Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3′UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3′UTR of all four distantly related ecological virus subgroups . RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3′UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3′UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3′UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.RNA secondary structures in the 3′untranslated regions (3′UTR) of the viruses of the family  Flavivirus and Pestivirus (family Flaviviridae) share a common genome organization and coding strategy Viruses in the genera Flavivirus is subdivided into 4 ecological subgroups that reflect their association with different hosts and different pathogenesis West Nile virus (WNV), Japanese encephalitis virus (JEV), Dengue virus (DENV) and Yellow fever virus (YFV) are all major MBFV pathogens, causing human infections on an epidemic scale in tropical and subtropical countries, in association with their natural invertebrate hosts, Aedes or Culex spp. mosquitoes The genus Tick-borne encephalitis virus (TBEV), Louping ill virus (LIV), Powassan virus (POWV), Omsk haemorrhagic fever virus (OHFV), Langat virus (LGTV), Kyasanur Forest disease virus (KFDV) and Alkhumra haemorrhagic fever virus (AHFV) TBFV are also human pathogens; their circulation in the environment is entirely dependent on ticks and they are geographically widely dispersed. In contrast to MBFV, the genomes of pathogenic TBFV show significantly higher homology (over 92% at the amino acid level), yet they display a variety of clinical syndromes in humans, including, sub-clinical infections, fever, bi-phasic fever, encephalitis and/or haemorrhagic fever. The major human pathogenic TBFV are Aedes or Culex spp.The life cycles of viruses in the other two flavivirus groups are limited to single host species and are not associated with human disease. The no-known vector flaviviruses (NKV) have only been isolated from rodents or bats whereas flaviviruses in the fourth group, yet to be classified, do not infect mammalian cells. They replicate only in mosquitoes Pestivirus is subdivided into Border disease virus (BDV), Bovine diarrhoea virus types 1, 2 and 3 (BVDV1–3), Classical swine fever virus (CSV), Hog Cholera Virus (HoCV), Giraffe pestivirus (GRFPV) and Reindeer pestivirus (RNDPV) Pestiviruses (PVs) infect and cause disease in farmed and wild ruminants, causing economic losses in the farming industry and also threatening many wildlife species. The genus Flaviviridae, the flaviviruses and pestiviruses demonstrate no significant sequence homology. However, using a unique approach to the construction of robust nucleotide alignments we have previously revealed unexpected homology in the 3′UTRs of the MBFV, TBFV, NKV and ISFV that are genetically only distantly related Flavivirus evolved by numerous duplications of a region of about 200 nucleotides (that we termed LRSs ie long repeated sequences) localized in the ORF region that encodes the C-terminal domain of NS5pol, the RNA-dependent RNA polymerase (RdRp). We demonstrated that almost intact LRSs were preserved in the TBFV group but only sequence remnants of the LRSs as short direct repeats (DRs) were detected in the MBFV, NKV and ISFV groups. However, although this was clearly relevant to the evolution of these viruses, the biological, epidemiological and pathogenic significance of sequence duplications remained unclear.Classified into two distinct genera within the family Here, using a unique research approach based on the construction of 3′UTR alignments and calibrated algorithms to produce MFold-generated RNA secondary structures and in association with functional studies, we reveal duplicated sequences and associated RNA structures in the enhancer region of the distantly related flaviviruses and pestiviruses. We also identify distinct RNA folding patterns in the conserved regions of the 3′UTR of TBFV species associated with different pathogenetic outcomes. A complex role for duplicated enhancer sequences and RNA conformations is proposed, one in the dimerization of accessory proteins for the efficient assembly of replicase complexes and the second in sequestering numerous factors of antiviral immunity (proteins and miRNA) that are important determinants of virus survival in the natural habitat.The alignments for 3′UTRs of TBFV, ISFV and pestiviruses were constructed using ClustalX available in the BioEdit suite of programs http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-FormThe secondary RNA structures for individual virus 3′UTRs were predicted using MFOLD 3.2 accessed at The secondary RNA structure predictions for selected TBFV virus species are shown in polA comparative alignment of TBFV 3′UTRs  was consSL2 is a third highly conserved promoter element which plays a key role in disruption of the dsRNA panhandle to facilitate the molecular switch from the circular to linear form of the genome during RNA synthesis in vitro but its loss attenuates replication, largely due to a reduced level of RNA synthesis The flavivirus enhancer is located between the stop codon and promoter  and S2. Although conservation of the TBFV 3′LSH has been reported Different conformations were apparent in the SL5-SL6 region for OHFV  and S2 a63G64→G63C64) restored the LIV DB2 .Aedes species cells Culex species cells Kamiti River virus (KRV) and Cell Fusion Agent virus (CFAV) 3′UTRs have two almost identical DRs The ISFVs include two subgroups of viruses that have been shown to replicate only in mosquitoes, or mosquito cell cultures. Thus far, they appear to reproduce either in As no previous predictions of secondary RNA structures for the ISFV have been performed, structures in the ISFV 3′UTR were predicted using MFold as shown in Culex flavivirus (CxFV) are shown in The MFold-predicted RNA structures for KRV, CFAV and Remarkably, a significant secondary RNA structural duplication of the 3′LSH-Y1 region was observed precisely between the two “halves” of the KRV 3′UTR. The 3′CPN of the 3′LSH top loop, CACCG, was homologous to the sequence CUUCG localised in an apical position of SL16. The Y1 structure was homologous to Y5, with notably similar short signals UUU and AUG exposed at the top of the loops. These data confirm the previous suggestion that the KRV 3′UTR was formed as a result of self-duplication of a sequence of ∼600 nts The most striking feature noted for each ISFV was the unusually large number of duplicated short signals, about 3–4 nucleotides in length, exposed as top loops. Some of these signals were even tri- and quadruplicated  and Culex-associated (CxFV) ISFV was low (26–38%) but nonetheless alignment between all ISFV 3′UTRs was possible . One CxFV strain isolated from Culex tritaeniorhynchus (QBV) was more distantly related (55% homology). The major diversity between CxFV stains was observed in the internal hypervariable region between nucleotides 1–313. It contained long insertions/deletions, which contributed to the overall low Culex-specific 3′UTR homology. Two groups of the CxFV 3′UTR were identified, one closely related to the CxFV strain isolated in Japan (referred to here as CxFV J) and the second represented by QBV isolated in Vietnam.The homology between the entire 3′UTR of possible . The 3′UAedes-associated (KRV/CFAV) and Culex-associated (CxFV), and could reflect the result of independent duplication events based on the same 3′UTR region.A complication in the overall ISFV comparative alignments was short quadruplicate imperfect DRs of ∼25 nts present for CxFV (NC_008604) and homologous to R1/R2 of the KRV/CFAV group Culex-associated ISFV DRs1–4 and Aedes-associated R1/R2 was low, ranging between 29–43% again supporting the concept of an independent evolutionary origin for the Aedes R1/R2 and Culex DRs2–4 duplications.The homology between KRV/CFAV R1/R2 and CxFV DRs1–4 was calculated  as definAedes- and Culex associated ISFV in this region  which probably initiated evolution of the long hepacivirus-like 3′HVR The 3′HVR bears traces of numerous re-iterations most likely resulting from RNA polymerase stuttering on short 1–3 nt sequences. Five discrete 3′HVRs, 3′HVR1–5, represent the pestivirus primordial lineage which has since given rise to pestivirus 3′UTR diversification. The 3′HVR1 may have formed initially by the numerous reiterations of the UA-dinucleotide observed in all virus species, with the longest stretches in BVDV1, probably representing the most ancient region. The original long poly(UA) sequence was further evolved by nucleotide substitutions, deletions and insertions resulting in the formation of the UAUUGUAUA block which was subsequently duplicated several times. Four such imperfect repeats are preserved in the BVDV1 3′HVR1 and regressed