealed a huge difference. Most mice (13 out of 15) from the NCAMhiCD44lo/– group had spontaneous metastases in the liver whereas only 1 out of 15 mice from the NCAMloCD44hi group displayed distant metastasis  and non-SCLC metastasizing cell (non-SMC), respectively.To test this, we then used Fluorescence Activated Cell Sorting (FACS) to isolate the NCAMors Fig. C. GenotyhiCD44lo/– expression pattern  signature has been recently established as an important index for SCLC metastasis . InteresSMC Fig. B. These Brg1, were markedly dysregulated between these two subpopulations . Real-time PCR quantification further confirmed the significant upregulation of Brg1 in SMC  mouse cohort and performed comparative analyses of tumorigenesis, SCN signature enrichment and metastasis in parallel with the RP model  network through the integrative analyses of RNA-seq and ATAC-seq data as previously described . We founely Fig. C. Moreovway Fig. D. Using Taz/Yap expression was regulated by Brg1. We found that Brg1 knockdown in SMC cells resulted in a significant upregulation of TAZ expression whereas the expression of YAP was downregulated , which might explain the reduced TAZ expression . In support of this, TAZ level was obviously downregulated in primary tumors at extensive stage . Moreover, knockdown of Arid1a or Arid2, another two important components of the SWI/SNF complex, obviously upregulated TAZ expression in SMC cells . However, YAP expression was only slightly upregulated with Arid2 knockdown, or even downregulated after Arid1a knockdown in SMC . Moreover, we performed BRG1 Chromatin Immunoprecipitation real-time quantitative PCR (ChIP-qPCR) analysis and found that BRG1 could bind to the promoter region of Taz . These results together demonstrate that TAZ is silenced during non-SMC-to-SMC transition through SWI/SNF-complex-mediated epigenetic reprogramming.We further asked whether Taz knockdown in non-SMC for allograft assay . Ectopic TAZ-4SA but not YAP-5SA [in vitro, indicative of the potential reversible transition from SMC to non-SMC   in SMC I. We fou YAP-5SA  expressiTAZ and SCN signature, and single-cell sequencing data of liver metastasis [TAZ expression (TAZlo) displays a significantly higher SCN score . Consistently, most SCN-signature-related genes, including ASCL1, INSM1 and CHGA, were significantly increased in TAZlo SCLC samples .To evaluate whether our findings are clinically relevant, we downloaded a public RNA-sequencing dataset of 112 human SCLCs  and analtastasis , to deteles Fig. C. Moreovens Fig. C, indicales Fig. C. Also, et al. single-cell RNA-sequencing data derived from SCLC liver metastasis [NCAM with concurrent low expression of CD44, resembling the SMC pattern  .SCLC is the most lethal form of lung cancer, characterized by highly metastatic capacity. A growing body of evidence based on mouse models has demonstrated that SCLC is highly heterogeneous with distinct subpopulations playing different roles during malignant progression and metastasis –15. In tRP model, we use both NE marker NCAM and mesenchymal marker CD44 to do the immunostaining and FACS analyses, and identify the NCAMhiCD44lo/– cells as the SCLC metastasizing cells. A previous study shows that mouse SCLC cells contain both NE and non-NE subpopulations [hiCD44lo/– cells harbor strong metastasis capability in allograft assay, and the tumors metastasize into multiple distant organs including the lymph node, lung and liver. Since the SMC defined here also expresses classical NE biomarkers, we reason that the NCAMhiCD44lo/– cells might belong to the NE subpopulation, but with higher metastasis potential. In other words, the NCAMhiCD44lo/– cells might represent the highly metastatic subpopulation of the NE subtype. In addition, we used primary NCAMhiCD44lo/- cells and demonstrated their robust metastatic capability, whereas Calbo et al. [To assess SCLC heterogeneity in the ulations . Howeverulations . In conto et al.  employedWe also find that phenotypic transition from non-SMC to SMC contributes to SCLC metastasis, which closely links cancer plasticity and malignant progression. Indeed, recent data also show, during SCLC drug resistance acquisition, that Notch signaling promotes the transition from an NE to non-NE subtype and thus provides a niche for resisting drug treatment . A similThrough integrative analyses of gene expression profiling and chromatin accessibility, we find that SWI/SNF complexes play an important role during non-SMC-to-SMC transition. Although the SWI/SNF complex is generally considered to be tumor suppressive , our resBrg1 is knocked down in SMC. Similar findings are also observed when Arid1a or Arid2 is knocked down. Consistently, Brg1 knockout in an RP mouse upregulates TAZ and significantly inhibits SMC appearance and SCLC metastasis. Moreover, we find that low TAZ expression is associated with SCN signature enrichment. In agreement with these observations, previous studies have shown that high YAP/TAZ expression correlates with decreased NE markers [We further find that TAZ is an important downstream mediator of the SWI/SNF complex during SCLC phenotypic transition. Although both YAP and TAZ are significantly upregulated in non-SMC, only TAZ is significantly upregulated when  markers , and YAP markers . Meanwhi markers ,50–52. OOur findings from loss-of-function and gain-of-function experiments support the tumor-suppressive role of TAZ in SCLC. YAP/TAZ is well established as oncogenic driver. Nonetheless, accumulated evidence has recently revealed the tumor-suppressive function of YAP/TAZ in multiple cancer types . For insTrp53L/L, Rb1L/L [Brg1L/L [Rb1L/L/Trp53L/L (RP) and Rb1L/L/Trp53L/L/Brg1L/L (RPB) cohorts. All experimental mice were maintained on a mixed genetic background as previously described [6 p.f.u.) by intratracheal intubation [Mice were housed in a specific pathogen-free environment at the Shanghai Institute of Biochemistry and Cell Biology, and treated in accordance with protocols conforming to the ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . Conditional knockout mice including [Brg1L/L  alleles escribed . Mice attubation  to allowtubation . The prit test or chi-square test. Kaplan-Meier analysis with log-rank test was used to assess patients’ survival between subgroups. P value <0.05 was considered to be statistically significant.Statistical analyses were carried out using SPSS 16.0 or GraphPad Prism 5/7 software . The significance of differences was determined using a two-tailed Student's Sequence data have been deposited in Gene Expression Omnibus (GEO) with the primary accession codes GSE158091 (ATAC-seq of SMC and non-SMC), GSE158290 (RNA-seq of SMC and non-SMC) and GSE158293 (RNA-seq of SMC-Ctrl and SMC-TAZ-4SA).nwab232_Supplemental_FilesClick here for additional data file.


