RNA-binding proteins (RBPs) play vital roles in many processes in the cell. Different RBPs bind RNA with different sequence and structure specificities. While sequence specificities for a large set of 205 RBPs have been reported through the RNAcompete compendium, structure specificities are known for only a small fraction. The main limitation lies in the design of the RNAcompete technology, which tests RBP binding against unstructured RNA probes, making it difficult to infer structural preferences from these data. We recently developed RCK, an algorithm to infer sequence and structural binding models from RNAcompete data. The set of binding models enables, for the first time, a large-scale assessment of RNA structure in the RBPome.We re-validate and uncover the role of RNA structure in the RPBome through novel analysis of the largest-scale dataset to date. First, we show that RNA structure exists in presumably unstructured RNA probes and that its variability is correlated with RNA-binding. Second, we examine the structural binding preferences of RBPs and discover an overall preference to bind RNA loops. Third, we significantly improve protein-binding prediction using RNA structure, both in vitro and in vivo. Lastly, we demonstrate that RNA structural binding preferences can be inferred for new proteins from solely their amino acid content.By counter-intuitively demonstrating through our analysis that we can predict both the RNA structure of and RBP binding to these putatively unstructured RNAs, we transform a compendium of RNA-binding proteins into a valuable resource for structure-based binding models. We uncover the important role RNA structure plays in protein-RNA interaction for hundreds of RNA-binding proteins.The online version of this article (10.1186/s12864-018-4540-1) contains supplementary material, which is available to authorized users. Protein–RNA interactions play vital roles in many processes in the living cell. These effect of a wide variety of cellular processes, including RNA replication, repair, recombination and post-transcriptional regulation . More thVarious high-throughput experimental techniques have been developed to measure protein-RNA binding. The CLIP-seq protocol and its variants measure protein-RNA binding in vivo, but they all suffer from noise and technological biases , 4. In vHere, we show, for the first time on a large scale, that RNA structural variability exists in unstructured RNA probes, and that it can be used to significantly improve the accuracy of binding predictions in various analyses. First, we show, via different approaches, that structure variability exists in the data, and that it correlates with protein binding. Then, we newly apply our recently-developed method RCK to catalog binding preferences of RNA-binding proteins on a large-scale. Third, we demonstrate how RNA-binding structural preferences learned in vitro improves binding prediction, both in vitro and in vivo. Lastly, we demonstrate that using RNA structure improves machine-learning methods for inferring RNA-binding preferences based solely on amino acid sequence. Taken together, these results further highlight the important role RNA structure plays in the RBPome.We first sought to test how much structure variability exists in unstructured RNA probes, which are the basis of the largest compendium of protein RNA-binding measurements . UnstrucOur results newly confirm on a large scale that unstructured probes are far from being unstructured. While the probes tend to have lower base-pairing probabilities than random probes, there exists significant structural variability in them. Indeed, more than 75% of unstructured probes have a non-zero probability of residing in a low-energy conformation, implying that some structure exists, albeit with smaller probability . Correlation of UUUUU base-pairing probabilities for random probes, using the same binding affinities, was r = 0.012 . These results show that variability at RNA structure binding sites is correlated with experimental binding affinities, and can potentially aid in learning RNA sequence and structure binding preferences of RBPs. We note that the correlation might be so slight due to the low base-pairing probability and structural variability in the RNAcompete probes.To demonstrate that this variability is correlated with protein-binding affinity, we focused on the binding affinity of the well-studied HuR protein. The HuR protein is known to preferably bind uridine-stretches in unpaired RNA structural context . Thus, wWe newly measured the distribution of structural preferences for a large compendium of RNA-binding proteins (RBPs) . Thanks Here, we perform what we believe to be the first rigorous test of the assumption that many RBPs prefer to bind unpaired regions , 12. ForWhile RBPs prefer to bind unpaired regions as a whole, some prefer to bind either loop or external regions; both regions are unpaired, yet loop regions are surrounded by a stem of paired nucleotides. To examine the preference of RBPs to bind loop or external regions, we measured the ratio of binding to loop regions as compared to external regions. Our results show that proteins vary in binding preference, and many do not demonstrate a strong preference ; however, overall more proteins in this dataset prefer to bind loop regions than external regions Fig. . We obseWe gauged the contribution of RNA structural preferences in binding prediction. We employed a comprehensive dataset of both in vitro and in vivo data. For in vitro prediction, we used 244 RNAcompete paired experiments, where we trained a model based on one half of a pair and tested on the other half of the same pair, resulting in 488 predictions . Predictp-value = 5.85·10− 82, Wilcoxon rank-sum test). When we assigned random structure probabilities, this improvement disappeared     Fig. , and simWe specifically focus on two RBPs, HRNPK and PUM2, for which structural information contributed most to in vivo binding prediction accuracy. HNRNPK is an hnRNP family protein that plays diverse roles in multiple processes . Ray et The second protein assessed in the current study, PUM2, is a sequence-specific RNA-binding protein that acts as a post-transcriptional repressor . AccordiWe explored the ability to predict RNA-binding preferences from amino acid sequence alone. This capability would enable binding prediction for mutated proteins (e.g. from genetic variation) as well as proteins with no experimental validation. In a recent study, Pelosoph et al. developed AffinityRegression, an algorithm for learning and predicting amino-acid k-mer associations with DNA and RNA k-mers . These aWe sought to improve AffinityRegression’s performance by including RNA secondary structure in the model. We augmented the RNA k-mer space with structure-based probabilities based on five contexts: paired, hairpin loop, inner loop, multi loop and external. We applied the same tests as used by AffinityRegression to gauge performance: Pearson correlation between predicted probe intensities to the measured ones, and area under the precision-recall curve (AUPR) for ranking 1% of top probes compared to (i) 50% of bottom probes, and (ii) 99% of bottom probes.p-value = 6.79·10− 23 Wilcoxon signed-rank test). Performance also improved significantly when gauged by AUPR criteria . When RNA structure probabilities are assigned randomly, we see no significant improvement when utilizing RNA structure  . While our models only address local RNA structure, and thus may miss more complex structures of long RNA molecules found in vivo, we were able to significantly improve in vivo binding prediction using structural information as opposed to using sequence scores alone. Detecting binding preferences to large RNA structures is still a major challenge as computational RNA structure prediction has been shown to be less accurate for long RNA molecules in vivo . Still, We wish to expand the RBPome prediction by augmenting it with new protein-RNA binding models and more accurate RNA structure predictions using experimental and computational advancements. We plan to expand our dataset of binding models by developing algorithms to learn RNA structure and sequence preferences from RNA bind-n-seq, a new technology to measure protein RNA-binding in vitro . We alsoIn this work we have taken the first steps in exploring the role of RNA structure in the RBPome on a large scale. We have demonstrated the importance of RNA structure in various applications of protein-RNA interactions. We expect the insights from this study and our resource to aid the research community in making significant advances in understanding the critical roles of protein-RNA interactions.We measured the structure of two RNA probe sets. One is the set of 241,357 RNAcompete probes, containing 219,990 unique sequences . The repw and structural context a with the highest binding score. We calculated the binding scores for k-mer w in the other structural contexts. The relative weight of those scores are the structural preferences assigned to that protein. For Fig. We explored the overall statistic of structural binding preferences amongst the set of RNA-binding proteins in the RNAcompete dataset . We usedWe tested the benefit of adding structure to the models for in vitro and in vivo binding prediction. We used the set of learned models from the RCK website . For in We used the original code of AffinityRegression with some modifications . For theAdditional file 1:A) Distribution of probe unstructured probabilities. Random probes are less likely to be unstructured, but more than 75% of unstructured probes have smaller-than-one probability of being unstructured. B) Distribution of nucleotide base-pairing probabilities. In random probes they are more skewed towards 1, but in unstructured probes they span the entire probability range of . (PNG 52 kb)Figure S1 Structure in unstructured probes. Additional file 2:A) RNA structural binding preferences do not improve in vitro binding prediction when random structure probabilities are assigned. Correlation results over 488 paired experiments reveals that RNA structure does not improve binding prediction when structure probabilities are assigned randomly. B) RNA structural binding preferences do not improve in vivo binding prediction when random structure probabilities are assigned. AUC results of 96 paired eCLIP and RNAcompete experiments over 21 joint proteins demonstrate that RNA structural binding preferences learned from in vitro data do not correlate well with protein-RNA interactions measured in vivo when structure probabilities are assigned randomly. (PNG 85 kb)Figure S2 Additional file 3:A) When we add RNA structural features to the sequence k-mer space of AffinityRegression, but assign structure probabilities randomly, we do no predict binding any better than using sequence features alone. B) When we add RNA structural features to the sequence k-mer space of AffinityRegression, but assign structure probabilities randomly, we do not predict the top-bound probes as compared to unbound probes any better than using sequence features alone. (PNG 68 kb)Figure S3 There is no improvement in binding prediction from amino acid sequence by utilizing RNA structure with random structure probabilities.