sequence remnants are also observed in other pestiviruses. Further evolution included the loss of these repeats in some pestiviruses and their replacement by other sequences. For example, in the BVDV2 3′HRV1, the intercalation of AAA, followed by the further duplication of new-formed sequence blocks, resulted in the formation of two perfect DRs UGUAAAUA. Similar numerous reiterations around the tetra-nucleotide UUUA formed the 3′HVR4 . All pesThe 3′HV5 was formed by sequences observable only in the BVDV1 group. Two short identical DRs (CUACCUCAA) are found in the 3′HV5 and downstream region boxed in  and appeOther notably long (60 nts) DRs have been observed in GRFPV C of top loop 2 are conserved between the major pestiviruses, including HoCV, CSFV, BVDV1 and BVDV2. However, some differences were also observed; the RNDPV SL2 folded into a DB-like structure  yet the The 3′VR and 3′HRV1–4 fold into SLs that are very divergent in number and conformation between the pestiviruses. A notable common feature in this region is the presence of duplicated structures or short repeated linear signals of loops. Most remarkably, 2 identical 60-nt long GRFPV DRs  form twoIn vitro experiments with BVDV1 replicons have demonstrated that the region identified here as 3′VR-3′HRV1–5 binds a set of cellular proteins, among them a complex of nuclear factors associated with dsRNA (NFAR) proteins. These proteins also interact with the hairpin in the 5′UTR thus enabling a protein bridge between the 5′ and 3′ UTRs. Such a cyclisation of the pestivirus genome, with no direct RNA-RNA contact between 3′UTR and 5′UTR but mediated by the NFAR complex, was proposed to regulate viral translation and replication. It was suggested that this acts as a safety-lock system to prevent collisions between translation and replication complexes moving in opposite directions Despite significant sequence and length divergence, the structure-function organization of the 3′UTR for pestiviruses and flaviviruses share common features. Both genera display the highly conserved terminal hairpin-like SL1. Flaviviruses and some pestiviruses display 3′CPN in a highly conserved apical position of the SL1 and deletion of the SL1/3′LSH abolishes pestivirus/flavivirus infectivity via reduced RNA synthesis, highlighting its role as an essential region for viral RNA synthesis.Flaviviruses in each of the 4 ecological groups display different levels of structural conservation upstream of the SL1 The alignment of pestiviruses revealed that non-cytopathic PV1 contained numerous deletions within the conserved part of the SL1-SL2 region Other similarities in terms of flaviviruses and pestivirus structural organization are the presence of long and short DRs, particularly in the internal 3′UTR regions, significantly divergent even among closely related viruses. In each group, duplicated sequences are exposed as linear signals in top loops, with the length and identity of each sequence specific only for a distinct virus group adapted to a particular host.Flavivirus and Pestivirus genera suggests a biological significance for sequence and structural duplications as viruses adapt to their hosts. Numerous repeated sequences were also detected in the 3′UTR of alphaviruses although their structures and functions remained undetermined The molecular mechanism of RNA duplications/deletions is common to all RNA viruses, a property of the RdRp A third possibility is linked to a subgenomic flavivirus RNA (sfRNA), an almost intact 3′UTR region generated as the product of 5′-3′ XRN1 RNase processing of virus genome RNA during replication of TBFV and MBFV The normal pathway for the interaction of siRNA with target mRNA includes the assembly of RISC (RNA-stimulated silencing complex) This hypothesis also explains the differences detected between RNA structures of OHFV and AHFV  as a groInsect immunity is increasingly recognised as inhibitory RNA silencing In conclusion, numerous duplicated RNA structures were identified in the enhancer regions of the 3′UTR of distantly related flaviviruses and pestiviruses. Analysis of virus sequences indicated that duplicated homologous RNA structures have emerged independently on numerous occasions in different virus groups of both genera as the result of convergent evolution. Identifiable as redundant for laboratory maintained viruses, these structures are highly conserved implying their significant biological role, rather likely as enhancers of virus replication and/or antagonists of cellular antiviral innate immunity. The latter possibly play a role in direct contact between virus and host molecular elements, acting as adaptors of virus replication cycles to the molecular machinery of the particular host, with its mi/siRNA and thus determining virus host range and its pathogenetic characteristics.Figure S1Predicted secondary RNA structures of TBFV 3′UTR. Images were produced for A) TBEV, B) LGTV, C) OHFV, D, E) LIV and F) POWV by MFold using MDBP = 80 and annotated with LRSs, 3′CYCL (brown line), a 3′CPN (dark pink background) and a conserved hexanucleotide (red background). Viruses and corresponding accession numbers are indicated. Promoter is enclosed in a red-lined box. The colour/box codes on the left top corner designate RNA conformations of TBFV that are conserved throughout the Flavivirus genus. Identical sequences of duplicated RNA conformations DB1 and DB2 and other repeated sequences are indicated by identical colours.(PDF)Click here for additional data file.Figure S2Alignment of TBFV 3′UTR (to view with magnification 130–200%). Viruses are identified by abbreviated names and accession numbers. The RNA conformations (SLs and Y-shaped) and top loop regions are outlined by square and semi-oval brackets respectively and are enumerated in correspondence with (PDF)Click here for additional data file.Figure S3Predicted RNA structures of the ISFV. Images for 3′UTR for A) KRV, B) CFAV and C, D) CxFV were produced using the MFold MDBP = 80 and annotated with features of (PDF)Click here for additional data file.Figure S4ISFV 3′UTR alignments (to view with magnification 130–200%). Viruses are identified by the abbreviated names and accession numbers. The R1 and R2 correspond to 67-nt long DRs of the KRV and CFAV identified in A. The alignment between 3′UTR of Aedes-  and of Culex-associated (red letters) ISFV annotated with RNA conformations  displayed in B. Alignment between TBFV and ISFV annotated with secondary RNA structures as scheduled in Figures S1 and S3 in the region of the CxFV 5′DR1-3′LSH. Red semi-oval brackets specify loops with sequences similar between TBFV and ISFV. The regions of CxFV DR1 and TBFV LRS2-LRS3 are indicated. C. Alignment between TBFV LRS3–4 and ISFV DRs1–4/R1–2. The loop sequences of TBFV DB1 and DB2 are enclosed in ovals. Solid lines, with appropriate colour code, outline the positions of the DB-like and SLs encoded by the repeated regions; the arrows indicate the extension of the DB-like structure outside the alignment region shown.(PDF)Click here for additional data file.Figure S5Convergent duplications of flavivirus and pestivirus sequences in the 3′UTR. A. Independent origin of Aedes (KRV/CFAV) R1/R2 (red-box) and Culex DRI-DR4 (black-box) duplicated ISFV sequences. Virus 3′UTR sequences are enumerated from the stop codon of the single ORF. The CxFV 3′LSH and 3′Y-1 structures are mapped to the alignment. The Culex- and Aedes-specific gaps are enclosed in grey areas. The full-length of the Culex- associated ISFV 3′UTR is presented whilst only aligned fragments of KRV and CFAV 3′UTR are shown. B. Sequence identity of GRFPV duplications.(PDF)Click here for additional data file.Figure S63′UTR alignment of pestiviruses (to view with magnification 130–200%). Viruses are designated by accession numbers and abbreviated names. The variable (3′VR and 3′HVR1–5) and the conserved (3′CR) regions are indicated. The DRs are shown by double-headed arrows or boxed, with an appropriate colour code. The RNA conformations (SLs and Y-shaped) and the top loop regions are outlined by square and semi-oval brackets respectively and are enumerated as in (PDF)Click here for additional data file.Figure S7Predicted secondary RNA structures for the pestiviruses. Images were produced by MFold using MDBP = 60 for A) HoCV; B) CSFV; C) RNDPV; D) GRFPV; E) BDV; F) BDVD2; G)BVDV1. Viruses are identified by the abbreviated names and accession numbers. The SLs are numerated and annotated with the conserved 3′CPN (highlighted in red), conserved loop 2 sequences (highlighted in bright green) and conserved ssRNA region (blue line). The DRs of each pestivirus are highlighted in identical colours.(PDF)Click here for additional data file.Table S1List of abbreviations.(PDF)Click here for additional data file.