To evaluate the similarity of quantitative dual-energy computed tomography (DECT) parameters between the primary breast cancer lesion and axillary lymph node (LN) for predicting LN metastasis.This retrospective study included patients with breast cancer who underwent contrast-enhanced DECT between July 2019 and April 2021. Relationships between LN metastasis and simple DECT parameters, similarity of DECT parameters, and pathological and morphological features were analyzed. ROC curve analysis was used to evaluate diagnostic ability.P < 0.001–0.004). The shortest diameter of the LN  and the similarity parameter of 40-keV attenuation  were independently associated with LN metastasis compared to simple DECT parameters of 40-keV attenuation . The AUC value of the similarity parameters for predicting metastatic LN was 0.78–0.81, even in cohorts with small LNs (shortest diameter < 5 mm) .Overall, 137 LNs (39 metastases and 98 non-metastases) were evaluated. Significant differences were observed in some pathological  and morphological characteristics , most simple DECT parameters, and all DECT similarity parameters between the LN metastasis and non-metastasis groups (all, The similarity of the delayed-phase DECT parameters could be a more useful tool for predicting LN metastasis than simple DECT parameters in breast cancer, regardless of LN size. Globally, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths in women . The morDual-energy CT (DECT) is a technology that collects data from two different energy spectra. Therefore, the difference in attenuation at the two energies can be used to differentiate and quantify material decomposition . Based oP < 0.001) [In previous studies of DECT for breast cancer, Zhang et al. reported the diagnostic performance of preoperative metastatic sentinel LNs (SLNs), and the accuracy of the venous phase slope of the spectral Hounsfield unit curve (λHU), one of the quantitative DECT parameters that represents the slope of the CT value between 40 and 70 keV for detecting metastatic SLNs, was 90.5%; moreover, the venous-phase λHU had a higher specificity and accuracy than the morphologic parameters (< 0.001) . Volterr< 0.001) . In earlDECT parameters can be used to predict LN metastasis or tumor histology in breast cancer –20. HoweThe research ethics committee of the Graduate School of Medical Sciences approved the study protocol . All study procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation , as determined by the Helsinki Declaration. The requirement for informed consent was waived owing to the retrospective study design.n = 63) because surgery was performed at another institution (n = 2), no surgery was performed for advanced cancer (n = 8), or LN surgery was not performed in ductal carcinoma in situ (DCIS) (n = 20); patients who underwent NAC with no fine-needle aspiration cytology (FNA) of axillary LNs (n = 33); patients with unreliable pathological results (n = 6); patients diagnosed with N1mi (n = 3); patients who had LN metastasis pathologically that could not be identified by normal preoperative examination imaging (n = 7) and patients with axillary LNs or breast primary lesions that were too small or unclear to place regions of interest (ROIs) (n = 16).In total, 232 consecutive patients with newly diagnosed breast cancer underwent preoperative DECT from July 2019 to March 2021  using the fast-kilovoltage-switching dual-energy CT imaging scan mode with fast tube voltage switching between 80 and 140 kVp. Non-enhanced chest-abdominal DECT was acquired first. After the non-contrast CT scan, the non-ionic contrast medium was injected at a patient weight-dependent dose of 600 mg iodine/kg in 1 min via the antecubital vein. At 120 s after the start of the contrast injection, delayed-phase contrast-enhanced DECT was performed. The other CT scanning parameters were as follows: noise index, 10 at 5-mm thickness ; iterative reconstruction, advanced statistical iterative reconstruction  of 30%; slice thickness, 1.25 mm; slice interval, 1.25 mm; and helical pitch, 0.508:1.DECT data were analyzed using a gemstone spectral imaging (GSI) viewer on a workstation . Two radiologists  performed the measurements of primary breast cancer lesions and axillary LNs. Two radiologists placed circular ROIs on the solid areas (avoiding necrotic and cystic portions) of the tumor and LN at the slice with the largest size of the lesion. The longest and shortest diameters, hilum of the LN, and longest diameter of the primary lesion were also evaluated.DECT quantitative parameters, including the attenuation at 40 and 70 keV, IC, water concentration (WC), and effective atomic number (Eff-Z), were calculated using the GSI viewer at the workstation. The attenuation at 70 keV was selected because 120-kVp scanning in conventional polychromatic images has an average energy of approximately 70 keV , and at The rate of difference (ROD) between the DECT parameters of the primary lesion and that of the LN was calculated using the following formula: The histological diagnosis was performed by a single pathologist with 19 years of experience in breast histological evaluation. A core biopsy of the tumor and FNA of the LNs suspected of metastasis was performed for diagnosis. The nuclear grade and immunohistochemical data, including the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) were obtained from patients’ medical records.Axillary LN dissection or SLN biopsy was performed according to the stage of the patients. SLNs were identified by using a combination of radioisotope labeling and the blue-dye method during surgery, and all patients underwent preoperative lymphoscintigraphy in combination with single photon emission CT (SPECT)/CT scan. Details of the excised LNs  were recorded. We determined metastatic LNs on the DECT images in the following two ways: identified using US images of LNs that were positive for US-FNA, or identified using SPECT/CT of preoperative lymphoscintigraphy in the patients with positive SLN. If none of the excised LNs were deemed metastatic, all the corresponding LNs on the DECT images were considered non-metastatic. One LN was analyzed per patient.U test, Fisher exact test, or chi-squared test. Multivariable binary logistic regression analyses were used to determine better DECT parameters for predicting LN metastasis. A multivariate analysis with forward elimination was performed with variables that had P values < 0.001 in univariable analysis, and a multivariate model was constructed with pathological and morphological characteristics from lowest P value as the pathological and morphological model. The number of variables was considered from the number of patients, statistically. The variables that had a significant difference in the pathological and morphological model (shortest diameter of the LN) and those with the lowest P value among the DECT parameters (attenuation at 40 keV) and the ROD of DECT parameters (the ROD of attenuation at 40 keV) in univariate analysis were selected and used (DECT parameters with the shortest diameter of the LN model). The Spearman rank correlation coefficient was used to evaluate the correlation between primary breast cancer and the axillary LN. Since the ROD between the DECT parameters of the primary lesion and that of the LN were too small for multivariate analysis, we used a value subtracted from 1 and multiplied the value by 10 [DECT parameters, pathologic characteristics, and morphologic parameters were compared between the LN metastasis and LN non-metastasis groups using the Mann–Whitney ue by 10 . ROC curP < 0.001), nuclear grade (P = 0.04), ER status (P < 0.001), and Ki67 index (P < 0.001) of breast cancer were higher in the LN metastasis group than in the LN non-metastasis group, and no significant difference was observed in age, histologic type, PgR status, or HER2 status between the groups. The ROI sizes were 43.0 ± 34.1 mm2 and 13.9 ± 19.3 mm2 in breast cancer lesions and axillary LNs, respectively.Finally, 137 patients were included. The mean age of the patients was 59.7 years (range 33–87 years). There were 110 (80.3%) patients with IDC, 7 (5.1%) patients with ILC and 12 (8.8%) patients with DCIS. The study included 22 patients who underwent NAC preoperatively, and all these patients had positive results for FNA of axillary LNs. LN metastasis (LN metastasis group) was confirmed in 39 patients and 98 patients had no LN metastasis pathologically (LN non-metastasis group) . The number of LNs without hilum was significantly higher in the LN metastasis group than in the LN non-metastasis group (P < 0.001).Regarding the morphologic characteristics of axillary LNs, the means of the shortest and longest diameters in all patients were 4.6 mm (range 2–17 mm) and 10.9 mm (range 6–32 mm), respectively. The LN size was larger and the ratio of the longest and shortest diameters was smaller in the LN metastasis group than in the LN non-metastasis group , attenuation at 70 keV (P < 0.001), λHU (P < 0.001), IC , and Eff-Z .Significant differences were found between the two groups in each of the following DECT parameters: delayed-phase attenuation at 40 keV (P < 0.05) in the LN metastasis group. However, no correlation was found in the LN non-metastasis group .There was a moderate correlation between DECT parameters  for the axillary LN and primary lesion  in the univariate analysis , and that among the ROD of DECT parameters was for the ROD of attenuation at 40 keV (P = 2.2 × 10–8).The ROD between the DECT parameters of the primary lesion and that of the axillary LN, which reflects the similarity between primary lesion and axillary LN, was significantly smaller in the LN metastasis group than in the LN non-metastasis group for the following parameters: delayed-phase attenuation at 40 keV and 70 keV, λHU, IC, and Eff-Z . The absence of LN hilum was also associated with LN metastasis . In the multivariate analysis that included the shortest diameter of the LN, attenuation at 40 keV, the ROD of attenuation at 40 keV (DECT parameters with the shortest diameter of the LN model), ROD of attenuation at 40 keV  together with the shortest diameter of the axillary LN  were independently associated with LN metastasis. Attenuation at 40 keV was not significantly associated with LN metastasis  , similar results were obtained .As shown in Figs. In this study, the quantitative DECT parameters, including the λHU, IC, and attenuation values at 40 keV and 70 keV, were useful for predicting LN metastasis, as previously reported . HoweverMultivariate analysis showed that the shortest diameter of the LN was most associated with LN metastasis. This finding was different from that in another study, in which the venous-phase λHU was more significant than the morphologic parameters . DiffereUS or MRI is useful conventionally to detect axillary LN metastasis of breast cancer –25. LN mIn metastatic LNs, tumor cells migrate via the afferent lymphatic vessels into the subcapsular sinus, and the growing metastasis replaces the LN tissue . Althougn = 3) were excluded from this study, potentially causing selection bias. However, the number of patients with N1mi was small, so the impact is limited. Fourth, we only investigated one axillary LN per patient, because it is difficult to link all excised LNs on CT images. Additionally, in the SLN positive cases, we were almost certain the LNs on DECT matched LNs pathologically diagnosed as metastasis, but not absolutely. Finally, the similarity of DECT parameters between the primary lesion and metastatic LN has little evidence to date. The similarity between the tissues of LN metastasis and primary breast cancer is not unclear. Further studies are required.Our study has some limitations. First, this was a single-institution retrospective study with a small number of patients. Therefore, the data distribution was unbalanced. Second, axillary LNs without metastasis were too small to place the ROI, so some cases were excluded. Third, cases of N1mi (In conclusion, the similarity of the delayed-phase DECT parameters could be a more useful tool for predicting LN metastasis than assessing the simple DECT parameters in patients with breast cancer, regardless of the size of the LNs.


Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5′-AAAU-3′ RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3′ extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an ‘open’ to a ‘closed’ state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.In  Arabidopsis, HEN1 SUPPRESSOR1 (HESO1) and URT1 (UTP:RNA uridylyltransferase) act cooperatively on unmethylated miRNA uridylation to trigger their degradation (Non-templated uridine addition to the 3′ end of RNA (RNA uridylation), aside from canonical poly(A) tails, has emerged as an important post-transcriptional RNA modification due to the fast development of RNA-sequencing technologies in recent years . 3′ uridradation . HESO1 aradation ,11. In bradation . AlthougSchizosaccharomyces pombe Cid1  size >13–15 As to size distribution of poly(A + U) centered at 16 nt to allow the effective binding by Poly(A) Binding Protein (PABP)  ,9. Interin vitro ,22.HESO1, the functional paralog of URT1 in Arabidopsis, has been proven to add a long U-tail to the 3′-end of miRNAs both in vivo and in vitro . In addiin-vitro transferase assay, we first identified that URT1 preferred to add two uridines at the 3′-end of poly(A). Furthermore, we determined the structure of URT1 in complex with a 5′-AAAU-3′ RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis revealed that the URT1–AAAU interaction at -1 position is crucial for URT1 to discriminate between purine and pyrimidine, and further control the optimal number of uridine added to 3′ extremity of poly(A) as two. Site-directed mutagenesis of key residues followed by in-vitro transferase assays and in-vivo functional complementation tests validated this finding. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an ‘open’ to a ‘closed’ state. Further molecular dynamic simulation supports an open-closed conformational selection model employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Our research reveals that the crucial structural factors determining the di-uridylation activity of URT1 may serve the effective functional reformation of URT1 and potentially lead to the illustration of the biological significance of dual TUTases (HESO1/URT1) in Arabidopsis.In this research, using an Escherichia coli Gold (DE3) cells for expression. For overexpression of wild-type and mutant proteins, cells were cultured in LB medium at 37°C until OD600 reached 0.8–1.0 before isopropyl β-d-1-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM was added and then cultured at 16°C for 24 h.The enzymatic domain of URT1 (residues 410–764) was amplified by PCR using synthetic gene as the template and was subcloned into pET28a vector with a 6 × His tag at the N-terminal site. All mutants were obtained by PCR and MutanBEST kit (Takara Bio Inc.), and verified by sequencing. Plasmids was transformed into The cells were collected by centrifugation and suspended by binding buffer , followed by sonication lysis and centrifugation at 12 000 rpm for 30 min at 4°C. The supernatant was purified by Ni-NTA column (Qiagen) and further purified by Superdex™ 200 pg (16/60) . The purified proteins were then dialyzed against storage buffer  and concentrated to 3.5 mg/ml.For the crystallization of URT1 complexed with RNA, purified URT1 D547A mutant, whose catalytic activity was abolished , was conhttp://www.pymol.org/).X-ray intensity data of the crystals were indexed, integrated, and scaled by HKL2000 package . The com2, 5 mM DTT and 5% (v/v) glycerol. 100 nM 13-nt poly(A) or its variants (synthesized from Takara Bio Inc.) with Cy5 probes labeled at the 5′ end was mixed with 50 nM protein and 1 mM UTP. The reaction was stopped at a series of time points  by adding EDTA . The product was denatured at 95°C for 2 min, incubated on ice for 2 min, and analyzed with 20% denaturing polyacrylamide gels. RNA bands were imaged on a Typhoon FLA 7000  by detecting Cy5, and quantified using ImageQuant TL . Rolling-ball algorithm was used for background subtraction.Purified URT1 wild type and its mutant were prepared for oligouridylation activity assay which was performed at 25°C in a buffer containing 20 mM Bis-Tris, pH 6.0, 100 mM NaCl, 10 mM MgCl2 and 1 mM UTP. The reaction was stopped by the addition of EDTA  after incubation for 15 or 30 min at 25°C. The product was denatured at 95°C for 2 min, incubated on ice for 2 min, and analyzed with 20% denaturing polyacrylamide gels.The enzymatic assay in the presence of a trap (competitor) was performed by pre-incubation of URT1 with Cy5-labeled 13-nt poly(A) for 20 min on ice, after which a 100-fold molar excess of the same but unlabeled poly(A) was added as a trap at the same time as the reaction was initiated using 10 mM MgClp35S:: URT1-GFP) was described previously  at 22°C.Wild-type URT1 in the binary vector pMDC83 +2U bands in our assays is that the addition of the first two uridines by URT1 could proceed in a processive manner while further uridylation is distributive. To testify this possibility, we re-performed the enzymatic assay in the presence of a trap . SpecifiWe previously determined the structures of URT1 (410–764) and its complex with UTP using X-ray crystallography . URT1 wi+1) of RNA stretch adopted very similar conformations for its base and sugar ring as those of the UTP molecule in the URT1-UTP structure (PDB ID 6L8K)  , indicat9 Figure . For theresearch , while tresearch . These rresearch .0) representing the 3′ extremity of the mRNA poly(A) tail. Among the URT1 residues surrounding A0, N552 provided the specific contact with the adenine by forming a hydrogen bond between its side-chain group and purine N3, while V532 made a hydrophobic interaction against the base  was perfectly sandwiched by the side-chain groups of Y592 from the central domain and L527 from the catalytic domain  was located at the exit of the catalytic groove of URT1, making no obvious interactions with the protein  mainly through its RNA recognition mode at the –1 position  in URT1 responsible for its di-uridylation activity, we re-analyzed the URT1–AAAU complex structure. URT1 distinguishes itself from other TUTases with reported protein–RNA complex structures  with D700 from the central domain  for further simulation. To obtain statistically meaningful results, we performed three independent 2-μs aMD simulations for apo-form URT1 and URT1–AAAU, respectively. From each simulation, a trajectory containing 2000 conformations extracted every 1 ns were used for analysis. The three aMD trajectories were combined, and thus the 6000 conformations were used for analysis. The Cα–Cα distance between R531 and D700 was calculated to measure the open/close of URT1 conformation. Figure To further investigate whether URT1 employs conformational selection or induced fit as its RNA binding/catalytic mechanism, we performed the accelerated molecular dynamics (aMD) simulation of the URT1 system see . Since tFrom our simulations, the apo-form URT1 is mainly in an open state, but it can occasionally sample the closed state with a population of 3.3% from 9 to 16 Å. Although URT1–AAAU is mainly in a closed state, it can sample the open state with a population of 10.2% from 21.8 to 30.0 Å. That is to say, there is an overlapping region of distance distribution between apo-form and RNA-bound URT1. Therefore, our results indicated that a dynamic conformational equilibrium exists between the open and closed states for the URT1 system, and RNA binding may shift the equilibrium from the open to the closed state via a ‘conformational selection’ mechanism.In addition, we performed a parallel simulation on R531A mutant to analyze its effects on URT1 conformations   were individually transformed into hen1-2 heso1-2 urt1-3. For each construct, multiple independent T1 plants were examined. As a control, wild-type URT1 restored the silique length to the hen1-2 heso1-2 level, but could not further reduce it , the A0 binding mutants (N552A), the A-1 recognition mutants (Y592A), and the conformation change mutants (R531A) were significantly impaired or completely abolished rescuing the silique length phenotype. Y599A even had longer siliques than hen1-2 heso1-2 urt1-3. Considering that URT1-3 was not a null allele  . To asse heso1-2 . Wild-tyt Figure  9). Con. Conurt1Schizosaccharomyces pombe Cid1 and Arabidopsis URT1 add a minimal number of uridines (one or two) to the 3′-ends of mRNAs or miRNAs in vivo, while others are often associated with the insertion of a polyuridylation chain to the RNA substrate  polymerases (PAPs) and dedicated to catalyzing the uridylation of RNA substrates. Among all identified TUTases from different species, ubstrate ,21,22,36ubstrate ,37,38. Iubstrate . Althougd hsTUT7 ,20, diffreported . Intrigureported . WhetherArabidopsis. In fact, in-vivo studies indicated that, among all the mutants we tested, L527I exhibited the strongest effect in rescuing the hen1-2 heso1-2 urt1-3 phenotype  at the 3′ end of RNA substrates in e Figure , consiste Figure . Given tAt last, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity, based on our findings in this research Figure . First, Coordinate and structure factor for URT1–AAAU complex have been deposited in the Protein Data Bank under accession code 7XS4.gkac839_Supplemental_FileClick here for additional data file.