We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV These advantages are particularly relevant for low-abundant targets1. However, there is currently little information available specifically concerning the rationale and efficacy of reference gene normalisation to correct for variable quality of input RNA in RT-dPCR. Such variation is more likely when sampling from disease-associated or tumour tissues that incorporate inherent necrosis or where significant areas are affected by mechanisms leading to cell death6. Under such conditions, the degradation of cellular RNA can be up-regulated by specific signalling pathways9, or as a consequence of therapeutic drug response10.Recent studies have shown that reverse transcription dPCR (RT-dPCR) technology can overcome the adverse impact of a range of background sample contaminants and PCR inhibitors. This enables the generation of reliable data that, in some cases, may not require normalisation to reference genes13. Studies that caution the use of degraded RNA in downstream applications highlight the characteristically unstable nature of mRNA and its susceptibility to RNA hydrolases (RNases), particularly during the acquisition, handling and storage of tissue samples. Transcript-specific degradation has also been described as a consequence of delayed post-excision or post-mortem RNA preservation13. However, the integral quality of resource tissue, particularly of valuable human pathological samples, provides an equally important barrier to molecular analysis. The underlying challenge researchers face when working with pathological, often rare, study samples is the acquisition of quality, biologically meaningful data from an adequate number of samples4.The pitfalls of using low quality mRNA in quantitative molecular analyses are well-established14, a granulomatous lesion generally associated with more severe rheumatoid arthritis. A feature of rheumatoid nodules is the “natural” presence of areas of necrotic tissue, accompanied by varying degrees of cellular apoptosis, both of which are an inherent part of the inflammatory process15. RNA from these areas is unavoidably extracted and consequently present as a variable component of nodule tissue RNA used for downstream studies. Fragmented RNA of at least 100 nucleotides in length contributes to the total RNA concentration measurement prior to RT-dPCR16, although because many transcripts may not be sufficiently intact for complete reverse transcription, correction is required to ensure that the final data reflects gene expression in functional, non-necrotic areas of the tissue.A focus of our investigations into chronic inflammation has concerned pathogenic mechanisms within the rheumatoid subcutaneous noduleHere we consider the application of nanofluidic partitioning chip-based dPCR to quantitative gene expression in tissues complicated by necrosis. We sought to utilise this technique to exploit the increased detection sensitivity and precision capabilities attributed to dPCR, when compared to qPCR, while maintaining an existing, well-validated, sample processing workflow. Results show that measures of the expression of appropriate endogenous control genes, like GAPDH and HPRT1, are proportionate to the level of mRNA of quality sufficient to allow reliable reverse-transcription. We also sought to establish suitable inclusion criteria for samples according to the accuracy of normalisation. This approach limits the undesirable impact on target gene expression values that can arise when working with tissues containing inherent necrosis, while still maintaining the high level of detection sensitivity of the digital technology.17.Retrospective histological analysis showed that all nodule tissues used for RNA extraction contained palisading monocyte/macrophages, focused on a central necrotic area (data not shown). This is a characteristic feature of granulomatous rheumatoid nodules, that we have previously described200 metric, a measure of the percentage of RNA transcripts >200 nucleotides in length. We compared the DV200 metrics measured in nodule RNA samples with those in synovial membrane RNA, finding greater variability in RNA fragmentation in nodules . Both tissue types undergo similar handling suggesting that the quality of nodule RNA reflects in vivo cellular processes characteristic of these pathological, necrotic lesions.RNA quality analysis revealed that RNA purified from nodule tissues was variably fragmented between samples, despite consistent and best practice for sample handling and RNA preservation Fig. . The extles Fig. . Consequp < 0.0001) between combined expression levels for these two endogenous control genes and the DV200 metric  and with the proportion of intact starting RNA   Fig. . Interes14) Fig.  legend.Fper se. Therefore, irrespective of quality, nodule RNA is not expected to contain transcript that might significantly influence measures of PRTN3 expression. We measured PRTN3 expression in the spiked samples using RT-dPCR. Predictably, when assessed per unit of starting RNA, the absolute copies of PRTN3 expression reduced as the proportion of fragmented RNA increased  in nodules sed Fig. . Normalised Fig. . HoweverT) value of ~37 by qPCR.We investigated IL27A gene expression in rheumatoid nodule tissues using dPCR, applying normalisation and appropriate corrections for RNA fragmentation. Within our nodule sample set (n = 27), absolute measures of IL27A expression ranged from 0.25 to 14.16 copies/ng RNA. As a point of reference, a sample returning absolute measures of 0.63 copies of IL27A/ng RNA using dPCR corresponded to an average cycle threshold .Based on DVles Fig. . Normalifait accompli, with potential confounders associated with available human tissue, including characteristics of any inherent pathological processes, likely to influence the quality of the tissue and yield of accurate molecular results. This study examined the characteristics of human rheumatoid subcutaneous nodules, an inflammatory lesion with inherent necrosis, associated with granulomatous inflammation. The goal was to identify those features that determine the suitability of variably necrotic tissue for downstream molecular analysis, particularly the impact on the detection of low abundance gene expression using digital PCR.Key factors in conducting research in human disease are both the availability and quality of appropriate tissue samples. This combination is often a 18. Effectively, measures of RNA integrity generate exclusion criteria that dictate the suitability of samples. The dilemma is the application of these criteria to tissues where there is inherent necrosis as part of the underlying pathological process, which is likely to impact on RNA integrity. In rheumatoid subcutaneous nodules, necrosis is a focus of the inflammatory granuloma. However, tissue necrosis is common during the development of a wide range of diseases, varying in aetiology that includes a “programmed” or “regulated” process, activated in response to death receptor ligands or cellular stress21, or through a contribution to the cytotoxicity of many chemotherapeutic agents in cancer10. In such settings, restricting molecular analysis to high quality tissues limits the available tissue and is unlikely to reflect the true pathological process in operation.The critical influence of RNA integrity on downstream molecular analysis is well recognised. Development of measures of RNA integrity from the long-time standard based on 28 S to 18 S ratio, to a predictive model of RNA integrity, the RIN, now provides a much more reliable and robust measure that subsequently correlates with downstream analysis of gene expression200 metric, indicative of the proportion of extracted RNA transcripts greater than 200 nucleotides in each sample. This metric correlated significantly with expression levels of the endogenous control genes GAPDH and HPRT1, measured by dPCR. This indicates that amongst tissue samples where RNA fragmentation has been naturally generated in-vivo, the downstream quantitation of target gene expression predominantly reflects the levels present in that portion of RNA containing the most intact transcripts.Our results show that despite nodule tissue samples being harvested and treated using optimal RNA-preservation procedures, the presence of inherent, necrotic tissue contributes considerable amounts of highly fragmented RNA. In order to gauge the extent of RNA fragmentation, we considered the DV22. Using experimentally generated mixtures of RNA and dPCR assays for proteinase 3 (PRTN3) expression, our results show that normalisation of gene expression data to endogenous control genes’ expression, also accounts for the proportion of intact transcript present amongst highly fragmented RNA. Further, the normalisation of target PRTN3 expression in THP-1 cell RNA, was effective in samples containing as much as 50% fragmented nodule RNA. However, when the proportion of fragmented RNA increased beyond 50%, and up to 75%, normalisation tended to slightly over-estimate target gene expression. Thus, when considerable RNA fragmentation is present, the expression of endogenous control genes is affected and becomes a less accurate reflection of the intact, measurable portion of RNA. Consequently, normalisation becomes less reliable. Our data suggest that implementing a 50% fragmentation threshold, circumvents any problems associated with more fragmented RNA in pathological samples.Reference gene normalisation is recommended for gene expression analysis using alternative dPCR approaches, thereby circumventing any impact from RNA sample contaminants200 metric >50% to ensure reliable normalisation, we excluded 9 of 27 samples from the final data set but subsequently revealed meaningful values for IL27A expression in those remaining. We conclude that normalisation of target gene levels to suitable endogenous controls in RT-dPCR gene expression studies is appropriate when applied to pathological samples where inherent RNA fragmentation is present. The generation of reliable, normalised data also depends on the level of fragmentation within samples, and may require the implementation of appropriate exclusion criteria. In that regard, we do anticipate similar normalisation strategies could be applied when assessing gene expression in formalin-fixed, and paraffin-embedded (FFPE) tissues, where RNA fragmentation and low RIN scores23 are an inevitable consequence of the treatment. A more likely factor limiting the assessment of gene expression within FFPE tissues is chemical modification of the RNA, fragmented or not. Consequently, adherence to the recommendations for processing and storage of FFPE tissue that is destined for gene expression analysis23 and careful selection and validation of appropriate endogenous control genes24 are additional factors for consideration.We evaluated nodule RNA samples for IL27A gene expression, comparing absolute  and normalised outcomes of dPCR. In these samples, expression of this cytokine gene was generally low by conventional qPCR. Applying the criterion of a DV25, undergoing elective surgery for nodule removal. The patient cohort comprised those of age 64 ± 9 years (mean ± SE), disease duration 21 ± 3 years, with the majority positive for both rheumatoid facter and anti-citrullinated peptide antibodies. Five patients provided multiple nodules, and 3 further patients multiple synovia, either at the same or different times. The study was approved by the New Zealand Health and Disability Multi-Regional Ethics Committee (MEC06/02/003). All patients provided informed written consent prior to tissue donation and all experiments were performed in accordance with relevant guidelines and regulations.Subcutaneous nodule tissues (n = 27) or joint synovial tissues (n = 27) were obtained from patients (n = 42) with rheumatoid arthritis (RA), as defined by 1987 American Rheumatism Association Classification criteriaNodule and synovial membrane tissues were processed immediately following surgical excision in an RNase-free environment or collected into RNAlater (Invitrogen) at 4 °C. Apportioned