Endothelial DLL4 plays an important role in controlling of tumor angiogenesis, which is required for tumor invasive growth and metastasis. However, the regulation of DLL4 in clear cell renal cell carcinoma (ccRCC) has not yet been systematically elucidated.We performed bioinformatical analysis to explore miRNAs targeting DLL4. miR-30a was selected as a representative to validate its functional association in endothelial cell. Then, the expressions of DLL4 and mature miR-30a from 90 cases of ccRCC and 28 cases of nonmatched adjacent non-tumor tissues were measured by quantitative real-time PCR. Finally, the expression of miR-30a was correlated with DLL4 expression, tumor features (metastatic condition and microvessel density), and patient metastasis-free survival. The univariate and multivariate analyses were performed to select the risk factors associated with hematogenous metastasis, respectively.miR-30a negatively regulated DLL4 and inhibited the proliferation and migration of endothelial cells. DLL4 was up-regulated in ccRCC and further increased in hematogenous metastatic cases, while miR-30a was down-regulated in tumor tissues and further decreased in hematogenous metastatic ccRCC . Additionally, expression of miR-30a was inversely correlated with expression of DLL4 and microvessel density . Low-level miR-30a also indicated a higher probability of developing metastasis . Most importantly, miR-30a expression was an independent predictor of ccRCC hematogenous metastasis by the univariate analysis and binary logistic regression model (both p<0.05).Down-regulated miR-30a in ccRCC was associated with tumor hematogenous metastasis through increasing microvessel density by targeting angiogenesis-specific DLL4. Renal cell carcinoma (RCC) accounts for about 3% of adult malignancies and causes about 90,000 deaths worldwide annually. Although the mechanism of metastasis is not fully understood, metastasis is generally accepted to depend on tumor angiogenesis. microRNAs (miRNAs) were shown to negatively regulate gene expression at the post-transcriptional level by binding to the 3′ untranslated region (3′-UTR) of target mRNAs. We hypothesized that miRNA-30a might be a new regulator of DLL4 and might play an important role in angiogenesis and tumor progression. To test this hypothesis, we performed luciferase assays to determine whether miR-30a bound to the 3′-UTR of DLL4 and whether miR-30a expression was associated with tumor microvessel density (MVD) and hematogenous metastasis status. We also performed metastasis-free survival analysis to further correlate expression of miR-30a with ccRCC hematogenous metastasis.Written Informed Consent was obtained from all patients. This study was approved by the Protection of Human Subjects Committee, Chinese People’s Liberation Army (PLA) General Hospital.A total of 90 cases of ccRCC (Summarized in http://mirdb.org/miRDB/), TargetScan (http://www.targetscan.org/), and PicTar (http://pictar.mdc-berlin.de/) were used to predict miRNAs targeting DLL4 and the binding regions.miRNA target predicting algorithms miRDB  or negative control (0.6 µg) was co-transfected with DLL4 3′UTR plasmid (0.2 µg) and pRL-TK plasmid (0.05 µg) for 48 hours. The Dual-Glo Luciferase Assay System  was used according to the manufacturer’s instructions. Luminescence was measured on a Centro XS3 LB960 luminometer  and firefly activities were normalized to renilla activities for each transfected well. Every experiment was performed in triplicates and repeated 3 times.The full-length human DLL4 and its corresponding empty vector PCMV6-XL6 were purchased from the OriGene Company (USA). miR-30a plasmid and its negative control vector pcDNA3.0 were provided by Prof. Guo . miR-30a inhibitor and scramble miRNA were purchased from GenePharma . miR-30a inhibitor: 5′-CUU CCA GUC GAG GAU GUU UAC A-3′, scramble miRNA: 5′-CAG UAC UUU UGU GUA GUA CAA-3′. The 3′-UTR of DLL4 mRNA was cloned downstream of a firefly luciferase gene of pMIR-REPORT plasmid  to construct the DLL4 3′-UTR vector. pRL-TK plasmid  was encoded with the renilla luciferase gene. Transfections were performed with Mega-Tran 1.0 transfection reagent  as previously described. Total RNA extraction was performed as previously described. The anti-DLL4 antibody was obtained from Abcam (USA) and the anti-CD34 antibody was obtained from Epitomics (USA). Protein extraction and Western blotting were performed by using standard techniques described previously. Cell proliferation was analyzed by the MTS assay with the CellTiter 96 Aqueous One Solution . Cell migration assay was performed with an 8 µm–pore size membrane Transwell apparatus . These techniques were carried out as previously described. Formalin-fixed, paraffin-embedded tumor sections that had been confirmed by hematoxylin and eosin staining were deparaffinized and incubated overnight at 4°C with rabbit anti-CD34 antibodies . Secondary detection was carried out with an anti-rabbit peroxidase polymer detection kit (Vector Laboratories). The staining signal was detected with a 3,3′-daiminobenzidine substrate kit (Vector Laboratories). Images were acquired with an Olympus IX81 microscope . The images of tumor sections were taken at 200× magnification or more. MVD was quantified by counting the number of microvessels in five selected fields distributed in four quadrants and the middle of the image.The relative quantitation of gene expression detected by real-time PCR was log10 transformed and analyzed by student t test or ANOVA. Linear or rank correlation analysis was performed to determine the correlation between the gene expression levels. The univariate analysis and binary logistic regression model were performed to select the risk factors associated with hematogenous metastasis, respectively. Briefly, all covariables were transformed into binary data, and the tumor metastasis status (hematogenous metastasis or not) was selected as dependent variable. The enter method was used in this model. The Kaplan-Meier method and log-rank test were used to estimate and compare the probability of metastasis-free survival. The significance level was set at p<0.05.To identify the potential miRNAs targeting DLL4, we started with multiple prediction programs and found that miR-30a was one of the miRNAs with the highest possibility of targeting DLL4. The matching consequences of DLL4 3′-UTR and miR-30a predicted by these programs, were shown in In the present study, the proliferation capacity of HUVE-C cells detected by the MTS assay was enhanced by overexpressing DLL4 . HoweverTo date, the regulation of DLL4 by miR-30a in ccRCC has not been well demonstrated. Thus, 90 cases of ccRCC and 28 cases of adjacent non-tumor tissues were collected to examine the expressions of miR-30a and DLL4. Compared with non-tumor tissues, miR-30a expression, detected by real-time PCR, decreased about 10 fold in ccRCC, whereas DLL4 expression was elevated approximate 20 fold , and thuWe then determined whether miR-30a was associated with tumor metastasis. Tumor samples were divided into 3 groups according to the metastatic status. Non-metastatic (NM) samples were obtained from primary sites without lymphatic or distant metastases. Lymphatic metastatic (LM) samples were obtained from primary sites with lymph node metastasis, and hematogenous metastatic (HM) samples were obtained from primary sites in the presence of distant metastases but absence of lymph node metastases. The data showed that miR-30a expression decreased form NM and LM groups to HM group, whereas DLL4 density and MVD increased in HM group. However, there was no statistically significant difference between NM and LM groups in these variables . FurtherFor further evaluating the association of miR-30a with hematogenous metastasis, a multivariate logistic regression model was constructed by considering the clinicopathological features. These features involved patients’ characteristics , tumor features , and miR-30a expression. As shown in Although emerging evidence shows that anti-angiogenesis therapy can partially decrease cancer-specific mortality, the regulation of tumor angiogenesis in mediating cancer metastasis remains unexplored. In the current study, we showed that miR-30a directly targeted DLL4 and decreased tumor MVD, which suppressed ccRCC hematogenous metastasis. Low-level miR-30a was associated with shorter metastasis-free survival. Therefore, early identification of patients at high risk for cancer metastasis by detecting miR-30a expression can identify patients for suitable treatment and may decrease metastasis-specific mortality.The endothelium-specific Notch ligand DLL4 is well documented to play important roles in embryonic vascular development and tumor angiogenesis. miRNAs negatively regulate gene expression by pairing to the target gene mRNAs. The widespread and comprehensive use of microRNA microarrays has enabled the identification cancer-specific microRNAs as potential biomarkers for diagnosis, prognosis and targets for therapy. In summary, miR-30a was down-regulated in RCC and further decreased in RCC with hematogenous metastasis. Mechanistically, down-regulation of miR-30a increased microvessel density by targeting DLL4. Clinically, low-level miR-30a indicated a higher probability of developing metastasis and shorter metastasis-free survival.Figure S1The association of DLL4 density with microvessel density.(DOC)Click here for additional data file.Table S1Real-time RT-PCR Primers.(DOC)Click here for additional data file.


