Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic  Arabidopsis thaliana and that this SINE RNA interacts with HYL1, a double-stranded RNA-binding protein required for the production of microRNA and trans-acting small interfering (tasi)RNA. We mapped the binding site of HYL1 to a SINE RNA region that mimics the hairpin structure of microRNA precursors. We also found that HYL1 induces conformational changes upon binding to RNA substrates. These data suggest that SINE RNAs modulate the activity of RNAi pathways in Arabidopsis.Short interspersed elements (SINEs) are transposable elements in eukaryotic genomes that mobilize through an RNA intermediate. Recently, mammalian SINE RNAs were shown to have roles as noncoding riboregulators in stress situations or in specific tissues. Mammalian SINE RNAs modulate the level of mRNAs and proteins by interacting with key proteins involved in gene transcription and translation. Here we show that constitutive production of a plant SINE RNA induces developmental defects in  Short Interspersed Elements (SINEs) are repetitive sequences, present in the genome of most eukaryotes and ancestrally derived from small functional RNAs  Evaluating SINE impact on genome structure and gene expression has been the subject of numerous investigations in the past 20 years  under the control of its natural promoter in Arabidopsis and followed SINE RNA production and maturation in two independent transgenic lines Arabidopsis lines can induce severe developmental defects. The SINE-induced phenotypes are similar to several RNAi mutant phenotypes. We show that SINE RNAs interact with a subset of highly divergent dsRBPs and affect the production of different families of small RNAs and the accumulation of their corresponding mRNA targets. Our results suggest that SINE RNAs influence the activity of a subset of dsRBPs and consequently, influence a variety of basic cellular processes including RNAi.The Arabidopsis thaliana transgenic lines transformed with the founder SB1.7 (na7) locus from Brassica napusFourteen The SB1.7 locus contains transcriptional cis-enhancer motifs that allow the SINE to partially escape transcriptional repression in its natural host Arabidopsis, SB1 transcription is associated with delayed growth and flowering time, abortive siliques, partial sterility, reduction of leaf and root size, leaf serration associated with a downward curvature, and partial loss of apical dominance  pathways, respectively  and short precursors (pre-miRNAs) emplates . This reXenopus laevis proteins, xlADAR1 and xlRPBA, were shown to bind efficiently to short stem-loop RNA structures containing bulges and mismatches in vivoRecently, double-stranded RNA binding domains from two Arabidopsis SINE RNA was used as a substrate  responsible for these phenotypes, our data raise the possibility that an interaction between SB1 SINE RNA and a subset of dsRBPs, some of which are involved in RNAi, could contribute to the developmental defects. Using a double-stranded RNA binding domain from in vitro studies, we propose that SB1 RNAs interact in vivo with HYL1, and consequently modify the steady-state level of several miRNAs and tasiRNAs. In this scenario, tasiRNA accumulation would be indirectly affected because tasiRNA biogenesis relies on miRNA-guided cleavage (miR173 targets TAS1 and TAS2 and miR390 targets TAS3) in vivo, such competitions likely are influenced by the varying tissue/spatial distributions of different miRNAs:HYL1 complexes, which would probably effect the capacity of SINE RNA to modulate the production of a given miRNA in a given tissue. In conclusion, we propose that SB1 RNA compete for several dsRBPs, not only HYL1, and that these competitions likely accounts for the extent and unusual characteristics (such as the variable penetrance) of the SINE-induced phenotype.Based on our in vitro, HYL1 binds efficiently different imperfect double-stranded RNA molecules, including the rapeseed SB1 . On the other hand, DRB4 binds perfect linear RNA duplexes formed by the action of RDR6 on a single stranded primary transcript in vivo, HYL1 preferentially interacts with imperfect double-stranded pri- and pre-miRNAs and not perfect