tissues destined for gene expression studies were snap-frozen and stored in liquid nitrogen.20 (Invitrogen) as per the manufacturer’s protocol to generate cDNA. RNA fragment and DV200 metric analysis were conducted by NZ Genomics Ltd using an Illumina Fragment analyser or Agilent BioAnalyzer and associated software.The RNeasy Mini Kit (Qiagen) was used according to manufacturers’ recommendations, with DNase digestion either on-column using the RNase-free DNase Set (Qiagen) or post-extraction with PerfeCTa DNase I, (Quantabio). Eluted RNA concentration was determined using a Qubit Fluorometer (Invitrogen) with low or high sensitivity Qubit assay kit (Molecular Probes). Up to 1000ng of total RNA was added per 20ul reaction with Superscript III Reverse Transcriptase (Invitrogen) and primed with Oligo (dT)Digital PCR (dPCR) was performed using the Quantstudio 3D Digital PCR system (Applied Biosystems) including the chip loader, ProFlex thermal cycler, and chip reader. Reactions were prepared in 15ul volumes with Quantstudio 3D Digital PCR Mastermix v2 (Applied Biosystems), Taqman gene expression assays , and appropriate sample input amounts . Reactions were loaded onto Quantstudio 3D 20 K V2 dPCR chips (Applied Biosystems) and cycled as follows: PRTN3 10 min at 96 °C, 39X , 2 min at 55 °C, held at 10 °C; all remaining assays: 10 min at 96 °C, 39X , 2 min at 60 °C, held at 10 °C. Prior to running duplex reactions, assays were validated to ensure the duplex readings were consistent with respective single-plex assay readings over a range of input cDNA concentrations (data not shown). Quantstudio 3D AnalysisSuite Cloud Software  with Poisson Plus quantification algorithm (version 4.4.10) and 95% CI was used. Imported eds files were individually assessed and fluorescence thresholds set manually to generate transcript copies/ul, then converted to copies/unit RNA.26. An average coefficient of variation (CV) of 23.3%, and M value of 0.658 for the two genes was generated.To achieve data normalisation, copies/unit RNA of the target gene were multiplied by the corresponding normalisation factor  and final data displayed as normalised copies/unit of RNA. The stable expression of the two endogenous control genes was previously assessed among equivalent nodule samples using qPCR with qbasePLUS v2.1 (Biogazelle)200 = 10%) from 4 rheumatoid nodules, then generated a series of samples containing set proportions (100–0%) of good quality THP-1 cell RNA , spiked with increasing proportions of the pooled, highly fragmented nodule RNA (0–100%). This nodule RNA contained negligible values for averaged GAPDH and HPRT1 expression (0.0017 copies/pg RNA).Available data from RNA-seq analysis identified high expression of the proteinase 3 gene (PRTN3) in unstimulated THP-1 cells . In comparison, PRTN3 expression within nodule tissues was low . This contrast in expression was used to validate the use of normalisation to account for variable fragmentation within sample RNA. We pooled naturally, highly fragmented RNA , was reverse transcribed and expression of GAPDH and HPRT1 endogenous control genes and the PRTN3 target gene measured with RT-dPCR.Samples identified as having high, medium or low IL27A expression levels by dPCR were compared for IL27A expression in routine qPCR. Ten uL reactions containing Quanta ToughMix (Quanta Biosciences), Taqman Assay (Hs00377366_m1 (VIC)), and cDNA representing 31.25ng starting RNA, were assayed in triplicate using a Roche LC480 (Roche Molecular Systems) and standard cycling and detection protocols.200 metrics between 27 nodules and 27 synovia, following a D’Agostino & Pearson test establishing normal distribution (p > 0.05). The geometric mean of the GAPDH and HPRT1 expression was correlated with the associated DV200 metric values using a two-tailed pearson correlation test with 95% CI for log-transformed nodule data (n = 27) and spiked samples (n = 5) . Absolute and normalised IL27A gene expression values were compared using a two-tailed non-parametric Mann-Whitney test, following assessment with D’Agostino & Pearson normalisty test .Graphpad Prism V7.0b (Graphpad Software) was used for statistical analyses. An unparied t test with Welch’s correction for unequal variance was used to compare the DVNodule RNAseq datasets referred to during the current study are not yet publicly available, as complete data analysis is part of on-going research. Information therein is available from the corrsponding author on reasonable request.Supplementary Figures


Lymphatic metastasis contributes to the poor prognosis of laryngeal squamous cell carcinoma (LSCC). This study aimed to investigate the roles of two metastasis suppressor genes, KAI1 and nm23, in lymphangiogenesis and lymph metastasis of LSCC.A total of 45 LSCC patients were enrolled in this study. The positive expression rates of KAI1 and nm23 protein were detected via immunohistochemistry in 45 LSCC and 22 normal laryngeal mucosa adjacent to LSCC. Micro-lymphatic vessel density (MLVD) was detected via immunohistochemistry with the specific antibody D2-40. Associations between KAI1 and nm23 expression and clinical characteristics of LSCC were then evaluated.P < 0.05). Significantly lower positive rates of KAI1 and nm23 were found in LSCC with lymphatic metastasis than those without lymphatic metastasis (P < 0.05), whereas MLVD was negatively correlated with the expression of KAI1 and nm23 (P < 0.05). However, no significant associations were found between KAI1 and nm23 expression and clinical characteristics of LSCC .The positive expression rates of KAI1 and nm23 were significantly lower in LSCC than normal laryngeal mucosa (Both KAI1 and nm23 can inhibit lymphangiogenesis and lymphatic metastasis in LSCC. Laryngeal squamous cell carcinoma (LSCC) is a common tumor of the head and neck, and accounts for 5% of all systemic malignant tumors . Early sKAI1 is a metastasis suppressor gene belonging to the transmembrane 4 superfamily. KAI1 regulates cell movement, metastasis, and proliferation by regulating cell adhesion . The lowRecently, various factors have been revealed to be closely related with lymphatic metastasis of LSCC, such as Notch 2 , TRPP 2 n = 22) were used as control. This study was approved by the local Institutional Review Board, and informed consent was obtained from all subjects.A total of 45 LSCC patients without history of radiotherapy and chemotherapy were screened from the Department of Otolaryngology in Qingdao Municipal Hospital between 2002 and 2008. LSCC was diagnosed via histopathological examination, and the tumor stage was determined according to the tumor node metastasis (TNM) classification published by Union for International Cancer Control (UICC). The clinical characteristics of these patients, including sex, age, tumor location, differentiation grade, and T-stage, were recorded. LSCC tissues isolated from surgical resection were paraffin-embedded, sectioned, and preserved in the Department of Pathology. Normal laryngeal mucosa adjacent to LSCC  at 4 °C and washing in phosphate buffered solution (PBS), secondary goat anti-rabbit antibody was applied according to manufacturers’ recommendations . To detect the expression of KAI1, nm23, and D2-40, diaminobenzidine substrate  was used and counterstained with hematoxylin . Images were taken under a light microscope with a Photometrics  Coolsnap camera using Image Master version 5.0.We first dewaxed 3-μm paraffin sections of selected LSCC tissues in xylene followed by a graded series of ethanol washes. After washing with distilled water thrice, the samples were incubated in 10 mM sodium citrate buffer at 95 °C for 30 min for antigen retrieval. Then, these sections were incubated in 3% HKAI1 and nm23 were mainly expressed in the cell membrane and cytoplasm as granular brown substances , and tisD2-40 was also mainly expressed in the lymph vessel as a granular brown substance. In each sample, two regions at ×100 containing the largest number of D2-40 positive lymph vessels were selected as hot spots, and the vessels were counted in five random regions of each spot under the microscope at ×200 magnification . MLVD was calculated as the mean number of D2-40 positive lymph vessels in two hot spots.t test and were expressed as mean ± standard deviation. Qualitative data were compared using χ2 test and were expressed as number (percentage). A P value less than 0.05 was considered statistically significant.Statistical analysis was performed by using SPSS version 17.0 . Quantitative data were compared between different groups using P < 0.05) (Table P < 0.05)   (Table P < 0.05)  Fig. . Moreove5) Table . Further5) Table .Table 3TP > 0.05) (Table To further identify the effects of KAI1 and nm23 on LSCC, the relations between KAI1 and nm23 expression and clinical characteristics of LSCC patients were evaluated. The results showed no significant differences in terms of sex, age, tumor location, differentiation, and T-stage in LSCC patients either positive or negative to KAI1 and nm23 P > 0.0 (Table 5Metastasis is the main cause of the low curative rate of oncologic tumors. Angiogenesis and lymphangiogenesis are early events in tumor metastasis, and the therapeutic effects of angiogenesis are limited by the close relationship between the vascular system and lymphatic system . In thisKAI1, a metastasis suppressor gene, is involved in the regulation of LSCC metastasis . KAI1 exnm23 is also an important metastasis suppressor gene involved in the regulation of LSCC metastasis . The expKAI1 and nm23 regulate tumor metastasis through various molecules and pathways. KAI1 can regulate tumor metastasis through p130CAS-CrkII , p53 3434, and SKAI1 and nm23 were found to play a role in the inhibition of lymphangiogenesis and lymph metastasis in LSCC. However, they were not significantly related with the clinical characteristics of LSCC. Further researches on the effects of KAI1 and nm23 in LSCC and their related mechanisms in the inhibition of lymphangiogenesis and lymph metastasis using large samples are needed.


Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact DOI:http://dx.doi.org/10.7554/eLife.21510.001 Based on sequence alignments, helicases were subdivided into six superfamilies (SF) which come in two different flavors . Most meThe SF2 is by far the largest superfamily, comprising a vast number of RNA helicases with implications in numerous RNP metabolism pathways, such as translation initiation and termination, pre-mRNA editing and splicing, mRNA export as well as ribosome biogenesis . The cenThe latter mentioned helicase, Prp43, is an outstanding member of the DEAH-box subfamily since it has implications in different substantive cellular processes. The first reported function of Prp43 is its involvement in pre-mRNA splicing. In the course of this process, Prp43 acts at the latest stage of the splicing cycle and it is required to dismantle the intron-lariat spliceosome into the excised lariat and the U2•U5•U6 snRNPs , 2016a. S. cerevisiae five G-patch proteins were identified out of which four interact with Prp43. In the context of splicing, Prp43 function is modulated by Ntr1 (Nineteen complex-related proteins 1)  . During teins 1) . Moreoveteins 1) . The remteins 1) .modus operandi of the DEAH-box helicases and thus of Prp43 is still elusive to date. This is primarily attributable to a lack of crystal structures of members of the DEAH-box subfamily in an active state and with bound interaction partners. Recently, the crystal structure of the related DExH-box helicase MLE , which belongs to another DEAH/RHA subfamily, provided a first insight into how this helicase might function. The complex of MLE with a U10-RNA and ADP•AlF4- led to a proposed mechanism of how RNA is translocated through its interior tunnel driven by conformational changes of a Hook-Loop in the RecA2 domain  as well as one of these structures show Prp43 with an additional U7-RNA. The Prp43•U7-RNA•ADP•BeF3-complex unveils the structural basis of why Prp43 can bind RNA in a sequence-independent manner. The Prp43•ADP•BeF3- structures illustrate how Prp43 binds and releases