Importin-α1 belongs to a subfamily of nuclear transport adaptors and participates in diverse cellular functions. Best understood for its role in protein transport, importin-α1 also contributes to other biological processes. For instance, arsenite treatment causes importin-α1 to associate with cytoplasmic stress granules (SGs) in mammalian cells. These stress-induced compartments contain translationally arrested mRNAs, small ribosomal subunits and numerous proteins involved in mRNA transport and metabolism. At present, it is not known whether members of all three importin-α subfamilies locate to SGs in response to stress.in vitro.Here, we demonstrate that the oxidant diethyl maleate (DEM), arsenite and heat shock, promote the formation of cytoplasmic SGs that contain nuclear transport factors. Specifically, importin-α1, α4 and α5, which belong to distinct subfamilies, and importin-β1 were targeted by all of these stressors to cytoplasmic SGs, but not to P-bodies. Importin-α family members have been implicated in transcriptional regulation, which prompted us to analyze their ability to interact with poly(A)-RNA in growing cells. Our studies show that importin-α1, but not α4, α5, importin-β1 or CAS, associated with poly(A)-RNA under nonstress conditions. Notably, this interaction was significantly reduced when cells were treated with DEM. Additional studies suggest that importin-α1 is likely connected to poly(A)-RNA through an indirect interaction, as the adaptor did not bind homopolymer RNA specifically bona fide SG components under different stress conditions. Furthermore, importin-α1 is unique in its ability to interact with poly(A)-RNA in a stress-dependent fashion, and in vitro experiments indicate that this association is indirect. Collectively, our data emphasize that nuclear transport factors participate in a growing number of cellular activities that are modulated by stress.Our studies establish that members of three importin-α subfamilies are  The appropriate response to environmental or disease-related stress is essential for the survival of all organisms. To cope with these challenges, the formation of cytoplasmic stress granules (SGs) is one of the conserved strategies in eukaryotes Although SGs appear as well-defined cytoplasmic compartments, they are highly dynamic; moreover, their composition can differ according to the stressor and cell type SGs are not the only cytoplasmic RNA/protein foci; for example, under normal and stress conditions processing bodies (PBs) are present in the cytoplasm, where they can act as mRNA silencing sites Among nuclear transport factors, the importin-α family participates in nuclear transport, spindle formation, ubiquitin-mediated protein degradation and the reassembly of nuclear envelopes after mitosis Caenorhabditis elegans, Drosophila and other model systems Seven importin-α family members have been identified in humans so far KPNA2); this adaptor belongs to the α2 subfamily and shuttles between the nucleus and cytoplasm under normal growth conditions. At steady-state, the protein locates predominantly to the cytoplasm and nuclear envelope The best-studied member of the importin-α family is importin-α1 -RNA interaction with in vitro homopolymer binding assays indicate that the protein does not bind RNA directly. Taken together, our results provide new links between the nucleocytoplasmic transport machinery, RNA metabolism and the stress response.With our current contribution, we provide several lines of evidence that connect nuclear transport factors to cytoplasmic SGs and RNA metabolism. In particular, we defined the oxidant-induced changes as they relate to transport factor localization and RNA-association. Our work demonstrates that not only importin-α1, but also importin-α4, α5 and importin-β1, but not CAS, locate to SGs under different stress conditions. By contrast, none of the transport factors was detected in PBs under normal or stress conditions. Moreover, we identified importin-α1 as a novel protein that associates with poly(A)-RNA 2. To induce the formation of SGs, established conditions were used; oxidative stress was generated with 0.5 mM sodium arsenite for 30 min HeLa S3 cells were grown in Dulbecco’s modified eagle medium (DMEM) containing antibiotics and 8% fetal bovine serum. Cultures were maintained in a 37°C incubator with 5% COCells were grown to 70% confluency on poly-lysine coated cover slips. After treatment, immunofluorescent staining was performed essentially as published The association of importin-α family members or importin-β1 with SGs was evaluated using the SG marker HuR as a reference. For each transporter three independent experiments were performed for every stressor. SGs were identified based on HuR and the presence of individual nuclear transporter was determined. To this end, 30 SG-positive cells were randomly selected, and all of their SGs were scored. Data for importin-α1, α4, α5 or importin-β1 are depicted in the results section.7.4, 0.1% Triton-X, 10 mM EDTA, 10 mM vanadyl adenosine complex), containing a protease inhibitor cocktail HeLa cells were grown to 70% confluency and treated with ethanol (control) or DEM. Cell extracts were prepared in binding buffer , poly(U), poly(C) or poly(G) homopolymers were coupled to BrCN-activated sepharose (Santa Cruz Biotechnology) following standard procedures. To monitor unspecific binding to the resin, the same procedure was applied to BrCN-activated sepharose, but without addition of a homopolymer.E. coli and purified with Ni-NTA agarose (Qiagen). Purified proteins were dialyzed against 20 mM Tris HCl, pH 7.4, 150 mM NaCl and 2.5 mM MgCl2 and employed for in vitro RNA homopolymer binding, essentially as described 2, 0.5% NP-40, 1 mM vanadyl adenosine complex, protease inhibitors) containing 200 µg BSA/ml. In control experiments, homopolymers were digested with micrococcal nuclease  for 1 h at 37°C and then blocked overnight with BSA as described above. Blocked resin (poly(A), poly(U), poly(C), poly(G)-sepharose or non-conjugated sepharose) was incubated for 10 min at room temperature with 5 µg purified protein and 100 µg BSA in 500 µl BB. Unbound material was removed by centrifugation , and resins were washed once with BB containing 2 mg/ml heparin  and once with BB/heparin/1 M NaCl . Bound protein was eluted with twofold concentrated gel sample buffer by incubation at 95°C for 15 min and the resin was removed by centrifugation . Eluted and unbound material was analyzed side-by-side by Western blotting with antibodies against importin-α1 or the His6-tag.His6-tagged importin-α1 or poly(A)-binding protein (plasmid encoding poly(A)-binding protein was kindly provided by Dr. N. Sonenberg) were synthesized in Western blotting and ECL with HRP-conjugated secondary antibodies followed standard procedures t-test, all data are shown as averages+SEM.For Western blot analyses results were normalized to controls; three independent experiments were carried out for each treatment. Significant differences were identified by Student’s We and others have shown previously that stress interferes with nuclear import, in part by retaining importin-α1 in the nucleus KPNA3) and α5 (KPNA1), which are representatives of subfamilies α3 and α1, respectively. To test whether importin-α4 or α5 is present in DEM-induced SGs, we located these proteins by imunocytochemistry, with two different SG markers, HuR  cellulose, and bound material was analyzed by Western blotting. In these experiments, importin-α1 associated with the oligo-(dT) resin, suggesting that the protein interacts with poly(A)-RNA . By contE. coli. For example, this method was used to pull down purified poly(A)-binding protein in vitroE. coli exhibited no or only weak interactions with homopolymers in vitro.Data described in Importin-α plays an essential role in nucleocytoplasmic transport by serving as an adaptor between the cNLS and importin-β1. One of the immediate responses to stress is nuclear transport inhibition. We have shown previously that heat shock and DEM interfere with nuclear import and export by affecting multiple transport factors bona fide SG constituents. Importantly, their association with SGs is specific, because they do not concentrate in PBs under normal or stress conditions -RNA in growing cells. Second, posttranslational modifications of importin-α1 may be necessary for its binding to RNA, and these modifications are absent when importin-α1 is purified from E. coli. Third, importin-α1 recognizes specific RNA sequences that are not provided by homopolymers. Future studies will have to distinguish between these possibilities to determine the precise mechanisms that promote the interaction between importin-α1 and poly(A)-RNA in vivo.Aside from its accumulation in SGs under different stress conditions, importin-α1 associated with poly(A)-RNA Independent of the nature of its RNA-binding, the coincidence of importin-α1 release from poly(A)-RNA and SG association could suggest that the stress-induced dissociation of RNA/importin-α1 complexes is linked to SG recruitment. In support of this recruitment, SG components could provide binding sites for importin-α1; HuR is a potential candidate for this interaction, as it is an established binding partner of importin-α1 under nonstress conditions In an alternative model, nuclear transport factors may promote SG assembly by moving individual constituents to SGs. According to this idea, members of the importin-α family and importin-β1 will deliver material to the growing granule Collectively, our experiments provide novel insights into the biological roles of several members of the importin-α family, especially the multifunctional protein importin-α1. The newly identified ability of importin-α1 to associate with poly(A)-RNA is particularly interesting, because it is controlled by stress. Moreover, we identified additional members of the importin-α family, which belong to different subfamilies, as novel SG constituents. While these factors have an established role in nuclear protein transport or gene expression regulation Figure S1Specificity of anti-importin-α antibodies for immunolocalization. Primary antibodies against members of the importin-α family and isotype-specific IgG controls (for importin-α1 and α5) or pre-immuneserum (control for importin-α4) were tested under the same conditions. Staining was evaluated for ethanol and DEM-treated cells as described for (TIF)Click here for additional data file.Figure S2Western blot analysis determines the specificity of antibodies against nuclear transport factors and HuR. Crude HeLa cell extracts were tested with antibodies against nuclear transport factors, HuR or control antibodies as indicated. Negative control antibodies, either isotype-specific IgG or pre-immuneserum (PS), were used at the same concentration as primary antibodies. For each antigen, the same filter was probed with primary and control antibodies, with identical exposure times during ECL.(TIF)Click here for additional data file.