Many RNA-based drugs, both vaccines and non-vaccines, are under development or even approved. They include coding mRNAs and non-coding (nc) RNAs among them antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), micro-RNAs (miRNAs), small activating RNAs (saRNAs), RNA aptamers and RNA guides. According to the European Union (EU) legislation, these products can be currently categorized into different regulatory statuses, depending, for vaccines, on their target (infectious disease or not) and, for other drugs, on how they are obtained . This classification is fundamental to the type of marketing authorization (MA), and therefore to the controls to be performed, from preclinical stages through clinical trials to pharmacovigilance, to meet the safety requirements for patients. However, the current rules raise several problems, in particular the risk, because technology is evolving, to have similar RNA drugs being covered by very different legal statuses and the lack of international harmonization. The objectives of this study are (i) to review how RNA medicinal products are currently legally categorized in the EU and especially whether they fall under the status of gene therapy medicinal products (GTMP), a regulatory status belonging to advanced therapy medicinal products (ATMP), (ii) to discuss the issues generated by this classification, with a focus on the heterogeneity of statuses of these products, the differences with the American and ICH definitions and the potential impact on the safety requirements. While the first mRNA vaccines against an infectious disease have reached the market , 2, manyvs. non-infectious diseases and for non-vaccine drugs, on the type of RNA substance and its production , simple biological medicinal products or “simple chemical medicinal products”, depending, for vaccines, on their targets, infectious oduction –28. HoweFew regulatory studies are available to assist in the regulatory categorization of RNA-based medicinal products. They concern mRNA drugs, especially the classification of mRNA-based vaccines against infectious and non-infectious diseases  and the The objectives of this study are (i) to review how RNA medicinal products are currently legally categorized in the EU and especially whether they fall under the status of gene therapy medicinal products (GTMP), a regulatory status belonging to advanced therapy medicinal products (ATMP), (ii) to discuss the issues generated by this classification, with a focus on the heterogeneity of statuses of RNA-based medicinal products, the differences with the American and ICH definitions and the potential impact on the security requirements.There are currently several products containing RNA on the market, and many are under development, among them mRNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small activating RNAs (saRNAs), micro-RNAs (mi-RNAs), RNA aptamers and RNA guides. These medicinal products do not have the same legal status. First, we will remind the rules of classification in these statuses according to the EU legislation, then, we will analyze the statuses of approved or under development RNA-based medicinal products inside the EU.According to the EU regulation, the RNA-based drugs can be classified in various specific medicinal product statuses see .Medicinal products are defined in the consolidated directive 2001/83/EC . They inAlthough there is no legal definition of a “chemical medicinal product”, the basis of the EU drug regulation concerns chemical drugs. Over time, new statuses have emerged to implement additional safety requirements, such as biological medicinal products status.According to the Consolidated Directive 2001/83/EC, Annex I, Part I, §3.2.1.1.b) :A biological medicinal product is a product, the active substance of which is a biological substance.”“a substance that is produced by or extracted from a biological source and that needs for its characterization and the determination of its quality a combination of physico-chemical-biological testing, together with the production process and its control.”And a biological substance is defined as “The regulation mentioned four subcategories of biological medicinal products  of whichAny medicinal product consisting of vaccines, toxins, serums or allergen products.” As for vaccines, they are defined here as “agents used to produce active immunity, such as cholera vaccine, BCG, polio vaccines, smallpox vaccine.”According to the consolidated Directive 2001/83/EC cited above , Articlepreparations containing antigens capable of inducing a specific and active immunity in man against an infecting agent or the toxin or antigen elaborated by it”  are defined in Article 2 of Regulation (EC) No 1394/2007 as any of the following medicinal products for human use :-a gene therapy medicinal product (GTMP),-a somatic cell therapy medicinal product (sCTMP),-a tissue engineered product (TEP).Among these three statuses, GTMP is the most frequently used status for RNA-based drugs, but sCTMP also represents a possible status.Gene therapy medicinal products (GTMPs) have been defined in the Annex I, Part IV, §2.1 of the Directive 2001/83/EC consolidated .This definition is complex and involves several criteria :1)the drug must belong to the status of a biological medicinal product. The active substance must therefore be a biological substance,2)the drug must contain or consist of a nucleic acid, in other words, DNA or RNA, and this nucleic acid must be recombinant,3)the sequence must be administered to regulate, repair, replace, add, or delete a genetic sequence,4)the action must be therapeutic, prophylactic, or diagnostic and must be directly dependent on the nucleic acid used.“Gene therapy medicinal products shall not include vaccines against infectious diseases.”It should be noted that vaccines against infectious diseases are not included in GTMPs: Somatic cell therapy medicinal products (sCTMP) are also defined in the Annex I, Part IV, §2.1 of the Directive 2001/83/EC consolidated.1)As for GTMP, this definition is complex and involves several criteria : the dru2)it must contain a cell or a tissue, both terms having definitions of their own,3)Examples of substantial manipulations include cell expansion (culture), genetic modification of cells, differentiation/activation with growth factors” (these cells (or tissues) must either be substantially manipulated or not be intended to be used for the same essential function(s) in the recipient and the donor. In the first case, the concept of “substantial manipulation” is particularly important and examples are cited: “factors” .in vivo must be tested. For sCTMP or if cells are used in GTMP, the origin of the cells , the capacity to proliferate and/or differentiate and to induce an immune response, the level of cell manipulation, the combination of cells with bioactive molecules or structural materials, etc. must be controlled  to study their physicochemical and functional properties, such as molecular structure, protein modifications and biological activity , 34. In ntrolled .Among the medicinal products containing RNA, there are currently seven types of drugs that can be further subdivided into subcategories see :•mRNA•non-coding RNAs-Antisense oligonucleotides (ASOs)-Small interfering (si) and micro (mi)-RNA -Small activating RNA (saRNA)-RNA aptamers-RNA guides (gRNA)Some of these drugs have already been approved and many are under development .For each type, we will discuss the legal status according to the EU legislation.Among medicinal products containing mRNA, we can differentiate different subcategories:•the general case for mRNA in therapeutics,•3 specific cases:∘mRNA-based vaccines,∘mRNA-based cell therapies as “therapeutic vaccines” against cancer,∘mRNA-based CAR-T cells.Committee for Advanced Therapies  has ruled that an mRNA meets the definition of a gene therapy medicinal product (GTMP) (The t (GTMP) . In factt (GTMP)  or an anin vitro transcribed by recombinant enzymes. Moreover, the mRNA produced in this way is considered as a recombinant nucleic acid. This point will be further discussed paragraph “The classification of some RNA-based medicinal products in the GTMPs emphasizes the need for a definition of the term “recombinant” and an extension to other genetic techniques.” Finally, from the CAT’s point of view, the administration of mRNA corresponds to “the addition of a genetic sequence” (this is the justification given in the case of the drug for treating a glycogen storage disease) and its therapeutic action depends directly on the nucleic acid used.Indeed, these mRNAs are biological medicinal products as they are produced from a biological source, e.g., a DNA plasmid Although mRNA-based vaccines coding for pathogens or tumor antigens can be directly injected either to prevent or treat infectious diseases or to treat cancers, according to the EU, these two kinds of vaccines are not categorized under the same status. Indeed, there is a regulatory prohibition on including mRNA-based vaccines against infectious diseases, but not “cancer vaccines,” in the scope of GTMPs, making the term “cancer vaccines,” that is used in the scientific community –40, an iagents used to produce active immunity, such as cholera vaccine, BCG, polio vaccines, smallpox vaccine” . The competent authorities issue a “Batch Release Certificate” when the results are satisfactory. This is known as “Official Control Authority Batch Release” (OCABR).Two other explanations concerning public health could explain the special place of vaccines against infectious pathogens. The first relates to the target population: a very large healthy population, mostly including children. The second, which is a consequence of the first, is the specific regulation of vaccines, adapted to this mass use of a drug in a population. Let