complex RNA substrates due to massive rearrangements of the C-terminal domains which affect an opening of the interior RNA-binding tunnel which is formed by the two RecA-like and the C-terminal domains. The hydrolysis of an ATP molecule induces conformational rearrangements of the RecA-like domains which, in turn, allow the translocation of the RNA through the interior tunnel via the so far unrecognized RF motif present in a β-turn of all DEAH-box helicases. According to our results, the translocation mechanism of MLE is very unlikely to be compatible for Prp43. All proposed functional mechanisms of Prp43 are strengthened by functional analyses of structure-based mutants in this study.So far, three crystal structures of Prp43 in the post-catalytic ADP-bound state have been available from mophilum . The latmophilum . Owing t3- as well as a U16-RNA. For these crystallization approaches, an N-terminally truncated version of Prp43 from C. thermophilum was used (ctPrp43ΔN), which lacks the first 60 amino acids  two alternative conformations are observed  (high resolution). The second crystal form belongs to the hexagonal space group P65 which exhibited a resolution limit of 3.24 Å (3-(LR) (low resolution). This structure was refined to Rwork / Rfree values of 18.22% and 21.78%, respectively.In pursuance of analyzing how Prp43 binds to ssRNA regions within folded RNAs, we targeted at determining the pre-catalytic state of Prp43 before RNA binding. For this purpose, we subjected ctPrp43ΔN to crystallization trials in the presence of the ATP analog ADP•BeFd 19.88%  and is rf 3.24 Å  and thus7•ADP•BeF3- complex structure, the conformation of the two RecA-like domains in the ctPrp43ΔN•ADP•BeF3- complex is almost identical and they superpose very well with r.m.s.d. values of 0.34 Å (312 Cα) in case of ctPrp43ΔN•ADP•BeF3-(HR) and with 0.39 Å (318 Cα) for the ctPrp43ΔN•ADP•BeF3-(LR) structure .In comparison to the ctPrp43ΔN•Utructure . Howeverructures , which l7•ADP•BeF3- and the ctPrp43ΔN•ADP•BeF3-(HR) complex structures after alignment of the RecA-like domains revealed that the center of mass of the ratchet-like domain is shifted by 16.2 Å, and by 14.1 Å for the OB-fold domain. In the ctPrp43ΔN•ADP•BeF3-(LR) complex structure, these values are equal to 17.0 Å (ratchet-like) and 11.7 Å (OB-fold), respectively. The ctPrp43ΔN•ADP•BeF3-(HR) structure is in a marginally more closed conformation, hence the ctPrp43ΔN•ADP•BeF3- complex structures are not completely identical. They represent two slightly different snapshots of Prp43 which both might exist in solution and presumably are favored by the different crystallization conditions or different crystal packing. These two states of ctPrp43ΔN•ADP•BeF3- might also give a hint at how Prp43 switches into the closed conformation since in the ctPrp43ΔN•ADP•BeF3-(HR) complex structure one inter-domain contact is present between the RecA2 (Asp 486) and the ratchet-like domain (Lys 605). These interactions between sequentially distal residues might trigger the switching from the open into the closed conformation after the binding to RNA. The circumstance that both structures of the ctPrp43ΔN•ADP•BeF3- complex are in a similar, open conformation strongly suggests the possibility that the complex structures presented reflect the main conformation of Prp43 in an active state in solution. Owing to the fact that Prp43•ADP•BeF3- crystallized in different space groups and in divergent crystallization conditions, the open conformation in crystallo is highly unlikely to be a crystallization artefact. This assumption is additionally supported by the low number of crystal contacts for the ctPrp43ΔN•ADP•BeF3-(HR)  and the ctPrp43ΔN•ADP•BeF3-(LR) (1524.3 Å²/4.7%) complex structure.The opening of the RNA-binding tunnel appears to be the key mechanism allowing Prp43, and very likely numerous other DEAH-box helicases, to bind to complex folded RNA substrates. Thus, these rearrangements of the C-terminal domains are expected to be crucial for their unwinding function. The opening is feasible due to a large movement of the ratchet-like and the OB-fold domains. Rearrangements of the WH domain are less pronounced owing to the fact that the WH rather functions as a hinge region between the RecA2 and the ratchet-like domain. Comparison between the ctPrp43ΔN•UVia Ellman reaction, the number of cysteines was determined to be 8.5 ± 0.1 (ctPrp43) and 9.3 ± 0.1 (ctPrp43-IDSB), respectively. This allows us to conclude that the majority of ctPrp43-IDSB exhibits the internal disulfide bridge because oxidized cysteines cannot be detected by this method and only the nine cysteines, which are also present in the wild-type protein, were determined.To further strengthen the hypothesis that the opening of the tunnel by the displacement of the C-terminal domains is crucial for the helicase function of Prp43, we designed a mutant of Prp43 which allows us to trap the closed conformation by the introduction of an internal disulfide bond (ctPrp43-IDSB). For this purpose, one cysteine was introduced into the RecA1 domain and another one into the ratchet-like domain at exposed positions to maximize the number of formed disulfide bonds . The funin vitro helicase activity analysis of all Prp43 mutants. ctPrp43-IDSB exhibited an activity of 12.18 nM/min which equals about 1/5th of the wild-type Prp43 activity and thus is clearly impaired concerning its ability to unwind dsRNA. The remaining helicase activity of the mutant is most likely caused by the fraction of ctPrp43-IDSB which does not contain the internal disulfide bond. The activity of ctPrp43 and the ctPrp43-IDSB mutant were also analyzed for a physiological and more complex substrate RNP by using the S. cerevisiae ILS disassembly assays  is very similar to its ortholog from S. cerevisiae (2.43 min−1)  . ctPrp43−1) is similar to the one determined for wild-type ctPrp43. ctPrp43-IDSB is also stimulated by ctPfa1-GP and by U16-RNA in the presence of the ctPfa1-GP, but in the contrast to wild-type Prp43 also just by U16-RNA. These findings confirm that the difference in the helicase and the ILS disassembly assay between ctPrp43 and ctPrp43-IDSB is solely caused by the impairment of the unwinding capabilities. The RNA-induced stimulation of ctPrp43-IDSB in the trapped conformation might still be feasible due to the fact that a short single-stranded U16-RNA without any secondary structures was used for these assays. Binding to the entry or exit site of the RNA-binding tunnel, which does not require an opening of the tunnel, might be sufficient for the RNA-induced stimulation of the ATPase activity (see below). Furthermore, Prp43 in the trapped closed conformation appears to be more prone for the stimulation of the ATPase activity by RNA since the ctPrp43-IDSB mutant (24.99 min−1) is stimulated by U16-RNA while the wild-type protein is not (2.08 min−1). It is conceivable that the GP increases the number of Prp43 molecules in a closed conformation since in the presence of a GP also the wild-type protein is stimulated by RNA.The intrinsic ATPase activity of ctPrp43-IDSB  and ctPrp43ΔN•ADP•BeF3-(LR) are very similar, the analysis of the conformational switches was restricted to the comparison of ctPrp43ΔN•ADP•BeF3-(HR) with the previously published ctPrp43ΔN•ADP structure.To investigate the effect of ATP hydrolysis on the Prp43 conformation, the structures of the ADP and the ADP•BeF3-(HR) superimpose well with r.m.s.d. values of 0.36 Å (139 Cα) for the RecA1 domains and 0.44 Å (142 Cα) for the RecA2 domains. Superimposing the helicase cores consisting of both RecA-like domains shows that the RecA1 and RecA2 domains are rotated by ca. 4˚ and ca. 14˚, respectively. There is also a slight translational displacement of the RecA2 domain by a 2.7 Å shift of its center of mass with regard to the RecA1 domain. Depending on the ADP/ATP state, conformational changes of several of the conserved SF2 helicase motifs, most strikingly of those in the RecA2 domain are observed  binds a water molecule (H2O 388) and positions it in close spatial proximity to the BeF3-, which corresponds to the ATP γ-phosphate. Presumably, this H2O 388 performs the nucleophilic attack on the phosphorus atom of the γ-phosphate since it is in an almost perfect orientation for an SN2 substitution mechanism at 180° to the leaving group. In the ADP•BeF3- complex structure, this water molecule is additionally bound via the interactions with Gln 428 and Arg 435 from motif VI. The region containing the motif VI undergoes one of the most pronounced conformational rearrangements of the helicase core and it is shifted in the ADP-bound state 5.1 Å apart from the bound nucleotide. In the ATP-bound state, also interactions between Arg 432 and Arg 435 from motif VI and the phosphate(-mimic) groups are detectable. In the ADP-bound state, only Arg 435 of motif VI interacts with the 3’OH of the ribose. Another noticeable rearrangement can be observed for Phe 360 which is not part of a classical SF2 helicase motif. In the ADP-bound state, the adenine moiety interacts with Phe 360 via π-electron stacking and by cation-π-interactions with Arg 162. In the ATP-bound state, Phe 360 is shifted 5.4 Å apart from the base which is now solely stabilized by Arg 162. The rearrangements of the other conserved motifs are less distinct but still notably. In the ADP-bound state, the motifs IV and V are shifted by 3.3 Å and 2.9 Å, respectively, apart from the C-terminal domains.The individual RecA-like domains of ctPrp43ΔN•ADP and ctPrp43ΔN•ADP•BeFobserved . The conIn the course of NTP hydrolysis, the loop harbouring the conserved motif III, which is known to couple ATPase and unwinding activity , also un7•ADP•BeF3- complex structure. An interaction between the Hook-Loop and the RNA, as seen in MLE, is unlikely in Prp43 because the Hook-Loop residues point at the opposite direction and do not contact the RNA (3-(HR) complex structure. According to this structure, this β-turn is present as type I (47%) and type II (53%) (The Hook-Turn and Hook-Loop mutants were tested for their dsRNA unwinding capabilities. As shown in  the RNA . In cont the RNA  and presII (53%) .The assumption that the Hook-Turn is the key player of RNA translocation in Prp43 is additionally strengthened by the results of the ILS disassembly assay . The ctP16-RNA even with a higher affinity than the wild-type protein (KD 0.84 ± 0.05 µM vs. 4.98 ± 0.57 µM)  .