Current RNA isolation methods have limitations in their ability to yield good quality and quantity of RNA from plants that have high content of phenols, polysaccharides and storage proteins. Existing methods also do not eliminate accompanying chromosomal DNA in RNA preparation that causes false positives in gene expression studies. Standard isolation technique was modified for rapid and quick extraction of RNA, and lentil tissue most appropriate to extract good quality RNA was determined. The concentration of the phenol blocker polyvinylpyrrolidone in the extraction buffer was determined, DNase I was added to eliminate chromosomal DNA and the timing of this step was optimized. RNA up to 568 μg of RNA from 1 g of tissue was isolated from four different tissues of lentil in less than half the time typically required by reported methods. The method avoids the use of toxic phenol–chloroform, hazardous guanidinium thiocyanate (GTC) and laborious CsCl ultracentrifugation. Absorbance A260/A280 ratio of 1.9 and A260/A230 ratio of 2.7 reveal RNA to be of high purity. Modified method yielded RNA that was free from contaminants and suitable for RT-PCR and cDNA library construction. Lens culinaris ssp. culinaris) is a cool-season grain legume cultivated throughout world and in Indian subcontinent providing a vital source of dietary protein in human diets and straw for animal feed. It is a diploid (2n = 2× = 14), annual flowering self-pollinating crop with a genome size of 4 Gbp  of the supernatant was used for the quantification by taking absorbance at A260 nm. To eliminate chromosomal DNA contamination, 10 μL DNase I (10 mg/ml) was added to the solution and incubated at 37 °C for 30 min. Excess DNase I and contaminating proteins were removed by potassium acetate (pH 4.8) and ethnol precipitation.The plant materials were finely powdered by grinding in liquid nitrogen and to maintain uniformity 100 mg of fine powder was used as starting material. A modified borax decahydrate extraction buffer was used for the extraction of RNA from all the tissues of lentil. The buffer contained 0.2 M sodium tetraborate decahydrate, 30 mM Methylene glycol tetraacetic acid, 1 % (w/v) Sarkosyl and 1 % (w/v) sodium deoxycholate and the pH was adjusted to 9.0 by the addition of 1 M sodium hydroxide. For each tissue sample, 500 μL extraction buffer containing 6 % (w/v) polyvinylpyrrolidone (PVP) and 0.2 % (w/v) of the reductant dithiothreitol were added. The mixture was vortexed and incubated at 80 °C for 5 min. Subsequently, 1 mg of proteinase K was added to the hot solution and was incubated at 42 °C for 1 h after vortexing. To eliminate seed reserves, 50 μL 2 M potassium chloride was added and the samples were incubated on ice for 30 min followed by centrifugation at 10,000 rcf for 10 min. Approximately, 400 μL of the lower aqueous phase was transferred to a new tube. This eliminated the lipids, which are present in the upper phase. Nucleic acids were precipitated by the addition of 0.3 volumes of 120 μL of ice-cold 10 M LiCl. After adding LiCl, the suspension colour immediately turned milky white. The sample was incubated at −80 °C for 30 min and again centrifuged for 10 min at 10,000 rcf. The supernatant was discarded and the pellet was washed with 100 μL of 2 M LiCl. The pellet containing nucleic acids was re-suspended in 100 μL TA260/280 ratio was used to detect the contamination with proteins and A260/230 ratio was used to check for carbohydrate contamination. Quality and integrity of isolated RNA was verified by electrophoresing RNA on 1.5 % agarose gel and staining with ethidium bromide  and 0.5 U of Taq DNA polymerase. The temperature profile was as follows: initial denaturation at 95 °C for 4 min, 35 cycles at 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min followed by final extension for 7 min at 72 °C. A ‘control PCR’ was carried out with 0.5 μg of total RNA in the absence of reverse transcriptase to check for chromosomal DNA contamination.cDNAs were synthesized using 0.5 μg total RNA extracted from lentil roots, stems, leaves and etiolated seedlings by RT-PCR using 1st strand cDNA synthesis Kit . The cDNAs were PCR amplified (with expected amplicon of 360 bp) by lectin gene (NCBI Accession No AY547295) specific primers, a gene reported to encode an insecticidal protein against sap sucking insects. PCR reactions were carried out in a final volume of 25 μL reaction mixture containing 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgClUsing 10 μg total RNA-isolated from developing seed samples, the cDNA library was constructed with SMART cDNA library construction kit . The library was subsequently used for screening full-length lectin gene (unpublished data).Standard methods for RNA extraction A260/A280 and A260/A230 ratios of the RNA extracts were as high as 1.9 and 2.7, respectively, which indicates the presence of very low amounts of contaminating proteins, polysaccharides and polyphenol compounds. Purity of RNA samples (A260/A230 ratio) isolated from leaves was 1.9, while in case of stem tissues it was 1.7. The bands corresponding to 28S and 18S rRNA were significantly prominent in RNA extracted from leaves than other tissues indicating amenability of leaf tissues for the isolation of RNA. Gel electrophoresis of RNA samples isolated from all tissues resulted in firm and distinct bands of 28S and 18S rRNA and showed the RNA to be of excellent quality  through library screening approach (unpublished data).The above described method allows for the extraction of sufficient quantities of high-quality RNA in a much shorter time (4 h as compared with 18 h) than previously reported. This was achieved mainly by taking small amount of tissues and reducing the time required for the precipitation of nucleic acids with lithium chloride. A short precipitation time of 1 h resulted in an excellent quality of RNA. Overall, the described method achieved more than adequate yields for RT-PCR purposes. Therefore, reverse transcription followed by standard PCR was used to test RNA quality. After extraction of RNA from roots, stems, leaves, etiolated seedlings, cDNA was prepared by reverse transcription. Further, the single stranded cDNA was used as a template in standard PCR with gene specific primers to amplify an expected amplicon of 360 bp of lectin gene. Successful amplification of lectin amplicon AY547295) from all RNA preparations (Fig. 47295 froUnlike pre-existing protocols for phenolic tissues (Salzman et al.


During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5′LTR and activate polyadenylation in the 3′LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal.Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5′LTR. In contrast, polyadenylation at the 3′LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation.Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5’end of their RNA. At the 3’end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression. Most cellular mRNAs are polyadenylated. Polyadenylation (poly(A)) is provided, by four sequence elements: the polyadenylation signal (poly(A) signal), the cleavage site (poly(A) site), G/U-rich downstream elements (DSE), and upstream cleavage factor I binding sites (for review see -4). The The first such mechanism involves polyadenylation signals encoded upstream of the promoter start site. This simple and obvious type of regulation has been described for Rous sarcoma virus (RSV), mouse mammary tumour virus (MMTV), and human T-lymphotropic virus type 1 (HTLV-1). In these viruses, the essential poly(A) signals are localized in the U3 region -10. SincA second mechanism for suppression of polyadenylation involves elements downstream of the LTR. Splicing and polyadenylation occur co-transcriptionally and are coupled processes . It hThe third mechanism for polyadenylation suppression involves weak polyadenylation sites in both LTRs. The Moloney Murine Leukaemia Virus (MoMLV) harbours a weak poly(A) site, and sequences in the R region are required for poly(A) site regulation . In contenv region, initiate gene expression. The activity of both promoters is dependent on the viral transactivator protein Tas. FV pol is encoded by a specific spliced transcript. Furthermore, the structure of the FV R region is unique  differs from that of orthoretroviruses . Two proe Figure A 25]. T. Tenv ree Figure . The MSDpression ,30. The pression  . In cells transfected with the pHSRV13-SDm1 plasmid, neither gag-encoding genomic RNA nor pol or env RNA was retrieved  signal and site in each LTR. Consequently, both repression of the poly(A) signal in the 5′LTR and promotion of polyadenylation in the 3′LTR are required to express full-length 5′LTR-derived transcripts. Surprisingly, previous experiments showed that inactivation of the MSD by site-directed mutagenesis resulted in complete loss of ression , encoding 11 nucleotides complementary to U1snRNA, was generated as well  into the MSD at the 5′LTR Figure A. SDm2 aIn order to analyse repression of polyadenylation at the 5′LTR in a quantitative way and to exclude influences of the 3′LTR, we constructed reporter plasmids encompassing the complete pHSRV13 5´LTR encoding either the wild-type MSD or the SDm1 or SDm2 mutants in the pGL3 vector backbone. Thus, the U3 promoter drives firefly luciferase expression   primed RT-PCR was performed with RNA of cells transfected with the reporter plasmids  suppression, we performed experiments with a mutated U1snRNA that was complementary to 7 nucleotides of SDm2 Figure A. Expresd Figure A. The red Figure A. As destas-specific probe  site is independent of splicing but dependent on U1snRNP binding, a pGL3LTR reporter plasmid encoding an inactive splice donor mutant (SDm5) was constructed. This mutant encodes an ideal U1 binding site with the exception of the G/G dinucleotide. This dinucleotide was mutated to G/C, which has been shown to inhibit splicing Figure A, likelyTo confirm these results, Northern blotting analysis using a probe encompassing the R region of the pGL3SDm5- and SDm5+p(A)m-derived transcripts was performed. RNAs were extracted using an miRNA isolation procedure Figure B. The muTranscription, splicing, and