us mention the possibility, given by Article 114 of the consolidated Directive 2001/83/EC, for a Member State, in the interest of public health , to require the holder of an authorization for marketing to “In conclusion, it is clear that the specification “against infectious diseases” is especially important as vaccines that induce immunity to an infectious disease are excluded from GTMP scope, while mRNA-based “therapeutic vaccines” which are directly injected and induce immunity to a non-infectious disease will be considered as GTMP since they follow the criteria developed in paragraph a). This classification involves different constraints for development and marketing as only GTMP categorization requires specific tests specially to assess genome integration. This point will be discussed more generally for all RNA-based drugs in paragraph “The different RNA-based drug statuses between the EU and the USA need to be harmonized”.ex vivo with in vitro transcribed mRNA coding tumor antigens.Besides mRNA-based cancer “therapeutic vaccines” which are directly injected in patients, another approach consists in injecting autologous dendritic cells (DCs) which have been loaded in vitro transcribed autologous renal cell RNA and CD40L RNA. The EMA/CAT considered that the product falls within the definition of a sCTMP  mRNA construct, the half-life of RNA would have been far longer , resultiex vivo, are classified as GTMP. The previous decision concerning DC has strong implications since there are CAR-T cells under development that are modified using ex vivo mRNA electroporation (Another immunotherapy approach in cancer is based on the redirection of T cells against tumor cells by stable integration of a chimeric antigen receptor (CAR) which recognizes tumor antigens independently of MHC. Current CAR-T cells, which are genetically modified by adding DNA poration , 8. As sin vivo using mRNA lipid nanoparticles . Moreover, the controls exerted before administration cannot be the same: for example, while it is easy to control the protein translation of mRNA in a cell manipulated ex vivo before its administration to the patient, this is very complex if the mRNA is injected in vivo through LNPs. In the latter case, it is very difficult to know if the translation was efficient and did not generate errors.Thus, depending on the methodology, Antisense oligonucleotides are short nc single-stranded (ss) RNA sequences that modulate gene expression by binding to pre-mRNAs or mRNAs and acting by either occupancy-induced (pre)-mRNA RNase degradation or by occupancy-only mechanisms (steric blocking). They can also modulate RNA splicing to produce functional or preferred genetic products.Both small interfering RNAs (siRNAs) and microRNAs (miRNAs) which mimic natural miRNAs are short nc double-stranded RNAs that modulate mRNA expression in the cytoplasm. They recognize target transcripts via the RNA-induced silencing complex (RISC) leading to translation inhibition or mRNA cleavage. The development of miRNAs is less advanced than that of siRNAs.Small activating RNAs (saRNA) are nc short double-stranded oligonucleotides that have the same structure as siRNAs, however, their biological function is totally different , 49. IndAs far as we know, ASOs, siRNA, miRNA and saRNA are currently chemically synthesized. Thus, they are not biological medicinal products, and they can’t by classified in ATMPs. They can only pretend to the status of “chemical” (“simple”) medicinal products.However, bioengineered si- or miRNA  aiming at better mimicking physiological si- or mi-RNA molecules , 51 are RNA aptamers bind to and inhibit a wide variety of targets  by virtue of their tertiary structure. They are produced by chemical synthesis and thus categorized as simple medicinal products , 51.in vivo injection of mRNA  sequence to direct the Cas9 endonuclease to target a gene. While the guide RNA is produced mainly by chemical synthesis, it is the nuclease that determines the status of the drug, i.e., either a biological medicinal product when it is injected as a recombinant protein or a GTMP when it is produced by  of mRNA .ex vivo , or for ncRNAs, on their production . Concerning the latter, the evolution of technology makes things more complex, as similar RNA drugs may be classified in different statuses. This raises the question of the relevance of controls for development and marketing. In order to help to categorize RNA-based drugs according to the current rules, we propose a flowchart , whether they are loaded hart see .in extension of a vaccine or a GTMP. In the case where some RNA could be both chemically and biologically synthesized, this could lead to having two medicinal products with the same indication and the same composition (with the only difference being the source of the RNA), but which would not have the same status at all. This would be problematic and cruelly lacking in consistency. Thus, CAT explained: “long chain mRNAs cannot yet be produced via chemical synthesis. However, when this becomes possible, the regulatory status of such synthetic RNAs needs to be considered, as it should be avoided to have similar products being covered by different legal frameworks”  or synthetic DNA oligonucleotides”  RNA: ASOs, siRNA, miRNA and saRNA”). They will probably lead to a new class of iRNA molecules, similar to chemical iRNAs but which should be categorized as biological medicinal products and thus possibly as GTMPs. Therefore, according to CAT, considering RNA as a biological substance could prevent categorizing similar RNA drugs in very different statuses, depending on their mode of production. But one should be aware that this change in classification would not be without consequence since, if GTMP status is retained, these RNA-based drugs would then have to follow the very specific GTMP regulations (see below).This type of problem highlights the possible inadequacy of the European definition of a biological medicinal product. Indeed, it seems that the current definition of a biological medicinal product is no longer consistent with the scientific reality. Some products would deserve to be placed under the status of a biological medicinal product so that they can undergo the appropriate controls to ensure the protection and safety of patients.ad hoc rules, creating a sort of regulatory patch, to fit this or that product into the existing regulatory categories.A modification of the definition of a biological medicinal product, which is fundamental because it conditions its potential categorization in the GTMP status, so that it is no longer only based on its biological source but possibly on its control, would resolve this issue and avoid complex situations where the EMA and the European Commission have to issue numerous Thus, a biological medicinal product should be a medicinal product whose active substance:•is produced or extracted from a biological sourceand/or•needs for its characterization and the determination of its quality a combination of physico-chemical-biological testing, together with the production process and its control.o 1394/2007 and some CAT recommendations, it seems that the word “recombinant” should be understood as an artificial recombination of nucleic acids. For example, a chimeric adenovirus obtained “simply” by a process of bio-selection is not an ATMP since it was not manipulated and therefore the therapeutic action is not mediated by any recombinant nucleic acid  Neic acid .recombinant nucleic acid techniques involving the formation of new combinations of genetic material by the insertion of nucleic acid molecules produced by whatever means outside an organism, into any virus, bacterial plasmid or other vector system and their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation.”We can also refer to the definition of “recombinant techniques” in Directive 2001/18/EC on the deliberate release into the environment of genetically modified organisms: “Of note is that the US definition of recombinant nucleic acid is: “molecules that (a) are constructed by joining nucleic acid molecules and (b) that can replicate in a living cell.”in vitro from a DNA template) fulfill the definition of a GTMP, and so are considered as recombinant whatever they are self-replicating or not.Currently, the CAT considers that messenger RNAs (mRNA) that are produced biosynthetically . So, in the US, mRNA technologies should not be considered as gene therapy.This divergence results most likely from a different vision of gene therapy. In fact, the FDA relies mainly on the mechanism of action to define gene therapy, which requires an interaction with human DNA in the nucleus . ConversSo, such differences resulting from different visions of what mRNA and gene therapies are, lead to discrepancies in the status of products marketed on either side of the Atlantic. Indeed, as seen above, an mRNA-based drug will be a GTMP in Europe (except for a vaccine against infectious disease) while it will not be so in the USA .This divergence has consequences in terms of requirements for the Marketing Authorization to ensure the safety and security for patients (see below).The solution could come from the ICH which is in charge of harmonizing the MA files and requirements between its members. A draft of the ICH guideline S12 gives a definition of gene therapy that is quite far from the European and US definitions .Gene therapy (GT) products within the scope of this guideline include products that mediate their effect by the expression (transcription or translation) of transferred genetic materials. Some examples of GT products can include purified nucleic acid , microorganisms  genetically modified to express transgenes (including products that edit the host genome), and ex vivo genetically modified human cells. Products that are