−1, ctPrp43-HL: 6.19 min−1, ctPrp43-HT&HL: 7.61 min−1) exhibit a level of intrinsic ATPase activity comparable to wild-type Prp43  and U8-RNA is available making a direct comparison with Prp43 very feasible (PDBid: 3o8r) , which stacks on the nucleotide at the 3’ end, is not conserved in DEAH-box helicases.Having unraveled the conserved interactions with RNA between the two members of the DEAH/RHA subfamilies, the question arose whether these interactions are present also in the related family of viral NS3/NPH-II helicases. Members of the NS3/NPH-II family also contain a helicase core composed of two RecA-like domains with the protruding β-hairpin in the RecA2 domain. In the NS3/NPHII helicase family there is only one C-terminal domain present, which is not homologous to any C-terminal domain of the DEAH/RHA helicases, and no RNA-binding tunnel is formed. Owing to the fact that numerous NS3 helicases are encoded by human pathogenic viruses, such as hepatitis C, Zika or yellow fever, these helicases have been subject of intensive studies and various crystal structures are available. The comparison with Prp43 was restricted to the NS3 hepatitis C virus (HCV) helicase, as NS3 HCV binds RNA also in a sequence-independent manner and a crystal structure with a bound ATP-analog (ADP•BeFd: 3o8r) . The comd: 3o8r) . In NS3 3- has not been observed in any other crystal structure of a DEAH- or DExH-box helicase, which demonstrates that Prp43 exhibits a higher degree of flexibility as initially expected from the previously determined crystal structures of Prp43•ADP complexes.With the set of new Prp43 structures novel insights into the mechanism of RNA loading are provided. Considering the fact that the RNA-binding site of Prp43 is in its interior tunnel and the circumstance that the substrates of Prp43 are highly folded RNA networks, RNA loading to Prp43 is not trivial. The Prp43 target sites in the spliceosome and in the pre-ribosomal subunits are surrounded by complex tertiary structures without single-stranded overhangs. Here, we could demonstrate that in the ATP-bound state Prp43 is capable of adopting an open conformation in which numerous interactions between the RecA-like domains and the C-terminal domains are disrupted converting the RNA-binding tunnel into a groove and thus enabling Prp43 to bind to single-stranded RNA regions within folded RNA stretches . This re3- and of MLE (chain A) with the transition state analog ADP-AlF4- superpose very well with an r.m.s.d. value of 0.70 Å for 235 Cα  (ctPrp43ΔN) were expressed and purified as previously described . The mutC. thermophilum were identified by BLAST search against the complete C. thermophilum genome  (Agilent Technologies). Cells were lysed in 50 mM Tris/HCl pH 7.5, 500 mM NaCl and 10 mM EDTA by microfluidization and soluble protein was separated by ultracentrifugation. The N-terminally tagged GST-fusion proteins were loaded onto Glutathione Sepharose 4B  in lysis buffer and thoroughly washed with an additional 2 M LiCl. After protein elution in 50 mM Tris/HCl pH 7.5, 500 mM NaCl, 2 mM MgCl2 and 30 mM reduced glutathione, GST-tag removal was realized by incubation with PreScission Protease (1:100 (w/w), GE Healthcare). Final polishing was obtained by size exclusion chromatography  and a second GST affinity chromatography step, both in 10 mM Tris/HCl pH 7.5, 200 mM NaCl and 2 mM MgCl2.The homologs of the G-patch proteins Pfa1 and Ntr1 from m genome . The idevia the sitting-drop vapor-diffusion technique by mixing equal volumes of protein solution and crystallization buffer. ctPrp43ΔN•ADP•BeF3-(LR) was crystallized at a concentration of 5 mg ml−1 in 40% (v/v) pentaerythritol propoxylate (5/4 PO/OH), 6% (v/v) ethanol, 16% (v/v) glycerine and 100 mM Tris/HCl pH 8.0 at 20°C. ctPrp43ΔN•ADP•BeF3-(HR) crystallized at 4.5 mg ml−1 in 35% (v/v) 2-methyl-2,4-pentanediol, 1.67% (w/v) PEG 4000 and 100 mM Na HEPES pH 7.0 at 4°C. Crystals of the ctPrp43ΔN•U7-RNA•ADP•BeF3- complex were obtained at 20°C by mixing ctPrp43ΔN at 3 mg ml−1 with a 2.5-fold molar excess of poly-U16-RNA  and 2.5% (w/v) PGA-LM, 13% (w/v) PEG 8000 and 100 mM Na Cacodylate pH 6.5. For all crystallization attempts, ADP was used at a tenfold molar excess with respect to ctPrp43ΔN, BeSO4 with a twentyfold and NaF at a sixtyfold excess.Complexes of ctPrp43ΔN were crystallized 3- crystals did not require additional cryo-protection. Diffraction data were collected at 100 K on beamline P13, PETRA III, DESY  and were processed with the XDS package  glycerine. Both ctPrp43ΔN•ADP•BeF package . Data co7-RNA•ADP•BeF3- was solved by molecular replacement using Phaser as implemented in the CCP4 suite and the ctPrp43ΔN•ADP (PDBid: 5d0u) structure as a search model  were determined by means of molecular replacement with Phaser using the atomic coordinates of ctPrp43ΔN•U7-RNA•ADP•BeF3- as a search model which was split in three distinct parts. The RecA1 with the RecA2 domain, the ratchet-like domain linked to the OB-fold and the WH domain as last item were placed in a stepwise fashion to obtain a successful replacement solution. Subsequent iterative cycles of automated refinement with PHENIX and manual model building in Coot were carried out before model quality assessment in MolProbity. The structure of ctPrp43ΔN•ADP•BeF3-(HR) was refined to Rwork and Rfree values of 17.67% and 19.88% and of ctPrp43ΔN•ADP•BeF3-(LR) to 18.22% and 21.78% with 97.34% and 96.28% of all residues in the most-favored regions of the Ramachandran plot, respectively. Both structures do not exhibit any Ramachandran outlier. Detailed refinement statistics are summarized in Both crystal structures of ctPrp43ΔN•ADP•BeFPyMOL  and Chimera . Reactions were supplemented with 250 nM NADH, 500 nM phosphoenolpyruvate, 6–8.3 U/ml pyruvate kinase  and 9–14 U/ml lactic dehydrogenase . ctPrp43 and mutants were used at a concentration range between 0.25–10 µM. G-patch proteins and U16-RNA  were each added in five-fold excess over ctPrp43. kcat and KM values were determined according to Michaelis and Menten equation.The ATPase activity of ctPrp43 and all of its mutants was monitored in an NADH-dependent enzyme coupled assay by recording the decrease of NADH absorption at 340 nm as initially described by Agarwal and coworkers . All rea2 and 1 mM ATP at 20°C and recorded on a Fluoromax III (Horiba Jobin Yvon). ctPrp43 and ctPrp43 mutants were used at a concentration of 250 nM, G-patch proteins at 1.25 µM and the dsRNA  at 500 nM. The excitation wavelength was set to 643 nm and the emission was measured at 673 nm with a slit setting of 2.5 nm/2.0 nm. The initial slope (ΔF/s) of each reaction, which is the maximum reaction velocity, was determined in three independent measurements per sample ) . Reactiovia fluorescence anisotropy measurements using a Fluoromax VI (Horiba Jobin Yvon). 5’ 6-carboxyfluorescein-labeled U16-RNA  was used at a concentration of 0.1 µM and ctPrp43 or ctPrp43-HT at concentrations between 0.01 µM to 50 µM in the presence of a 100-fold excess of AMPPNP . RNA-binding assays were performed in 25 mM Tris pH 7.5, 150 mM KCl and 3 mM MgCl2 at 25°C. The excitation wavelength was set to 490 nm and the emission was measured at 517 nm with a slit setting of 5 nm/5 nm. Data points were analyzed as a set of triplicates and for each sample, and the anisotropy was determined as the mean value of ten individual measurements. The measured anisotropy was normalized with respect to the sample with a concentration of 0 µM ctPrp43 or ctPrp43-HT.RNA binding of ctPrp43 and ctPrp43-HT was analyzed act ΔPrp2 complexes were assembled in heat-inactivated extracts from the yeast strain prp2-1 . The matrix was washed twice with 10 ml GK75 buffer. To obtain ILSs  and the reaction volume was adjusted to 400 µl with GK75 buffer; then 40 µl of 10x ‘rescue’ solution (200 mM KPO4 (pH 7.4), 10 mM MgCl2, 20 mM ATP, 10% PEG 8000) were added to the reaction that was incubated at 23°C for 45 min. After thorough mixing, the reaction was incubated at 23°C for 45 min. Matrices were subsequently washed 3 times with 10 column volumes of GK75 buffer. Then a 10-fold molar excess of recombinant Prp22 was added and the volume was adjusted to 400 µl with 1x ‘rescue’ solution prepared in GK75 buffer. After thorough mixing, the reaction was incubated at 23°C for 15 min. The supernatant (containing the released ILS) was collected, and GK75 buffer was added to the matrix to a final volume of 400 µl. After gentle mixing and repeated centrifugation for 1 min at 2000 rpm the supernatant was collected and then loaded onto linear 10–30% (v/v) glycerol gradients containing GK75 buffer. Samples were centrifuged for 16 h at 21,500 rpm in a TH660 rotor (Thermo Scientific) and harvested manually from the top in 23 fractions of 175 µl. Fractions were analyzed by Cherenkov counting in a scintillation counter. Peak fractions containing ILSs were pooled and the glycerol concentration was adjusted to 5% with GK75 buffer without glycerol.Yeast Bn prp2-1  by incubn prp2-1 . Samplesain ILSs , Bact ΔPTo dismantle purified ILSs , samples7•ADP•BeF3- (PDBid: 5lta), ctPrp43ΔN•ADP•BeF3-(HR) (PDBid: 5ltj) and ctPrp43ΔN•ADP•BeF3-(LR) (PDBid: 5ltk) have been deposited in the Protein Data Bank.Coordinates and structure factors of ctPrp43ΔN•U In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by James Manley (Senior Editor) and three reviewers, one of whom, Timothy W Nilsen (Reviewer #1), is a member of our Board of Reviewing Editors.Thank you for submitting your article "Structural insights into the mechanism of DEAH-box RNA helicases" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you will see all were generally positive about the work but also raised some concerns. In particular, it is necessary to test the RNA binding affinity of the hook loop mutants to ensure that the effect on translocation is not simply because of failure of substrate binding. Another point that must be addressed is to put your work in context with work published on DEAH proteins found in flaviviruses. In addition to these points please deal with the other concerns raised as thoroughly as possible.Reviewer #1:Ficner and co-workers report the crystal structure of the DEAH RNA helicase Prp43 with bound RNA and an ATP ground state analog. Compared to a previously reported structure of Prpr43 with ADP (but without RNA), the new structure reveals a significant conformational change that is interpreted as facilitating RNA loading and movement of the RNA relative to the protein. The authors further identify a hairpin loop with a characteristic motif that is conserved among DEAH helicases. While the work focuses on structural data, the main structure-inspired claims are supported by biochemical and functional studies in yeast for several mutants.The new structure provides critical information for understanding helicases of the DEAH family, many of which participate in pre-mRNA splicing. In addition, the work highlights potentially important differences to a recently reported structure of the related MLE helicase bound to RNA. The manuscript thus makes the point that even within the DEAH/RHA family differences exist with respect to the RNA binding mode.While the data and the overall information mark an important contribution to our understanding of RNA biology, especially pre-mRNA splicing, the presentation leaves room for improvement. The writing is verbose, and the manuscript would probably benefit from tightening of the prose. In addition, the figures do not always convey a clear point and are at times hard to follow  that is important for the translocation. Although there is currently no structure for the transition-state of the reaction, the authors include biochemical studies with structure-based mutants that support their proposed mechanism (with the caveat that an important control is missing – see below).This mechanism proposed for Prp43 is different from that proposed in a previous study reporting the structure of a DEAH/RHA helicase, MLE, bound to RNA and ADP-AlF, e.g. in the transition-state of the reaction. In general, I find interesting that two helicases that are thought to belong to the same family differ in mechanisms. Indeed, not only the translocation mechanism appears to be different, but also the unwinding mechanism and the RNA-binding specificity. Rather than playing down the previous work and boosting their own , the authors could elaborate positively on the different mechanisms of translocation, unwinding and specificity of the RHA-like and Prp43-like subfamilies of DEAH helicases.Specific points:The authors claim that this is the first structure of a DEAH helicase bound to RNA, and emphasize that the structure provides the first insight into the RNA-binding mechanism of a DEAH-box helicase. However, the RNA binding tunnel of Prp43 is essentially the same as previously reported for MLE. Indeed, the RNA seems to be less well defined in the electron density in the Prp43 structure, with fewer nucleotides and alternative conformations. The claim is therefore inappropriate.Since the 'hook-loop' has been defined before, the authors should refer to the corresponding Prp43 348-349 residues as 'hook loop 1' and to their newly identified Prp43 180-181 residues as 'hook loop 2', rather than the other way round.The important control that is currently missing is biochemical evidence that the proposed Prp43 hook-loop binds RNA. If the hook-loop mutant would be impaired in RNA binding, the impairment in unwinding cannot be interpreted as a failure of translocation, but simply as a failure of substrate binding. The authors should measure the RNA-binding affinity of the hook loop mutant and compare it to the wild type. The hook-loop mutant previously implied for translocation in the case of MLE, for example, can bind RNA but cannot unwind.How is the hook-loop