poly(A) addition are coupled processes . Since tRenilla and firefly luciferase genes in the pRL vector  signal only encompasses the R-region. To further investigate whether 3′polyadenylation is influenced by upstream sequences, a simplified reporter was constructed by inserting nucleotides −13 to +1 into the pGL3-CMV-RU5 clones described above Figure C, which To determine the differences in both polyadenylation and splice donor dependence at the 5′ and 3′LTRs, we analysed the RNA secondary structure of two RNA fragments, one representing the 3′LTR RNA (nucleotides −13 to +198) and one representing the 5′LTR RNA (nucleotides +1 to +198) by RNA SHAPE Figure A, the fiThe single 5’LTR mutation of SDm1 completely disrupts the local RNA fold and modifies the RNA structure to a 3’LTR-like fold  signal and is deleterious for p14 mRNA biogenesis -UTP  were included in the in vitro transcription assay. All RNA probes were purified by PAGE. The RPAs were performed using the RPAIII kit (Ambion) according to the manufacturer’s instructions.Synthesis of all RNAs used in this study was performed with SP6 RNA Polymerase (Promega). To obtain -end labeling of the primer, selective 2′hydroxyl acetylation analyzed by primer extension (SHAPE), and sequencing reactions were performed as described by Hartl et al. with minor modifications [-end of the RNAs  and the other binding to the central part of the RNAs . The plasmid pHSRV13 was used as a template for sequencing reactions to assign the SHAPE reaction products. The program Mfold [-type 3′ and 5′LTR (nucleotides 99 to +198) were constrained to ensure the integrity of the folding topology of these regions, while the folding of nucleotides −13 to +98 was unconstrained.RNA synthesis, 5′ications . RNAs spications -48. SHAPam Mfold -51 was uDSE: Downstream element; FV: Foamy virus; HIV-1: Human immunodeficiency virus type 1; HTLV-I: Human T-Lymphotropic virus type I; LTR: Long terminal repeat; MMTV: Mouse mammary tumour virus; MoMLV: Moloney murine leukaemia virus; MSD: Major splice donor; poly(A) signal: Polyadenylation signal; poly(A) site: polyadenylation cleavage site; PFV: Prototype foamy virus; RSV: Rous sarcoma virusThe authors declare that they have no competing interest.E-MS, R M, M J H, JB, H W, D F, and J L performed the experiments. JB, Je B and BMW contributed to the data analysis and the design of the study. JB wrote the manuscript. All authors read and approved the final manuscript.Supplementary material. Figure S1. SDm4 suppresses polyadenylation at the LTR poly(A) site efficiently. Luciferase assay of pGL3-LTR derivatives. BHK-21 cells were co-transfected with pGL3-LTR derivatives, a Tas expression plasmid and a CMV-driven Renilla-luciferase expression plasmid. Luciferase expression was measured 2d after transfection. The bars represent the mean of 3 independent experiments. The error bars indicate the calculated standard deviation. The experiment was repeated six times. FigureS2. Relative SHAPE intensities as a function of base position. The 5'LTR intensities are shown in white, the 3'LTR intensities in black bars. Bases with intensities higher than 0.3 were assumed not to be paired. The data are derived from a single SHAPE experiment. The experiment was repeated twice. The positions of the MSD and the polyadenylation signal are indicated. Figure S3. Calculated RNA structures of (A) SDm1, (B) SDm2, and (C) the Renilla luciferase-R-U5 construct using the constraints from the RNA SHAPE analyses .Click here for file


NxUGUNyUGU and identify a high affinity interaction (Kd ∼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3′ or 5′ terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5′ to 3′ orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5′-UGU Xenopus and the highly homologous Bruno in Drosophila  in humans, ETR-3-like factors, embryonic deadenylation element binding protein (EDEN-BP) in osophila , are widosophila . In miceosophila ,15 and hosophila . Homologosophila ,13,19–26Xenopus, CELF1 induces deadenylation and appears to play a key role in regulating poly(A) shortening  shortening in the 3′ untranslated region (3′ UTR) by recruiting the poly(A) deadenylase ribonuclease (PARN) (cis-acting AU-rich elements (AREs)  ,30. In te (PARN) ,12,31–35s (AREs) . In mamms (AREs) . CELF2 hs (AREs) ,37. Thess (AREs) .The CELF proteins consist of three 80–100 residue RNA recognition motifs (RRMs) all of which adopt the same structural topology with a four-stranded anti-parallel β-sheet packed against two α-helices ,40. The Although the individual domains of CELF1 have been studied on multiple RNA substrates and early investigations identified binding to CUG repeats ,42–45, Gc-fos and TNFα RNA substrates within AREs shown to enhance mRNA decay in HeLa extracts. In these studies, a synthetic GU-rich EDEN motif of 19 nt (EDEN19) derived from the 3′ UTR of Xenopus c-mos was shown to be a competitive inhibitor in in vitro deadenylation assays (in vitro assays which show that CELF1 enhances deadenylation of these RNA substrates. The observation that the poly(A) deadenylase PARN is co-immunodepleted with CELF1 strongly suggests that PARN and CELF1 form a protein-mediated complex  that permits simultaneous interaction of all three RRMs within a single 15-nt substrate. The PRE experiments demonstrate selective signal attenuation in NMR spectra that are consistent with binding selectivity and a non-sequential arrangement of RRMs on consecutive UGU sites along the RNA substrate.The structural basis for the binding of the full-length CELF1 to its target RNA substrate is still not fully understood. It is unclear whether all three domains of the CELF1 protein are simultaneously recruited to a single RNA-binding element, what the sequence requirements are for this interaction and whether there is selectivity in the arrangement of the three RMMs in this recognition process. Using a stable three-domain CELF1 construct (RRM123), we describe nuclear magnetic resonance (NMR) structural and biophysical studies based on chemical shift perturbation (CSP) analysis, paramagnetic relaxation enhancement (PRE) experiments with spin-labelled RNA substrates and binding affinity studies by isothermal titration calorimetry (ITC). A family of target RNA sequence of the form 5′-UGUn expansion in the mutant RNA is mediated primarily by the RNA-binding protein MBNL1 (muscleblind-like 1) -like 1) . The bin-like 1) , but theXenopus CELF1 was cloned into the pET28a vector. The RRM123 construct was produced by deletion of the codons for residues 215–384 using one-step PCR. The RRM23 construct was produced by the further deletion of the codons for residues 1–107 using the same method. All protein constructs were expressed in Escherichia coli BL21 (DE3) cells as fusions with an N-terminal histidine tag. Growths were carried out in Luria broth for unlabelled material and M9 minimal media for production of isotopically labelled protein. In all, 30 µg/ml kanamycin was used as an antibiotic control in all growths. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 30°C.A construct of full-length Cells were harvested by centrifugation and lysed by sonication. The lysis buffer for RRM123 was 25 mM potassium phosphate, 500 mM NaCl (pH 7.5). For wild-type CELF1, the lysis buffer was 25 mM potassium phosphate, 50 mM NaCl and 4 M urea. In all cases, a protease inhibitor cocktail (Roche) was added to the lysis buffer immediately before sonication. Cell debris was removed by centrifugation of the lysate, which was then applied to a His-Pur cobalt affinity column and left for 2 h at 4°C.This column was then washed with 25 ml of a high salt buffer  followed by a 10 mM imidazole wash to remove non-specifically binding proteins. RRM123 was eluted into 25 mM potassium phosphate, 200 mM NaCl and 500 mM imidazole (pH 7.0). Wild-type CELF1 was eluted in 25 mM potassium phosphate, 50 mM NaCl, 2 M urea and 500 mM imidazole. RRM123 was purified by gel filtration using a Superdex 200 column and desalted into water. Wild-type CELF1 was immediately desalted into water using a HiTrap desalting column (Amersham Biosciences). The eluted proteins were immediately frozen using liquid nitrogen and then lyophilised and stored at −20°C.2O (pH 7.0). The RRM123 construct contains seven Cys residues, which could potentially lead to aggregation through disulfide bond formation. NMR sample stability studies showed little evidence of significant degradation over NMR acquisition periods of >96 h even in the absence of DDT. Wild-type CELF1 required the addition of 200 mM urea to the NMR buffer for solubilization of the protein. Experiments were conducted at 25°C on an 800 MHz Bruker Avance III Spectrometer with cryoprobe. Protein concentrations of 200–250 µM were used for all RRM123 experiments. Wild-type CELF1 experiments used protein concentrations of ∼100 µM. Data were collected using Topspin 2.3 and 3.0 and processed in CCPN Analysis version 2.1.NMR samples of RRM123 were prepared by dissolving lyophilised protein in 25 mM potassium phosphate, 100 mM NaCl, 10% (v/v) Det al. methanethiosulphonate (MTSL) for 2 h in darkness at room temperature, following the procedure of Qin et al. . Unreact−1 was used. Fitting of the resulting data was carried out using the Origin 7.0 fitting program using various binding models to determine ΔH, ΔS, Kd and the stoichiometry for the interaction. Baseline correction was necessary for some data sets.ITC was carried out using a high sensitivity microcalorimeter driven by VPViewer2000 software (version 1.4.27) . All buffered solutions were degassed for 10 min in a Thermovac  before loading. The cell was filled with 1.4 ml of 12.5 µM RRM123 in 25 mM potassium phosphate, 100 mM NaCl (pH 7.0) in RNase free water. In all, 300 µl of 125 µM RNA in the same buffer solution was loaded into the syringe. All titrations were carried out at 25°C. After an equilibration period of 20 min, 30 injections of 10 μl were added at 600 s intervals. A constant stirring speed of 300 rpm was applied throughout the titration to rapidly mix the contents of the cell. A reference power of 5 μcals. All hydrogen atoms were introduced, and suitable linkers constructed between