intended to alter the host cell genome in vivo without specific transcription or translation  are also covered in this guidance.”The ICH, in its S12 guideline (still in draft form at this time) has introduced a singular definition of medicinal products for gene therapy: “So, this definition could resolve the question of the heterogeneity of similar RNA-based drugs being produced by a chemical or biological approach as the mode of production is not mentioned neither the question of recombinant nucleic acids. However, ncRNAs which do not mediate their effect by the expression (transcription or translation) of transferred genetic materials and do not alter the host cell genome are still not considered gene therapy whereas, according to the EU definition, they could be in the case where they are biologically produced and recombinant (BERAs).ex vivo genetically human modified cells, they are also considered gene therapy in the ICH definition. This could resolve the question of the different statuses for CAR-T cells depending on the strategy used to prepare them (see paragraph “Specific case of mRNA-encoded Chimeric Antigen Receptor”).Concerning Finally, direct in-body gene editing using CRISPR/Cas9, fall within the scope of the proposed ICH regulation whatever, once again, is the approach. Whereas according to EMA/CAT nanoparticles containing the gene editing components (CRISPR/Cas9 and the single guide RNAs) intended for the treatment of Recessive Dystrophic epidermolysis bullosa, do not fall within the definition of ATMP as the product does not contain an active substance which contains a recombinant nucleic acid administered to human beings with a view to regulating, repairing, adding, deleting a genetic sequence .In conclusion, this definition simplifies the categorization of mRNA-based drugs compared to the EU regulation and allows harmonizing the EU and US regulations, as it does not consider the origin of RNA. It also solves the question of genetically modified cells and of genome-editing methods see . Howevervs. non-infectious diseases – and for non-vaccine drugs, on the type of RNA substance and its production. For vaccines, although subject to discussion, the target criterion is easy to use for classification. Moreover, it is harmonized with the US legislation. On the contrary, for non-vaccine RNA-based drugs, the current evolution of technology in this field and the possibility to produce similar drugs using different approaches, make it necessary to amend the legislation to avoid similar products being covered by different legal frameworks. In particular, a clarification of the definition of a biological medicinal product considering not only the biological source of the product but its physico-chemical-biological control, and of a recombinant nucleic acid, could solve some problems. However, such modifications, in addition to not solving all the problems, do not address the issue of the international harmonization which is necessary. Indeed, there are currently major differences between the EU and the US legislations. In this respect, the proposed definition of a gene therapy product by the ICH represents an important step forward. The question of nc RNA-based drugs which are not concerned by this proposition should, however, be discussed with regard to the most important criterion to consider for a gene therapy drug, i.e., safety and protection of patients. According to the EU, GTMPs require specific tests or trials to evaluate the risk of germ-line transmission, insertional mutagenesis, tumorigenicity, and embryo/fetal and perinatal toxicity and, according to article 15 of Regulation 1394/2007 , may involve retrotransposons LINE-1 (long interspersed element-1), which are remnants of ancient retroviral infections containing endogenous RT; they represent about 17% of the human genome, among them few copies are active. These elements predominantly transpose their own copies; however, they can also promote the retrotransposition of other cellular RNAs, including short interspersed elements (SINEs) and less commonly, small nuclear non-coding RNAs and mRNA –66. Althprimum non-nocere should therefore be applied in regulatory matters.Thus, while the international harmonization proposed by the ICH seems inevitable and desirable, it will not solve all the problems of categorization. The forthcoming revision of European regulations, which should incorporate the ICH definition, could lead to a greater clarity in categorization, but vigilance will be required, since a minor change in the regulations could have unexpected cascading repercussions on future and past categorizations. The notion of One solution could come from drafting specific guidelines for RNA drugs. These guidelines could go beyond the differences in categorization and thus be applied across the board. Logically, they should make it possible to establish specific safety rules, particularly if two identical but differently produced RNAs are placed on the market.In addition to the guidelines already applicable according to the legal categorization, such guidelines could also ensure the safest possible development, by integrating the appropriate toxicological studies according to the risk of oncogenesis, integration, cell migration, etc.As far as production is concerned, the guidelines in force provide numerous guarantees, and the notion of risk-based approach (for ATMPs) allows for the closest possible adaptation to each medicinal product profile. However, specific guidelines for verifying the sequence produced and the three-dimensional conformation could perhaps prove useful.Finally, specific guidelines for vigilance could be interesting, in particular to ensure long-term monitoring. The reinforcement of the follow-up of chemically produced RNAs should be considered.The regulation of EU RNA-based medicinal products is very complex, and this is likely to become more pronounced with the evolution of technology. Moreover, there are profound disparities between the EU and the US classifications.The current EU regulation raises the question of a biological medicinal product which should be modified to consider not only the biological source of the product but its physico-chemical-biological control to include all RNA-based drugs whatever their production.Considering RNA-based drugs and gene therapy, the ICH definition proposal should allow simplification and harmonization between the EU and US regulations for mRNA-based drugs, among them mRNA-based cell therapies as well as for gene-editing therapies and facilitate marketing. However, it will not solve all the issues related to RNA-based drugs, especially since some of them are not covered by the definition, such as mRNA vaccines and ncRNA. Therefore, the drafting of specific guidelines could ensure patient safety by imposing specific controls.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins – Hfq, ProQ, and CsrA – have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism. The world of cellular RNAs is surprisingly rich in complexity and regulatory potential, with RNA molecules serving indispensable roles in sophisticated mechanisms controlling cellular homeostasis and adaptive responses. Starting with stable RNAs that support translation, like rRNAs and tRNAs, through mRNAs carrying information about proteins sequence, RNA species can guide or catalyse chemical reactions, and many encompass regulatory functions in controlling the expression of genetic information. Different RNAs have explicitly defined functions in the cell, with precisely specified fold, targets, and half-life. Although the pool is immensely diverse, the individual RNA species share in common protein companions that stand by and ensure their stability and facilitate their functions.RNA chaperones, the proteins that ensure RNA integrity, structure and function, are less diverse than the RNA molecules themselves, and a single chaperone is often capable of interacting with many different RNAs. In bacteria, RNA chaperones facilitate proper RNA folding, remodel existing states and expose crucial regulatory elements to assist in matchmaking between regulatory RNAs and their targets. Chaperones also protect bound RNA from ribonucleases and effectively steer RNA molecules to their destination . As suchThe biological necessity of RNA chaperones might be apparent when considering that RNAs are highly labile molecules and do not exist naked in the cell due to the high probability of rapid degradation by ribonucleases. In addition, RNAs are postulated to be prone to misfolding, and incorrect structures of RNA molecules can lead to significant inefficiency of RNA-dependent processes or aggregation . VariousOne of the most conserved and pleiotropic RNA chaperones in bacteria is Hfq. First described as an essential host factor of the RNA bacteriophage Qβ, Hfq was quickly recognised as an indispensable element of bacterial RNA metabolism. Hfq belongs to the widely occurring family of Sm and Sm-like (LSm) proteins, which places its evolutionary origins as far back as the common ancestor of bacterial, eukaryotic and archaeal lineages . Hfq haschiPQ operon in Salmonella [Hundreds of sRNAs have been reported to interact with Hfq which assists sRNA-mediated regulation through several mechanisms . Upon bilmonella .Escherichia coli and Salmonella enterica, but is also present in some gram-positive bacteria, where it participates in stress response and virulence [Staphylococcus aureus, Neisseria monocytogenes, and some clades among Bacillus and Clostridium genera [Staphylococcus aureus is not clear as the deletion of the chaperone does not impair sRNA mediated regulation in this bacterium [E. coli.About half of bacterial genomes encode Hfq or its close homologues , and funirulence ,18. Hfq irulence   and two (-RKER-) arginine residues, respectively, Hfq variants in some gram-positive bacteria with diminished RNA annealing activity, like Bacillus subtilis Hfq (-RKEN-) or S. aureus Hfq (-KANQ-), have lower arginine content.The core of Hfq forms a platform for RNA interaction with three RNA binding surfaces available, each with different affinities for specific RNA sequences . The prosRNAs have different modes of binding to Hfq and are generally classified into two groups – Class I or Class II – based on their proposed modes of interaction ,30. ClasE. coli cells, Hfq localises predominantly in the cytoplasm, while 10–20% of the molecules can be found in the nucleoid region [In exponentially growing d region ,34. Hfq d region . Single-E. coli cells are subjected to certain stresses, which often provoke an sRNA-mediated response, the diffusivity and cellular distribution of Hfq changes. Recent studies have shown that Hfq and sRNAs accumulate at the poles of E. coli cells when these are subjected to hyperosmotic stress [However, when c stress . Levels E. coli cells experience growth attenuation and activate the nitrogen regulation stress response [hfq bacteria. Overall, these findings suggest a compartmentalisation of ribonucleoprotein complexes, in which Hfq plays a central role, involved in the adaptative response to certain stresses through post-transcriptional regulation of gene expression.Another strong trigger of stress response is loss of nitrogen nutrient availability. Under nitrogen starvation, response . While cresponse . Under cresponse . These aresponse . Thus, tresponse ,41. Hfq E. coli cells depending on the growth conditions [Hfq is an abundantly expressed protein, with levels ranging 3,000–10,000 Hfq hexamers in nditions ,42. Howenditions ,43–47. Dnditions ,45,48. Fnditions . Throughnditions . Conversnditions .rpoS and dgcM [Hfq is known to mediate the imperfect base-pairing between sRNAs and their targets. In this regard, the chaperone could be aiding to surpass a kinetic barrier that otherwise prevents the duplex formation. Such kinetic barrier can be imposed by elements of secondary structure, like stem loops, present close to the sRNA binding site in the target mRNA. These can affect the speed of target recognition by the sRNA as well as the rate of successful RNA duplex formation . Recent and dgcM ,51.et al. evaluated the impact of overexpression of well characterised sRNAs on their respective target mRNAs, through transcriptomic analysis combined with the application of RIL-Seq (RNA interaction by ligation and sequencing) to determine the sRNA-mRNA pairs bound to Hfq under those experimental conditions [Changes in the levels of sRNAs do not affect all their mRNA targets in the same way. In a recent study, Faigenbaum-Romm nditions . The RILnditions . Their rnditions . NeverthtraJ [Legionella pneumophila [et al. discovered its homologues are present in many α, β and γ proteobacteria that do not encode ProP [ProQ is a FinO-like protein that was initially identified as a factor affecting post-translational activation of the osmoregulatory transporter ProP ,55. FinOtraJ . Anotherumophila . ProQ waode ProP   and a C-terminal domain (CTD), connected by a flexible linker of 50 amino acids which might contribute to RNA binding . The NTD patches . The preE. coli ProQ is mainly composed by β -strands that form a barrel-like structure, and the surface is overall electrostatically neutral [The C-terminal domain of  neutral ,62 , which mapped for the first time the  E. coli . ProQ re E. coli ,64. The  binding . The CTD binding  , are widely distributed in eubacteria. They can be found in gram-positive species, but are mostly distributed within the gram-negative γ-proteobacteria  was initially discovered as a carbon storage regulating protein. Its role in post-transcriptional regulation is determined by interactions with the 5’ UTR and coding regions of many mRNAs, which can result in either activation or repression of their translation. CsrA is the main player of the stationary-phase metabolite control system, but its activity modulates expression of different genes through the entire bacterial life cycle, regulating not only carbon metabolism but also motility, virulence, iron homeostasis, cell envelope integrity and biofilm formation. In e levels  and overbacteria . Some mer genome ,71. In Bs target .Pseudomonas spp., yet possesses a short α-helical insert between β strands 2 and 3 that stabilises the dimer [1 strand of one protomer links with the β5 strand of the other, forming a novel RNA binding surface [The structure of CsrA revealed a homodimer with each monomer composed of five antiparallel β strands linked with an unstructured C-terminus through a small α-helix ,74. The he dimer . Two RNA surface ,76,77  remains poorly understood. Different studies trying to unravel the functions of this unstructured region have come to contradictory conclusions. While some studies have indicated that deleting this region does not disrupt sRNA-mediated control of gene expression, others have found the CTD critical for this type of regulation . FurtherE. coli spans residues 66–102 and is highly enriched in acidic residues in the tip (-DSEETE), while in P. aeruginosa the tail is only 16 amino acids long and has no particular enrichment at the tip. A cross-comparison of over 220 Hfq proteins from different bacterial species divided the CTD into three regions [E. coli, the start of the tail is marked by a strongly conserved proline (P64) followed by a set of residues which also show a high conservation, especially R66, that pack against the core of Hfq. After the core packing region, there is a middle linker region (residues 73–96) which varies highly in length and sequence in different bacteria, and at the very end of the CTD occurs the acidic tip [The CTD of Hfq is intrinsically disordered and varies in length and amino acid composition across different bacterial species. For instance, the C-terminal tail in  regions . In E. cidic tip   does not seem to substantially alter the equilibrium dissociation constants of RNA to the proximal and distal faces [65 molecules show an increased affinity for RNA that interact with the chaperone through the rim, pointing to an inhibitory effect of the CTD. When the target RNA binds to the distal face, Hfq–RNA interactions appear to be immune to the autoinhibitory effects of the CTD [E. coli Hfq, R16, R19 or K47, weakens CTD-rim interactions, and changing the acidic residues in the tip of the CTD for non-electronegative ones disrupts them completely [Competitive binding experiments using a fluorescently labelled CTD peptide have shown that the acidic tip of the CTD could mimic nucleic acids and transiently interact with the rim of the Hfq core . Throughal faces . In cont the CTD . The str the CTD . Mutatinmpletely . Besidesmpletely ,95.In vitro experiments have shown that the capacity of ChiX (Class II sRNA) to outcompete DsrA (Class I sRNA), RyhB (Class I sRNA) and RprA (mixed Class I/II) for the binding to Hfq is negatively affected in Hfq65 compared to full-length Hfq [In vivo, the removal of the CTD of Hfq leads to a decrease in the accumulation levels of several Class II sRNAs, which lose their kinetic advantage over other RNA molecules, with regard to Hfq binding, and are more rapidly degraded than in the presence of the full-length chaperone [The capacity of RNA to compete against the CTD for core binding is directly correlated with the length of the nucleic acid and thus the rim appears to have low sequence-specificity . Howeverngth Hfq . In vivohaperone . Notwithhaperone . Additiohaperone .in vivo, has revealed that combinations of mutations in the CTD and the distal face or the rim have synergetic effects which points to a collaboration between the tail and these surfaces on E. coli Hfq [mutS mRNA by Hfq. For distal face function, the core packing region (residues 65–72) seemed to be the only required section of the CTD for Hfq-mediated regulations and the highly conserved R66 appeared to be the most critical residue. Conversely, full-length CTD of Hfq and particularly its tip appears to interact and cooperate with the rim to mediate the regulation of sodB by Class I sRNA RyhB.Furthermore, a recent study on the implications of the CTD coli Hfq . For insE. coli Hfq was fused to the CTD of B. subtillis Hfq (-KNVQLELE), shorter and slightly less acidic than that of E. coli, showed that B. subtillis CTD contacted the rim more often than E. coli CTD, but less strongly and with different amino acid preference [Caulobacter crescentus Hfq with a CTD which also has an acidic tip (-DADD) but is substantially shorter than that of E. coli Hfq (14 residues vs 36 residues). The comparison between the RNA binding activity of the C. crescentus variant and that of E. coli Hfq showed that the latter had a higher affinity for E. coli sRNAs, but also for sRNAs found in C. crescentus [C. crescentus Hfq was fused to the E. coli Hfq core, this chimeric variant had lower RNA binding affinities than the E. coli variant, being similar to the binding affinities of C. crescentus Hfq. This higher degree of autoinhibition by the CTD of C. crescentus Hfq also lead to a stronger inhibition of RNA annealing and could be explained by the higher local density of acidic residues at the tip.The determination of the properties that govern CTD-rim interactions poses the question on the role and mode of action of these tails in species in which Hfq has a different number of basic or acidic residues in the core rim or the C-terminal tip, respectively, or in which the length of the linker region in the CTD varies. The analysis of a chimeric Hfq variant in which the core of eference . A more escentus . When thE. coli Hfq. On the one hand, the initial part of the tail is proposed to participate in the function of the distal face of Hfq and mutations in R66 have been shown to affect the binding of Class II sRNAs and of mRNAs to this surface [Overall, to date, two main roles on RNA binding have been predicted for the CTD of  surface   at the ribosome-binding site: the binding of CsrA inhibits the translation of Hfq by impeding the ribosome to interact with the transcript without leading to mRNA degradation [Hfq is known to interact and interplay with other RNA binding proteins than ProQ, for example CsrA. So far, little is known about the functional meaning of the target overlap of these two riboregulators, nevertheless, some examples lead to speculation regarding the biological relevance of the interplay.  