of Prp43 connected with the nucleotide state? In particular, how does it sense the presence or absence of the γ phosphate of ATP?In The authors refer to Prp43 residues 527-640 as the ratchet domain, from the original Hel308 structure paper. From their mechanism, what is the evidence that this domain is ratcheting? If they have no evidence, they should consider a more cautious nomenclature.The title should more accurately define the content. Since it appears that the translocation mechanisms of Prp43 and MLE are distinct, the title should be more precise: 'Structural insights into the mechanism of the DEAH-box helicase Prp43'.Reviewer #3:In this comprehensive analysis of Prp43, the authors present a series of high resolution crystal structures of the protein, in various ligand-bound forms, as it samples conformational states that may play a role in RNA recognition and the function of Prp43 in splicing and translation. The quality of the data is particularly notable, and it is in large part due to the judicious choice of a stable thermophilic variant that can substitute for S.c. Prp43 in splicing assays. In addition, to trapping with specific ligands, the authors captured different conformational states by observing the molecule in different crystal forms. Particularly clever was the use of an engineered disulfide bridge to test the structure and functional attributes of the closed form, as this provided a very nice way to freeze the molecule in single state for solution experiments.Other positive attributes of the paper are that a dynamic RNA tunnel is clearly observed, and it involves interesting auxiliary domains that are not common to all SF2 proteins. In this way, it does provide insights into the molecular mechanism for Prp43 translocation and binding to RNA. The splicing experiments and other biochemical experiments were of very high quality and these were used effectively to establish structure-function relationships among domains and motifs of the protein.The major criticism of this work is that the investigators appeared to be functioning "in a bubble", seemingly unaware of the vast crystallographic and biochemical literature on translocation by very closely related proteins. This negatively impacts the paper in several important ways: A) The paper consistently misrepresents the novelty of the findings, seeming to imply that this is the first time translocation has been structurally characterized for a motor protein in the DEXH family, including NS3 from HCV. B) Important similarities and differences from other motors are overlooked, leading to a missed opportunity for understanding the function of individual motor components. Adding to the issue of novelty is that nucleotide-bound Prp43 has been previously structurally characterized in the absence of RNA ligand. So, this is not the first time we have visualized this particular protein. In the opinion of this reviewer, it appears to bind ssRNA in a nonspecific manner not too dissimilar from other SF2 proteins, so it is not entirely clear what we have learned. Without any comparison to related proteins (other than MLE), the authors make it difficult for the reader to ascertain the structural significance of the reported findings.Specific criticisms:1) The Introduction, last paragraph, subsection “Crystal structure of Prp43 with bound RNA in an active state”, second paragraph and subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, last paragraph, and many other places in the paper say this is the "first insight", "first insight into a genuine DEAH.." These statements, and those throughout the paper, misrepresent the significance of this work, which is certainly not the first structural insight into a member of this family, and it does not provide the first structural or mechanistic insight into translocation by the family members. Full-length HCV NS3 is in the same DEXH family and it has been the subject of two studies in which translocation was captured on polynucleotide substrates. Indeed, in Appleby , NS3 opening and closing, together with sequential translocation along polyuridine, was captured in an exhaustive series of crystal structures that provide a complete mechanistic picture of translocation by this family along RNA. Similar work was done by Gu and Rice, but using a DNA oligonucleotide bound to a truncated NS3 variant, which is less directly comparable with the present study. All of that work built upon incisive single molecule experiments that specifically monitored the translocation of NS3  and showed that it follows a mechanistic paradigm originally elucidated by Hopfner for Hel308. Indeed, it has been known for about ten years that SF1 and SF2 helicases translocate by opening and closing the RecA1 and RecA2 domains as a function of ATP binding and release. There are probably 50 papers on this. While HCV NS3 is formally a DECH variant, the related and structurally-characterized Dengue and YFV NS3 proteins are DEAH – so the significance of the third amino acid is not relevant to the argument.2) Subsection “Prp43 binds RNA in a sequence-independent fashion”, first paragraph. Please compare the RNA interaction network with that shown in Appleby 2011.3) Subsection “Crystal structure of Prp43 with bound RNA in an active state”, last paragraph and the corresponding figures. While there is much text devoted to the shape of an RNA "tunnel", the authors do not provide figures that really show what this tunnel looks like. Perhaps a space-filling rendering, of the cavity, with the RNA inside in mesh rendering, zoomed in, would help the reader visualize this feature. Again, the authors need to differentiate this cavity from the grooves that are formed upon opening and closing of NS3.4) Subsection “Crystal structure of Prp43 in an active state”, last paragraph. – The fact that the authors observe several different structures does not mean that the protein is inherently more flexible. They should not infer dynamic information from these results.5) Subsection “Crystal structure of Prp43 in an active state”, last paragraph. It is difficult for the reader to keep track of which complex is open and which is closed as a function of ATP analog, solvent molecules, or crystallization conditions. Specifically, which ligands trigger the open state? Which stabilize the closed state? The argument is quite opaque as written.6) Subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, first paragraph. What is the significance and meaning of ∆Fs for unwinding? This is not a term that is typically used within the helicase community. It is therefore not clear what it means, how it is quantitated, or what it reveals. Again, the authors seem to be in their own world, not even using the assays or terms that typify the field. Because of this, it is harder to make comparisons with previous work.7) Subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, first paragraph. The fact that opening and closing of the two RecA domains is linked to ATP binding and translocation has been demonstrated for a broad variety of SF1 and SF2 proteins. In addition to Appleby for SF2s, please check out Wei Yang's work on UvrD, or Lohman's collaborative work on Rep or UvrD. Recent work on RIG-I by Marcotrigiano would also be informative.8) Subsection “The pre- and post-catalytic state of Prp43”, last paragraph. There is no citation provided for previous work on Motif III. There are a number of key papers on a broad variety of SF2 proteins showing that Motif III coordinates ATP and RNA binding. For the most recent one, see Fitzgerald and Pyle, NAR 2016, and citations therein. This shows a structural basis for the involvement of Motif III.9) 10)  As you will see all were generally positive about the work but also raised some concerns. In particular, it is necessary to test the RNA binding affinity of the hook loop mutants to ensure that the effect on translocation is not simply because of failure of substrate binding. Another point that must be addressed is to put your work in context with work published on DEAH proteins found in flaviviruses. In addition to these points please deal with the other concerns raised as thoroughly as possible.We agree that it is necessary to show whether the effect of the hook-loop mutation on RNA unwinding is caused by reduced or lost RNA binding. We therefore performed RNA-binding studies by means of fluorescence anisotropy spectroscopy and the results were included into the revised version.We also added a detailed comparison of our Prp43 structures with the viral NS3 helicase, which is related to the spliceosomal DEAH-box helicases, but also has structurally important differences .Reviewer #1: […] While the data and the overall information mark an important contribution to our understanding of RNA biology, especially pre-mRNA splicing, the presentation leaves room for improvement. The writing is verbose, and the manuscript would probably benefit from tightening of the prose. In addition, the figures do not always convey a clear point and are at times hard to follow  that is important for the translocation. Although there is currently no structure for the transition-state of the reaction, the authors include biochemical studies with structure-based mutants that support their proposed mechanism (with the caveat that an important control is missing – see below).This mechanism proposed for Prp43 is different from that proposed in a previous study reporting the structure of a DEAH/RHA helicase, MLE, bound to RNA and ADP-AlF, e.g. in the transition-state of the reaction. In general, I find interesting that two helicases that are thought to belong to the same family differ in mechanisms. Indeed, not only the translocation mechanism appears to be different, but also the unwinding mechanism and the RNA-binding specificity. Rather than playing down the previous work and boosting their own , the authors could elaborate positively on the different mechanisms of translocation, unwinding and specificity of the RHA-like and Prp43-like subfamilies of DEAH helicases.We removed all “first” claims in order to prevent any potential inappropriateness. The differences in mechanisms and substrate specificity between the related RHA-like and DEAH-box helicases were discussed in more detail.Specific points:The authors claim that this is the first structure of a DEAH helicase bound to RNA, and emphasize that the structure provides the first insight into the RNA-binding mechanism of a DEAH-box helicase. However, the RNA binding tunnel of Prp43 is essentially the same as previously reported for MLE. Indeed, the RNA seems to be less well defined in the electron density in the Prp43 structure, with fewer nucleotides and alternative conformations. The claim is therefore inappropriate.According to the classification of helicases, MLE was considered to belong not to the sub-family of  DEAH helicases but rather to the closely related sub-family of RHA helicases. Due to the very high structural similarity, we followed the suggestion of the reviewer and removed the statement, that our structure represents the first structure of a DEAH-box helicase with bound RNA.Since the 'hook-loop' has been defined before, the authors should refer to the corresponding Prp43 348-349 residues as 'hook loop 1' and to their newly identified Prp43 180-181 residues as 'hook loop 2', rather than the other way round.In order to prevent any confusion with regard to the previous definition of the hook loop in the MLE structure, we introduced the name hook-turn for the region 180-181, as it is actually a β-turn, rather than a loop. The term hook loop is now used for the residues 348-350, which corresponds to the hook loop of MLE.The important control that is currently missing is biochemical evidence that the proposed Prp43 hook-loop binds RNA. If the hook-loop mutant would be impaired in RNA binding, the impairment in unwinding cannot be interpreted as a failure of translocation, but simply as a failure of substrate binding. The authors should measure the RNA-binding affinity of the hook loop mutant and compare it to the wild type. The hook-loop mutant previously