the RRMs where these were not present in the available PDB files. TIP3P water was used as an explicit solvent and was added in a truncated octahedron geometry to a distance of 9 Å around the protein. Monovalent (Na+) counterions were added to ensure overall charge neutrality of the RNA phosphate backbone. Energy minimization was carried out using the SANDER modules of the AMBER program. Molecular dynamics simulations were run without restraints for up to 2 ns at 300 K. The resulting models were viewed using Pymol  and for the full-length 489 residue CELF1 protein using methodology previously described . The isoc-fos, TNFα, c-jun and c-mos  in which deletion of the central guanine eliminates the possibility of three UGU binding sites. Both the binding affinity and stoichiometry  are closely similar to EDEN11. In both cases, a binding stoichiometry mid-way between 1:1 and 1:2 (protein:RNA) is consistent with each RNA substrate carrying only two viable binding sites to accommodate the three binding motifs of RRM123.The affinity and binding stoichiometry of RRM123 with a number of RNA substrates of the general sequence 5′-t pH 7.0 . In all n = 1.45 . As a coc-mos sequence, produced a binding stoichiometry closer to 1:1 (n = 0.79) and a 7-fold enhanced binding affinity (Kd = 0.33 ± 0.07 µM) compared with the EDEN11 GRE and EDEN-4U  consistent with the simultaneous interaction of all three RRM motifs on the same RNA substrate , suggesting some selectivity in how the three domains are arranged on the RNA target.We turned to the longer RNA substrates with greater separation between UGU sites. ITC analysis of EDEN19, derived from the natural  EDEN-4U . Putativubstrate . Switchie , which is 15-fold weaker than for EDEN-2U/4U . However, the 1:1 stoichiometry similarly suggests that all three RRMs are engaged in binding interactions with available UGU sites.We examined in more detail the effects of spacer separation between the second and third UGU sites while maintaining the optimum 2 U spacer for the tandem interaction of the two N-terminal RRMs . The seqEN-2U/4U . This is15N-labelled RRM123 with a number of RNA substrates by NMR at 800 MHz at protein concentrations of ∼200–250 µM . The 1H-15N transverse relaxation optimized spectroscopy (TROSY) spectrum of the 319 residue RRM123 is well dispersed at 298 K with narrow line widths indicative of favourable internal dynamics between RRMs (1H-15N heteronuclear nuclear overhauser enhancement (NOE) experiments show positive NOEs (>0.8) for the structured domains and zero or negative NOEs for the linker residues indicative of considerable flexibility, although in many cases, sequence specific assignments in the disordered region were not achieved.We examined the mode of interaction of een RRMs a. The sp15N-labelled RRM123 to further corroborate observed binding stoichiometries identified by ITC. The 11-mer GRE consensus sequence (UGUUUGUUUGU) was used as a control, representing the minimum binding element with three possible UGU binding sites in close proximity. In 1H-15N-TROSY spectra, a 1:1 ratio of RNA:protein resulted in extensive line-broadening across all three RRMs and the loss of many resonances, suggesting conformational exchange on an intermediate to slow timescale. However, increasing concentrations up to a 2:1 ratio of RNA:protein produced considerable sharpening, with bound peaks visible for all three domains. Residues G21 and C61 (RRM1), G113 and C150 (RRM2) and G278 (RRM3) are particularly sensitive to RNA binding and are all seen to undergo significant CSPs in the bound state , C150 (RRM2) and G278 (RRM3) particularly affected a–c, conf1H-15N-TROSY spectra of the complex showed even greater line broadening effects, which precluded an extensive CSP analysis. However, key resolved residues  within each of the three domains showed similar CSP effects to EDEN-2U/4U, as shown overlayed in 1H NMR spectra of RRM123 bound to EDEN-2U/HL revealed imino proton signals between 12.5 and 13.5 ppm that were consistent with four stable Watson–Crick base pairs within the hairpin double-stranded stem region. In both complexes, a combination of ITC and NMR data strongly support formation of 1:1 complexes in which all three RRMs simultaneously interact with the three UGU sites on a single RNA substrate.Titration experiments with EDEN-2U/HL d, in whi1H-15N CSP methods (A paramagnetic spin label (MTSL) was covalently attached to the RNA substrate through the incorporation of the nucleobase analogue 4-thiouridine (U*) . Relaxat methods ,58.Figu1H-15N TROSY spectra with those of the ascorbate-reduced RNA substrate with the spin label chemically quenched. The spin label produced a range of effects from either no attenuation to partial or complete signal broadening for a significant number of residues, suggesting a high degree of binding selectivity  HuD and Hrp1 RNA binding proteins bound to various different single-stranded substrates reveal RNAs with either a linear or kinked trajectory between RRM-binding surfaces . All of c-fos and TNFα AREs have been shown to enhance mRNA decay and contain multiple UGUA, UGUU and UGUG sequences in close proximity. The greater abundance of these sites than predicted from a random distribution suggests that these sequences contain high affinity CELF1-binding sites. A 19-nt fragment of c-mos (EDEN19) was shown to be a highly competitive inhibitor of deadenylation of c-fos and TNFα, suggesting that c-mos similarly contains one or more high affinity CELF1 recognition sites within the EDEN19 element  derived from the c-mos natural target sequence. We have shown from ITC and NMR experiments, including PRE effects using spin-labelled RNAs, that the RRM123 CELF1 construct is able to simultaneously bind to the three UGU sites. The high affinity EDEN-2U/4U RNA substrate leads to binding selectivity in which the three RRMs of CELF1 are aligned in a non-sequential 2-1-3 arrangement from 5′ to 3′ along the RNA target sequence.We have studied a number of mRNA substrates of the general sequence 5′-UGUNet al. (UGUUUGUUUGU). Thus, the GRE with sub-optimal spacing of UGU sites does not capture the full extent of CELF1-RNA recognition but may act as a key anchor point for the initial RNA interaction.In contrast, our NMR and ITC studies of the consensus GRE identified by Vlasova et al. , which cet al. ,41. We ssyn glycosidic conformation for the guanine base, more typical of left-handed Z-RNA helices (d ∼ 2–4 µM) compared with the other isolated RRMs (Kd ∼ 30–60 µM) (The recognition of UGU(U) motifs by the three RRMs is mediated by a common set of conserved residues on the surface of the β-sheet and adjacent loops that use a combination of base-specific hydrogen bonding and π-stacking interactions ,40. Furt helices . These d0–60 µM) .d ∼ 0.4–0.7 µM) to closely spaced UGU sites, realizing a >100-fold enhancement in affinity over the isolated domains (d = 0.52 ± 0.12 µM for RRM3 binding to a UGU(U) site. We conclude that the co-operative binding of RRM1 and RRM2, which leads to a high affinity interaction, together with the strong binding of RRM3 in isolation, ensures that both ‘ends’ of CELF1 are anchored to an RNA substrate with comparable affinity, of particular relevance in targeting dispersed UGU-binding sites and in the structural remodelling of pre-mRNAs.However, RRM1 and RRM2 are separated by a short 8-residue linker and the tandem interaction of the two RRMs leads to high affinity co-operative binding (K domains . This tad = 0.55 ± 0.08 µM) in a 1:1 binding event with NMR CSP effects consistent with all three motifs simultaneously engaging in RNA interactions. Bringing together distant UGU sites through RNA secondary structure formation may account for the observed interaction of CELF1 with mRNAs such as TNFα  with an additional 170 residues of flexible linker sequence was isolated in small amounts for RNA-binding studies with both EDEN-2U/4U and EDEN-2U/HL. Although the spectra of the wt-CELF1 in isolation were of high quality, owing to the intrinsic dynamics of the structure, RNA complex formation leads to extensive line broadening and poor resolution. Many of those residues that are expected to be perturbed , and a variety of residues usually associated with smaller CSPs show characteristic, although not readily quantifiable, binding effects that are consistent with all three RRMs of wt-CELF1 binding to these high affinity RNA substrates.Although we have worked largely with the truncated RRM123 construct in this study, for reasons of stability, in vitro .We also considered reports that differential phosphorylation of CELF1 has been implicated in regulating both protein–protein and protein–RNA interactions, with studies suggesting that phosphorylation of Ser28 in RRM1 of CELF1 increases RNA binding in vitro  and thatin vitro . To inveThe RNA mutation model for DM pathogenesis has shown that CUG expansion repeats in the 3′-UTR region lead to the formation of long double-stranded RNA hairpins that sequester RNA-binding proteins ,49. The The RRM is a ubiquitous RNA-binding motif that is used in numerous post-transcriptional steps in gene expression and as a regulatory repressor of alternative splicing events. The polypyrimidine tract-binding protein (PTB) is a typical well-characterized example with a similar architecture to CELF1 of multiple flexibly linked RRMs, which bind pyrimidine-rich motifs   within rin vitro assays for assessing mRNA decay in AU-rich mRNAs have shown that CELF1 binds specifically to mRNAs and stimulates poly(A) shortening in the 3′ UTR by recruiting the poly(A) deadenylase PARN , is well characterized structurally and functionally ,85. Struase PARN ,30. Althase PARN  and is case PARN  in whichBiotechnology and Biological Sciences Research Council (BBSRC) to M.S.S. and J.L. (BB/I011420/1) and studentship funding (to J.E.) from a BBSRC Doctoral Training Grant and from the School of Chemistry, University of Nottingham. The NMR facilities were supported by the University of Nottingham through the Capital Infrastructure Fund. Funding for open access charge: University of Nottingham.The authors acknowledge funding from the Conflict of interest statement. None declared.


Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAifMet) while binding of elongator tRNAMet proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAifMet becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine  S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator tRNAifMet as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in  Staphylococcus aureus causes a wide range of diseases and, together with coagulase-negative staphylococci (CoNS), accounts for approximately 30 per cent of all hospital-acquired infections de novo and staphylococci employ the trans-sulfuration pathway to generate methionine Bacillales are thought to control this pathway by SAM-binding S-box riboswitches in silico analysis predicts the presence of a T-box riboswitch in the 5′-untranslated region of the methionine biosynthesis operon (metICFE-mdh operon) in staphylococci Bacillus subtilis and other Firmicutes . We further show that, in staphylococci, T-box control of methionine biosynthesis has a key role in a complex regulatory network that also involves stringent response-mediated CodY regulation and RNA decay to tightly control this pathway.Staphylococci are important skin and mucosa commensals but also major human pathogens. The most pathogenic species metI, metC, metF, metE, mdh ) are organized in an operon-like structure and are annotated as SACOL0431 - SACOL0427 in S. aureus COL and as NWMN_0351 - NWM_0347 in S. aureus Newman, respectively, with metF being named metH in the latter strain  which partially overlapped with a strong Rho-independent transcription termination signal in the 3′-portion of the transcript  being identical and representing the initiator tRNAifMet. Binding studies using tRNAifMet, tRNAMet3 and tRNAMet4, respectively, with free 3′-CCA ends revealed that the met leader RNA interacted strongly with tRNAifMet while interactions with tRNAMet3 and tRNAMet4 proved to be weak  in the presence or absence of methionine. RNA was isolated in the early exponential (E1), exponential (E2) and early stationary (S) growth phase and analyzed by Northern hybridization using met leader RNA- and metI-specific DNA probes, respectively  or charged tRNAs (terminator formation), T-box riboswitches indirectly sense amino acid levels in the bacterial cell ectively . In the l growth . In contthionine . Upon mel growth . Interesmdh mRNA . InsteadS. aureus, many of which being involved in amino acid biosynthesis and transport met leader RNA and the recent identification of the methionine biosynthesis genes as direct CodY targets in S. aureusmet leader/metICFE-mdh transcription in more detail. A S. aureus codY deletion mutant was grown in CDM with and without methionine and analyzed for met leader and metICFE-mdh transcription by Northern blot hybridization. In the absence of methionine, a stronger met leader RNA signal was detected in both the wildtype and the codY mutant ppGpp synthetase domain in strain S. aureus Newman brnQ-1, which encodes a CodY-repressed BCAA permease, was included in the analysis. In the S. aureus wildtype, methionine depletion led to brnQ-1 induction along with met leader RNA and metICFE-mdh expression ppGpp synthesis may be required for efficient activation of the system. Deletion of codY resulted in a generally higher basal transcription of both brnQ-1 and met leader RNA in the presence of methionine, whereas metICFE-mdh transcription remained tightly controlled and switched off under these conditions ppGpp involving bifunctional RelA/SpoT synthetases/hydrolases (RSHs) and affecting/modulating many cellular functions. Recently, a link between CodY and the stringent response of pression . In contnditions . In the wildtype .metICFE-mdh mRNA is subject to rapid degradation. To investigate the possible involvement of specific RNases in this process, the stability of met leader RNA and metICFE-mdh was analyzed in S. aureus mutants that were deficient in RNase J2 and RNase III activity, respectively. For this purpose, de novo RNA synthesis was interrupted by the addition of the RNA polymerase inhibitor rifampicin to the cultures. Total RNA was isolated at different time points and subjected to Northern blot analysis  are rare among Bacillales they are more common in Lactobacillales. In both orders they are associated with methionine metabolism or transport (Met) are encoded by four distinct gene loci in the genomes of S. aureus and S. epidermidis. Two of them are identical and represent the initiator tRNAifMet, while the other two tRNAMet loci differ in their nucleotide sequence from each other and from the tRNAifMet. Surprisingly, we found a clear preference for interaction of the met leader RNA T-box with the initiator tRNAifMet  methionyl-tRNA synthetase, it is only tRNAifMet that is specifically recognized by the methionyl-tRNA-formyltransferase which then mediates N-formylation of methionine to produce fMet. At present, it is not clear how the observed specificity of the met leader RNA T-box for tRNAifMet is accomplished. An involvement of the putative specifier box in the 5′-region of the met leader RNA seems unlikely because all methionyl-tRNAs use the same anticodon, suggesting that other regions of the met leader RNA interact with structures that are unique to the initiator tRNAifMet. In line with this hypothesis, our data showed that tRNAifMet binding by the met leader RNA was not affected when the specifier box in the met leader was substituted with a cysteine-specific codon  biosynthesis in B. subtilis, respectively The data obtained in this study lead us to propose that a hierarchical regulatory network controls methionine biosynthesis in n needed . InteresS. aureus employs RNA decay mechanisms to quickly remove newly transcribed metICFE-mdh mRNA from the system, thus further limiting the risk of sustained (over)expression of genes involved in methionine synthesis. Although the complete mechanism and enzymes involved in the process still need to be established, the data give a first hint that RNase J2 participates in metICFE-mdh degradation  were found to be not degraded by RNase J1/J2 met leader RNA degradation -binding S-box riboswitches http://www.genome.jp/kegg/) and experimental research Bacillus, staphylococci may have only limited capacity to reuse or redirect methionine to other pathways because they lack both the methionine salvage and polyamine synthesis pathways  to produce the effector molecule required to stop methionine production. Alternatively, microorganisms that produce polyamines may need a larger SAM pool as precursor for the synthesis of these important compounds. Therefore, S-box control of methionine biosynthesis might be more effective in these organisms to ensure a constant SAM supply. More experimental work is needed to further substantiate these hypotheses.T-box control of methionine biosynthesis genes in staphylococci is an exception among pathways . Therefode novo methionine biosynthesis. It is tempting to speculate that overproduction (rather than lack) of methionine may be critical to staphylococci and, thus, this strict control of methionine de novo synthesis would not only save resources and energy but also meet the requirement to prevent methionine accumulation. While excess methionine biosynthesis might be critical for staphylococci under most conditions, de novo methionine biosynthesis becomes crucial for growth and survival when methionine supply is limited, for example, when entering the host during infection S. aureus methionyl-tRNA synthetase by an experimental compound has already provided evidence that methionyl-tRNA metabolism is a suitable anti-staphylococcal target in vitro, indicating that RNA-based drug targeting is a promising new avenue in medicinal chemistry Staphylococcus-specific antibiotics.The general life style of staphylococci provides easy access to methionine sources from the respective host they colonize or infect and, therefore, suggests that methionine supply may not be a limiting factor under normal conditions. The data presented in this paper provide first insight into the regulation of methionine synthesis gene expression in staphylococci and, more importantly, show that staphylococci have evolved special mechanisms to tightly restrict Bacterial strains, construction of mutants, growth conditions as well as RNA isolation and Northern blot procedures can be found in the SI nd Generation (Roche) was used for determination of transcript ends by synthesis of first-strand cDNA with reverse transcriptase (Fermentas) and the oligonucleotides listed in Table S4  and RNA quality was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies). The 5′/3′ RACE Kit, 2Table S4 .met leader RNA sequence of S. aureus COL was amplified by PCR with oligonucleotides T7-F_met-sRNA and R_met-sRNA  with the appropriate mutations were used as met leader RNA templates in tRNA binding assays.The S. aureus COL and oligonucleotides listed in Table S4  with oligonucleotides T7-F_met-sRNA and R_met-sRNA (Table S5). The products were used in IVT/tRNA binding assays in the presence of pre-formed tRNAs: 10 µl reaction volume consisted of T7 transcription buffer, 10 U RNase inhibitor, 6 U T7 RNA polymerase (Fermentas) and 0.5 mM NTPs. DNA templates of the met leader RNA and of pre-formed tRNA were adjusted to end concentrations of 8 nM and 50 nM, respectively, and the mix was incubated at 37°C for two hours. Samples were immediately separated on a non-denaturing 6% (w/v) polyacrylamide gel by electrophoresis at 4°C. Visualization was attained with the PhosphoImager (Fujifilm FLA-7000) and quantification of bands was achieved with the software Multi Gauge V2.2.tRNA templates were generated by PCR from genomic DNA of Table S4 . Four pi600) of 0.05 and grown with shaking at 220 rpm at 37°C to an OD of 0.5. The cultures were filtered over a 0.22 mm filter applying vacuum, washed twice with sterile phosphate buffered saline (PBS) and bacteria were resuspended in 15 ml CDM medium without methionine and grown for another 60 minutes in a 30-ml tube. Then rifampicin (500 µg ml−1) was added to the cultures. Before (0) and after 0.5, 2, 5, 10 and 60 minutes of rifampicin exposure, RNA was isolated and Northern blot analyses were performed as described in the Supporting Information . Known motifs are highlighted. Purple boxes contain conserved motifs identified in B. subtilis T-box systems (TIFF)Click here for additional data file.Figure S2Structural model of the full-length S. epidermidis RP62A met leader RNA. The secondary structure model of the met leader RNA was based on the structural determination of the B. subtilis tyrS T-box leader RNA B. subtilis T-box systems are indicated by asterisk. The first hairpin structure from nucleotides 20 to 125 includes several conserved structural motifs described for the B. subtilis tyrS T-box leader RNA, such as the GA-motif at the base, the AG-bulge and the S-turn motif opposite the potential specifier loop as well as the conserved sequence in the apical loop of stem I (TIFF)Click here for additional data file.Figure S3In vitroCys with SC1. binding assay of tRNAIn vitro transcription of the met leader RNA template was done in the presence of either tRNAifMet or tRNACys. On the left the wildtype sequence of the met leader RNA was used, on the right the construct SC1 with mutation of the specifier box from methionine (AUG) to cysteine (UGC) codon. The asterisk indicates the control reaction without tRNA present but [α32P]-CTP. The arrow indicates the binding interaction of tRNAifMet with the met leader RNA.(TIFF)Click here for additional data file.Text S1Additional material and methods information as well as Tables S1 to S5. Descriptions are given of the experimental setup concerning cell growth and viability in chemically defined medium, the construction of conditional RNase J2 (rnjB) and RNase III (rnc) mutants and details of the RNA isolation and Northern blot analysis protocols used here. Tables S1 to S5 list the bacterial strains and plasmids as well as all oligonucleotides used in this study.(PDF)Click here for additional data file.