and Hfq ,114. hfqsrlA transcript, which encodes a component of a d-sorbitol-specific phosphotransferase system. When Spot42 binds srlA, it negatively regulates the translation of srlA mRNA in a Hfq-dependent manner. CsrA has been recently shown to bind Spot42, preventing cleavage of the small RNA and leading to an enhanced repression of srlA. Furthermore, Spot42 is not as efficient as CsrB and CsrC in recruiting CsrA, so there is potential for graded effects depending on the relative abundance of these factors. The discovery of the interaction with the Spot42 has opened tantalising possibilities for a new role and mechanism of action of CsrA [Another example is the sRNA Spot42, which is bound both by Hfq and by CsrA ,116. Spo of CsrA ,116.P. aeruginosa the RNA-binding protein RsmA binds to nascent transcripts [hfq [estA) and monocistronic mRNAs (amrZ). By binding to the same transcript, RsmA and Hfq could act in combination to exert control on translation and abundance of transcripts in the cell: if RsmA and Hfq are sensitive to different environmental stimuli, this would enable the control of common transcripts in a synchronised way that reflects the effects of both chaperones [Finally, in nscripts , like hfpts [hfq . This coaperones .P. aeruginosa, i.e. the Hfq-Crc mediated translation repression system [P. aeruginosa, succinate, is depleted. When succinate is available, Hfq and Crc (catabolite control protein) mask ribosome-binding sites on target mRNA transcripts to prevent translation of genes involved in the uptake and metabolism of secondary carbon sources [amiE mRNA revealed for the first time how Hfq captures and presents substrate RNAs to Crc during CCR [amiE and more than 100 other metabolic mRNAs [amiE intermediate assembly by Krepl et al. revealed a putative mechanism for rapid cycling of RNAs on the Hfq distal side. Local perturbations in the form of syn-/anti flipping of the A-site base in its distal Hfq pocket could allow for rapid exchange for of RNAs on the Hfq surface, and more research will further explore this [A well-studied example of cooperative targeting of transcripts can be found in the main Carbon Catabolite Repression (CCR) regulon in n system ,119. CCR sources . Recent ring CCR  Fig. 6)P. aerugiic mRNAs ,118,121.ic mRNAs ,122. Notore this .Figure E. coli and many other bacteria, several of the key enzymes involved in RNA processing and degradation assemble to form a central multi-enzyme machinery in the cell, known as the RNA degradosome [RNase E is a key enzyme for RNA metabolism in many bacteria. It belongs to the RNase E/G enzyme family, with members present across the proteobacteria, actinobacteria and firmicutes phyla, and with homologues also found in cyanobacteria and plant chloroplasts. RNase E is an endoribonuclease catalysing the first RNA cleavage that initialise a complete degradation of transcripts ,124. Howradosome .in vivo in the absence of RNA molecules [The potential interplay between Hfq and the degradosome has been postulated in many studies. The interaction between RNase E and Hfq is RNA-dependent, and the two proteins are unlikely to directly interact olecules ,127–129.olecules . Moreoveolecules . This coolecules . The matPNPase is a conserved, ancient exoribonuclease that in bacteria processively degrades RNA molecules from the 3’ end either in isolation or in conjunction with the RNA degradosome. However, PNPase is more than just an RNA degrading enzyme, and its multiple functions include stable RNA processing and polymerisation of heterogeneous tails on existing RNA molecules . The repDeletion of PNPase causes increased stability of many transcripts, but, surprisingly, also destabilization of several sRNAs. The recent characterisation of the structure of a PNPase-sRNA-Hfq ternary complex by cryo-EM helped to understand the interactions underpinning this phenomenon  Fig. 8)Fig. 8). The PNPase-sRNA-Hfq complex brings together PNPase and Hfq. However, no contacts are made between these proteins and the ternary complex is maintained solely by interactions of both proteins with the same RNA molecule. This allows for some flexibility in the formation of protective complexes of this kind. In these assemblies, the sRNA interacts only with the RNA binding domains of PNPase, S1 and KH, being rerouted away from the catalytic core, where the active sites of the enzyme localise. Hfq binding on the other side, sandwiches the sRNA between both proteins and makes it to some extent inaccessible to other cellular components. As such, the Hfq-sRNA intermediate hijacks the PNPase RNA binding modules (KH-S1) for downstream regulatory processes (e.g. pairing with a target RNA) and bypasses the catalytic core entirely. Within this complex, however, the RNA must still be able to pair with its target transcript. Thus, mechanisms must be in place to allow for relaxation of the assembly when the target mRNA is encountered, although these are still to be characterised.E. coli, the main cold shock protein is CspA, and nine proteins from the CspA family have been identified, named CspA to CspI. Homologues of CspA are widespread in prokaryotes, acting in response to many different environmental stresses [Besides Hfq, ProQ and CsrA, other proteins with RNA chaperoning function can be found in bacteria, some of which are limited to certain RNAs, while others act on larger RNA pools. Among these proteins, cold shock proteins have an important role. RNA molecules have a natural ability to adopt more than a single stable conformation, and the adoption of a certain conformation depends on environmental conditions. Upon cold shock, the structure of RNA changes as with the drop of the temperature many RNA structures become more stable, changing RNA accessibility and fold. Under these conditions, cold shock proteins (Csp) exert their chaperoning activity and melt RNA secondary structures, restoring processes such as transcription and translation . In E. cstresses ,134.in vitro [E. coli as a homologue of the histone-like protein H-NS, was shown to be able to act as a non-specific RNA chaperone [in vivo substrate of this protein has been identified so far. StpA participates in the regulatory circuit of ompF expression by modulating the stability of the sRNA MicF that represses ompF translation [in vitro [The CspA fold is structurally similar to the fold of the S1 domain, an RNA binding domain identified in many proteins involved in RNA metabolism . Six copin vitro . Anotherhaperone . Howevernslation . StpA chin vitro .RNA fulfils a broad range of functions in the cell: from encoding the amino acid sequence in proteins, through acting as a building block for many molecular assemblies, catalysing reactions, transducing cellular signals, to being a regulator of many cellular processes. This versatility has arisen in the face of the labile character of RNA molecules, their chemical instability and susceptibility to ribonuclease attacks. Furthermore, RNA molecules can adopt more than one stable conformation and often get trapped in folding intermediate states. RNA chaperones can prevent or resolve such trapped states without requirement for ATP consumption. Instead, they simply bind and release an RNA molecule so that misfolded regions are destabilised, and the structural confinement resolved. They can also help in annealing two RNA molecules and stabilising bound RNA substrates to support their function .B. subtilis were shown to act as chaperones [Streptococcus pneumoniae, proteins with a KH domain, like KhpA and KhpB, could act as RNA chaperones [RNA chaperones constitute a very diverse group and are widespread across all domains of life. On the one hand, the list of bacterial proteins with chaperone activity is constantly growing. For example, three small proteins FbpA, FbpB and FbpC in aperones , not to aperones . Moreoveaperones . On the aperones .P. aeruginosa [Also increasingly apparent is the expanded network of cooperation and interplay between RNA chaperones and between the chaperones and other proteins involved in RNA metabolism. The recent discovery of the overlapping targetome of Hfq and ProQ suggests that these most common RNA chaperones could be able to support the same pathways or compete for their targets depending on the circumstances ,112,146.ruginosa . Moreovein vivo? How does it detect if the RNA structure is right? What is the explanation of the large discrepancy between the number of RNA chaperones in the cell and the number of RNA targets? When is a chaperone-bound RNA fully released? How is the RNA delivered to its destination? How does the pool of RNAs get accurately divided between cellular chaperones? How do the chaperones manage to coordinate their RNA targets without creating chaos by cross-interference? Despite some hints from bacterial physiology that indicate spatial separation of different processes in the cell or characteristics of RNA substrates bound by a particular chaperone, clear answers to these questions are yet to come.Although the field is mature, many questions remain unanswered, e.g. how does a chaperone find its RNA target