implied for translocation in the case of MLE, for example, can bind RNA but cannot unwind.We agree with the reviewer that the lack of dsRNA unwinding of the hook-turn mutant could be caused by a loss of RNA binding capability. We therefore measured the RNA binding of ctPrp43 and the ctPrp43 hook-turn mutant by means of fluorescence anisotropy. These experiments demonstrated that the binding of RNA to the mutated Prp43 is not impaired, and therefore the loss of unwinding activity is most likely caused by a loss of RNA translocation activity.These new results were included in the revised version of the manuscript.How is the hook-loop of Prp43 connected with the nucleotide state? In particular, how does it sense the presence or absence of the γ phosphate of ATP?The hook-loop (now hook-turn) is not involved in any contacts with the bound ATP molecule or in particular with the γ phosphate. Thus, there is no direct sensing of the nucleotide state by the hook-turn. The movement of the hook-turn is caused by the global rotation of the RecA1 domain. This information is now provided in the revised manuscript.In As suggested by the reviewer, the ATPase activity was also determined for ctPrp43-IDSB+U16-RNA as well as for all other ctPrp43 mutants and these data were added to the revised KM and Kcat have to be addressed. The KM value for ATP is lower in the presence of RNA, which means that the affinity of ATP is increased, hence there is still a cooperative effect of RNA and ATP binding. The Kcat value of the ctPrp43-GP is lower in presence of RNA, which appears to be puzzling. It is very difficult to explain this effect by the currently known structures, due to the fact that the G-patch protein is missing in all structures. As a consequence, the structural basis for the stimulation of ATPase and helicase activities by the G-patch proteins in not understood at all. Hence, one possibility is that the stimulating effect of the G-patch is reduced in case of the double hook-loop mutant upon binding of the RNA. However, this is pure speculation as no corresponding structural or biochemical data are available, and has therefore not been included in the revised version of the manuscript.Regarding the question concerning the reduced RNA-induced stimulation of the double hook-loop mutant the changes in both The authors refer to Prp43 residues 527-640 as the ratchet domain, from the original Hel308 structure paper. From their mechanism, what is the evidence that this domain is ratcheting? If they have no evidence, they should consider a more cautious nomenclature.The name ratchet domain was introduced with the first crystal structure analysis of Prp43 by Walbott et al. (2010) and He et al. (2010) and was based on the structural similarity to the DNA helicase Hel308.We fully agree with the referee that there is no evidence for a ratcheting function of this domain in Prp43, which is consistent with the Prp43-RNA complex structure, as no side chains of this domain bind to the RNA. The structural rearrangements of the C-terminal domains leading to the opening of the RNA-binding tunnel shows that most of the conformational changes occur mainly within the degenerate winged-helix domain, while the conformation of the ratchet domain remains unchanged. Hence, we prefer to keep these as two separate domains instead of using the term HA2 (helicase associated 2) domain, in which both domains are fused. We now denote the “ratchet domain” as “ratchet-like” domain in order to maintain the structural similarity to Hel308.In the revised version of the manuscript we included a clear statement, that the “ratchet-like” domain has no ratcheting function in Prp43.The title should more accurately define the content. Since it appears that the translocation mechanisms of Prp43 and MLE are distinct, the title should be more precise: 'Structural insights into the mechanism of the DEAH-box helicase Prp43'.We changed the title to 'Structural insights into the mechanism of the DEAH-box helicase Prp43' as suggested by the reviewer.Reviewer #3:[…] The major criticism of this work is that the investigators appeared to be functioning "in a bubble", seemingly unaware of the vast crystallographic and biochemical literature on translocation by very closely related proteins. This negatively impacts the paper in several important ways: A) The paper consistently misrepresents the novelty of the findings, seeming to imply that this is the first time translocation has been structurally characterized for a motor protein in the DEXH family, including NS3 from HCV. B) Important similarities and differences from other motors are overlooked, leading to a missed opportunity for understanding the function of individual motor components. Adding to the issue of novelty is that nucleotide-bound Prp43 has been previously structurally characterized in the absence of RNA ligand. So, this is not the first time we have visualized this particular protein. In the opinion of this reviewer, it appears to bind ssRNA in a nonspecific manner not too dissimilar from other SF2 proteins, so it is not entirely clear what we have learned. Without any comparison to related proteins (other than MLE), the authors make it difficult for the reader to ascertain the structural significance of the reported findings.10-ADP-AlF4- complex  all exhibit a conformation of the C-terminal domains which is similar among each other but in striking contrast to that of our new Prp43-ATP-analog complexes.All “first” claims were removed in our revised manuscript to prevent any potential inappropriateness. We added a detailed comparison of Prp43 with HCV NS3 and also deepened the one with MLE in the revised version of the manuscript. Similarities and differences between these helicases were worked out in more detail (for more details see below [specific criticisms point 1]) and we improved the visualization for this purpose (e.g. with the new Specific criticisms:1) The Introduction, last paragraph, subsection “Crystal structure of Prp43 with bound RNA in an active state”, second paragraph and subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, last paragraph, and many other places in the paper say this is the "first insight", "first insight into a genuine DEAH.." These statements, and those throughout the paper, misrepresent the significance of this work, which is certainly not the first structural insight into a member of this family, and it does not provide the first structural or mechanistic insight into translocation by the family members. Full-length HCV NS3 is in the same DEXH family and it has been the subject of two studies in which translocation was captured on polynucleotide substrates. Indeed, in Appleby , NS3 opening and closing, together with sequential translocation along polyuridine, was captured in an exhaustive series of crystal structures that provide a complete mechanistic picture of translocation by this family along RNA. Similar work was done by Gu and Rice, but using a DNA oligonucleotide bound to a truncated NS3 variant, which is less directly comparable with the present study. All of that work built upon incisive single molecule experiments that specifically monitored the translocation of NS3  and showed that it follows a mechanistic paradigm originally elucidated by Hopfner for Hel308. Indeed, it has been known for about ten years that SF1 and SF2 helicases translocate by opening and closing the RecA1 and RecA2 domains as a function of ATP binding and release. There are probably 50 papers on this. While HCV NS3 is formally a DECH variant, the related and structurally-characterized Dengue and YFV NS3 proteins are DEAH – so the significance of the third amino acid is not relevant to the argument.We added a detailed comparison of Prp43 and NS3 structures, which revealed important differences between both families of helicases:i) We removed all “first” claims in order to prevent any potential inappropriateness.ii) NS3 is lacking the three C-terminal domains common the DEAH/RHA family, hence the RNA-binding tunnel of Prp43 formed by the RecA domains and the C-terminal domains is not present in NS3.iii) NS3 contains one different C-terminal domain that is also involved in RNA binding. One important residue (Trp 501) of the NS3 CTD, which is critical for RNA binding and translocation, does not have any equivalent in Prp43  helicases, indicating significant differences in the mechanism of RNA binding and translocation.2) Subsection “Prp43 binds RNA in a sequence-independent fashion”, first paragraph. Please compare the RNA interaction network with that shown in Appleby 2011.We added a figure showing the RNA interaction network new  and we a3) Subsection “Crystal structure of Prp43 with bound RNA in an active state”, last paragraph and the corresponding figures. While there is much text devoted to the shape of an RNA "tunnel", the authors do not provide figures that really show what this tunnel looks like. Perhaps a space-filling rendering, of the cavity, with the RNA inside in mesh rendering, zoomed in, would help the reader visualize this feature. Again, the authors need to differentiate this cavity from the grooves that are formed upon opening and closing of NS3.We added a new figure  showing 4) Subsection “Crystal structure of Prp43 in an active state”, last paragraph. The fact that the authors observe several different structures does not mean that the protein is inherently more flexible. They should not infer dynamic information from these results.Actually, we intended to state that the rearrangement of the C-terminal domains was unexpected, as the several Prp43 structures from different organisms, which were obtained under different crystallization conditions, and as well as the related MLE structure, showed all the same conformation/position of the CTD with respect to the helicase core. It was not our intention to gain dynamic information from crystal structures.5) Subsection “Crystal structure of Prp43 in an active state”, last paragraph. It is difficult for the reader to keep track of which complex is open and which is closed as a function of ATP analog, solvent molecules, or crystallization conditions. Specifically, which ligands trigger the open state? Which stabilize the closed state? The argument is quite opaque as written.We used the term ‘open’ or ‘close’ with regard to the position of the C-terminal domains and the accessibility of the RNA-tunnel. The closed state was observed for the RNA/ATP complex and was well for the ADP complex. The open state was observed only for the ATP complex. Both states are independent of the crystallization condition or crystal packing, as for each state at least two independent crystal structures  are available.The crystallographic data suggest that the open state forms upon binding of ATP, while in the presence of RNA the closed state is stabilized. However, crystallization or crystal structures do not provide any information about the equilibrium of the different states in solution.6) Subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, first paragraph. What is the significance and meaning of ∆Fs for unwinding? This is not a term that is typically used within the helicase community. It is therefore not clear what it means, how it is quantitated, or what it reveals. Again, the authors seem to be in their own world, not even using the assays or terms that typify the field. Because of this, it is harder to make comparisons with previous work.We used the fluorescence-based helicase assay published by Belon and Frick in 2008. In the revised version of manuscript this assay is explained in more detail within the Materials and methods section.In order to compare the helicase activity of the mutants with respect to wildtype Prp43, we originally just showed the experimental data as ∆F/s. For the revised version these values were converted into an activity (nM/min) corresponding to the amount of dsRNA substrate unwound per minute.7) Subsection “Prp43 trapped in the closed conformation is impaired in its helicase activity”, first paragraph. The fact that opening and closing of the two RecA domains is linked to ATP binding and translocation has been demonstrated for a broad variety of SF1 and SF2 proteins. In addition to Appleby for SF2s, please check out Wei Yang's work on UvrD, or Lohman's collaborative work on Rep or UvrD. Recent work on RIG-I by Marcotrigiano would also be informative.We do not refer to the opening of the two RecA-like domains in the course of ATP hydrolysis. In our manuscript, these lines are dedicated to the opening of the C-terminal domains and thus the opening and closing of the RNA binding tunnel.We completely agree with reviewer that the opening/closing of the RecA domains is a very well-studied feature of SF1 and SF2 proteins. Since this concept is well established, and additionally not the main focus of our manuscript, we don’t want to extend the length of our manuscript by a review and comparison of all helicases.8) Subsection “The pre- and post-catalytic state of Prp43”, last paragraph. There is no citation provided for previous work on Motif III. There are a number of key papers on a broad variety of SF2 proteins showing that Motif III coordinates ATP and RNA binding. For the most recent one, see Fitzgerald and Pyle, NAR 2016, and citations therein. This shows a structural basis for the involvement of Motif III.We added to this part of our revised manuscript several citations for the work on Motif III. In our improved version, we now cite Schwer and Meszaros , Banroques et al.  and Fitzgerald et al. .9) We added a supplementary figure  showing 10) rd remark) to We tried to improve the quality of the figures throughout the whole manuscript. We added a schematic representation of the RNA-interaction network and a depiction of the RNA-binding tunnel (as suggested in your 3


Cis-acting RNA structures in the genomes of RNA viruses play critical roles in viral infection, yet their importance in the bipartite genomes of the picorna-like, plant-infecting comoviruses has not been carefully investigated. We previously characterized SLC, a stem-loop structure in the 5′ untranslated region (UTR) of the bean pod mottle comovirus (BPMV) RNA2, and found it to be essential for RNA2 accumulation in infected cells. Here we report the identification of SL1, a similar cis-acting element in the other BPMV genome segment - RNA1. SL1 encompasses a portion of RNA1 5′ UTR but extends into the coding sequence for nine nucleotides, thus was missed in the previous study. While the stems of SL1 and SLC share little sequence similarity, their end loops are of the same size and identical for 11 of 15 nucleotides. Importantly, SL1 and SLC are functionally interchangeable, and separate exchanges of the stem and loop portions were likewise well tolerated. By contrast, the conserved loop sequence tolerated minimal perturbations. Finally, stem-loop structures with similar configurations were identified in two other comoviruses. Therefore, SL1 and SLC are likely essential comoviral RNA structures that play a conserved function in viral infection cycles. The best known example of these RNA structures is probably the internal ribosomal entry site (IRES) element encoded within the 5′ termini of many picorna- and flavi-viruses, which enables efficient translation of virus-encoded polyproteins by guiding ribosomes directly to the start codon2. Long distance interactions between two or more secondary structure elements are also known to enhance cap-independent translation of viral genes, and to facilitate the synthesis of subgenomic RNAs of many viruses4, whereas various tRNA-like elements at the 3′ termini of genomic RNAs function to prime the synthesis of (−) strand replicational intermediates5. Additionally, many internally encoded stem-loop structures were shown to exert diverse functions ranging from templating the synthesis of uridylylated VPg , anchoring viral RNA-dependent RNA polymerase (RdRP), and serving as the initiation site of genome encapsidation8.The genomes of many single-stranded (ss), positive sense (+) RNA viruses fold into numerous secondary structures that play critical Comovirinae subfamily . Comoviruses and animal viruses of the family Picornaviridae are considered to be evolutionarily related as the genomic RNAs of all these viruses encode large, undisrupted polyproteins that are proteolytically processed to release multiple mature viral proteins10. Additional similarities between comoviruses and picornaviruses are: (i) they share similar, nonenveloped icosahedral particles; (ii) they harbor similar sized VPgs (between 22 and 28 amino acid [aa] long) that are much smaller than that of plant-infecting potyviruses (approximately 200 aa)11. However, while cis-acting RNA secondary structures have been systematically examined in poliovirus (PV) – a model picornavirus12, their involvement in comoviral infection cycles remain largely elusive.Despite their recognized roles in viral multiplication cycles, RNA secondary structures have so far been studied in only a relatively small number of model viruses. Conversely, their broader prevalence remains largely unknown for the vast majority of (+) RNA viruses, including viruses of the plant-infecting 13. BPMV is a bipartite comovirus with two single-stranded, (+) RNA segments, RNA1 and RNA2, that are both covalently linked to a VPg at the 5′ end, and polyadenylated at the 3′ end. Both RNA1 and RNA2, 6.0 and 3.6 kilobases (kb) in size, encode large polyproteins  as a modelins Fig. . All proins Fig. . RNA2 re 14 Fig. .Figure 113. SLC was initially presumed to be a structural element uniquely important for RNA2 replication, as its function was not restored by RNA1 5′ UTR replacement at the cognate location. Here we report the characterization of SL1, a novel hairpin structure within the 5′ terminus of BPMV RNA1 that is functionally exchangeable with SLC. We show that the stem portion of SL1 extends into the coding region of RNA1 polyprotein, contributing to the previous failure to complement SLC function with solely RNA1 5′ UTR. Importantly, both SL1 and SLC are required for the accumulation of their respective genome segments in infected cells, likely through their participation in the step of genome replication.We previously reported the characterization of a stem-loop structure, designated SLC, within the 5′ untranslated region (UTR) of BPMV RNA2, and found it to be specifically required for the accumulation of BPMV RNA2 in infected cells13  assembled by RNA1-encoded proteins13. However, upon closer inspection we were able to identify a novel stem-loop structure within RNA1 that spans from nucleotide number (no.) 336 to no. 376  should not affect the coding capacity of RNA1  differing from each other Fig. . As showTo further determine if the stem of SL1 needs to stay base-paired to maintain SL1 function, we generated three additional mutants, all of them in the BR2G-SL1 backbone Fig. . SpecifiThe 15-nt loops of SL1 and SLC are identical except for positions 2 to 5  to UAG Fig. . The SLCWe further interrogated the role of L6-L15 by making both nt substitution mutations (SLC-mL6/7/9 and SLC-mL11/12/13) and deletion mutations (SLC-Δ6-10 and SLC-Δ11-15). As shown in Fig. Cis-acting RNA structures play critical roles in the replication cycles of many (+) RNA viruses by regulating genome replication and transcription, viral protein translation, as well as the specificity of virion assembly9. However, the significance of such structures in the life cycle of bipartite comoviruses was not thoroughly investigated. Unlike other better studied (+) RNA plant viruses that rely on subgenomic RNAs for the translation of some more 3′ terminally located open reading frames, comoviruses adopt a gene expression strategy that involves the translation of long polyproteins from each of the two genome segments17. The polyproteins are then proteolytically processed to generate functional protein products. Another unique feature of comoviruses, but also of many animal viruses including polioviruses, is that the replication of their RNA genomes is primed by VPg, a small protein of fewer than 30 aas that is part of the RNA1-encoded polyprotein. VPg of animal-infecting picornaviruses is known to be uridylylated to become VPgpUpU. VPgpUpU then primes the synthesis of both (−) strand replicational intermediates from (+) strand genomes by pairing with the poly-A tail, and (+) strand progenies from (−) strand intermediates by pairing with two A residues at their 3′ ends18. It was further shown that the uridylylation of VPg depends on a stem-loop structure referred to as cis-acting element (CRE) that templates the addition of U residues to VPg by virus-encoded RdRPs18.13. Based on the fact that SLC lies within a region of RNA2 5′ UTR not exchangeable with RNA1 5′ UTR, we initially speculated that SLC might represent a structure unique to RNA2. However, this speculation has now been invalidated by the identification of SL1, a stem-loop in BPMV RNA1 that is structurally similar to SLC but requires a few nts at the beginning of RNA1 coding region for its integrity. Specifically, SLC and SL1 both have a 15-nt end loop, with 11 of the 15 nts being identical. Although the nt sequences of the SLC and SL1 stems are very different, they both form unbranched stems with few unpaired bases. The need for a base-paired stem in both SLC and SL1 was experimentally confirmed . Another possibility is that these stem-loop structures serve a role similar to that of CREs of animal-infecting picornaviruses. Several lines of evidence suggest that this is the case. First, the general structure of SLC and SL1 is very similar to that of CREs, in that they all have a long, unbranched stem and a large loop. In polioviruses, other enteroviruses, as well as rhinoviruses, the loops are mostly 14-nt in size, but can vary between 14 and 23 ntsImportantly, similar stem-loop structures can also be identified in both RNA segments of two other comoviruses – cowpea severe mosaic virus (CPSMV) and cowpea mosaic virus (CPMV). As shown in Fig. Intriguingly, the CPMV SL1 and SLC appear to diverge further from BPMV and CPSMV. First, unlike SL1 and SLC of BPMV or CPSMV that encompass nts in the vicinity of translational start point, the CPMV SL1 and SLC are both located within the coding regions of RNA1 and RNA2 ORFs. It is also remarkable that the sizes of CPMV SL1 and SLC differ by one nt (15 nts for SL1 and 14 nt for SLC), which could be due to the fact that the same nts are involved in both RNA secondary structures and protein coding, thus are severely restrained in their evolution. Finally, compared with those of BPMV and CPSMV, the loops of CPMV SL1 and SLC also contained a greater degree of sequence variations.To summarize, the evidence presented in this report suggests that SL1 and SLC of comoviruses are functionally analogous to CREs of picornaviruses. If this is true, the next question is why both genomic RNA segments of the bipartite comoviruses require a template-presenting structure for VPg uridylylation? It is possible that the VPgpUpU is generated only within the close vicinity of viral RNA, and immediately routed to the site of viral RNA replication, hence unavailable for use by a different genomic RNA segment. This would further suggest that the two genomic RNAs of comoviruses are replicated in separate replication complexes. In conclusion, our current study hints at the presence of CRE-like stem-loop structures in both genome segments of comoviruses. This work represents the first thorough examination of such structures in a comovirus. It advances our understanding of the mechanisms of comovirus replication, and should stimulate further investigations into these mechanisms.14. Constructs containing mutations within the SL1 and SLC regions were generated using PCR, with appropriate DNA oligonucleotides (sequences available upon request). The sequences of all mutants were confirmed with Sanger sequencing.Constructs BR1, BG2G, and BR2G-ΔSLC have been described in previous studiesPhaseolus lunatus cv. Anderson Bush) were purchased from Earl May Seed and Nursery . Bombardment experiments were carried out following the procedure described by Hernandez-Garcia et al. .