double-stranded PTGS precursors. Based on our RNase V1 mapping results, the basis of this selectivity could be the capacity of HYL1 to interact with single-stranded RNA regions  plates at 21°C under a 12-h light/12-h dark regime.The construction of the SB1 expressing transgenic lines RNA structures were predicted using RNA/DNA folding and hybridization software Mfold, version 2.3 Arabidopsis cDNA library (Stratagene) using primers designed according to the Arabidopsis sequence database. All PCR amplifications were performed using 5′-primers with a terminal BamHI restriction site in combination with a 3′-primer ending with a XhoI restriction site. After PCR amplification and BamHI/XhoI digestion, the coding sequences of HYL1 or DRB4 were cloned into the pGEX-5X-1 expression vector (Pharmacia Biotech). In the resulting constructs named pGEX-HYL1 or pGEX-DRB4, HYL1 or DRB4 are fused to the C-terminal end of GST. Prior to expression in bacteria, sequencing was performed to verify the sequence of the cDNAs and the translational fusions. To express the HYL1 or DRB4 recombinant proteins, pGEX-HYL1 or pGEX-DRB4 were transformed into Escherichia coli BL21 (DE3) cells. A single colony of E. coli cells containing a recombinant pGEX plasmid was used to inoculate 50 ml of LB medium containing 100 µg/ml ampicillin. Cells were incubated overnight at 37°C with vigorous shaking. Cultures were diluted 1∶100 into fresh LB medium containing 50 µg/ml carbenicillin and grown at 37°C with shaking until the A600 reaches 0.7–1. Recombinant protein expression was then induced by addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) followed by an incubation of 4 to 5 hours at 37°C. Induced cells were harvested by centrifugation at 7700 g for 10 min at 4°C and pellets were frozen at −20°C overnight. Bacterial sonication and batch purification of the fusion proteins using Glutathione Sepharose 4B were performed according to the manufacturer's protocol (Pharmacia Biotech).cDNAs encoding HYL1 or DRB4 were amplified by PCR from an in vitro transcribed from linearized T7 promoter-containing vector using recombinant T7 polymerase and resulting transcripts were gel-purified. SB1.7 RNA was then dephosphorylated using shrimp alkaline phosphatase (Roche) according to the manufacturer's protocol and 5′-end labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-32P] ATP . After labeling, SB1.7 RNA was purified using BD Chroma spin-30 columns (BD Biosciences Clontech). A pBluescript II SK vector (Stratagene) containing a short fragment (multiple cloning sites) between the T7 and T3 promoters was used to prepare substrates. The T3 transcript  was used as the ssRNA substrate. To make the dsRNA substrate, T3 transcripts were first kinase-labeled, and then T7 transcripts were hybridized with labeled T3 transcripts to form dsRNA. A BssHII fragment (173 base pairs) that included both phage promoters and the multiple cloning sites of the pBluescript II SK vector was gel purified, dephosphorylated, labeled with T4 polynucleotide kinase and use as dsDNA substrate. After labeling, dsDNA substrate was purified using Microspin S200 columns (Amersham). To obtain the ssDNA substrate, the labeled dsDNA was denatured by heating.SB1.7 was Binding assays were performed in 10 µl of a Mobility Shift Buffer (MSB) containing a final concentration of 10 mM Tris (pH 8.0), 25 mM KCl, 10 mM NaCl, 0.5 mM DTT, 10% glycerol, 100 µg/ml BSA. Approximately the same amount of each test molecule (10 ng) was used for each experiment; the amount of fusion proteins added is indicated in the figure legends. Binding was performed at 25°C for 5 min and then quickly cooled on ice. Binding reactions were then directly loaded onto a 4% polyacrylamide native gel. Electrophoresis was for two hours at 150 volts on a gel of 18 cm in length; running buffer was 1× TBE . Gels were dried and then subject to autoradiography. A small amount of bacterial RNase co-purify with the GST-HYL1 fusion protein and degrades the free (unprotected) RNA probes in in vitro transcribed and radioactively trace-labeled (for quantification purposes) from linearized T7 promoter-containing vector using recombinant T7 RNA polymerase and [α-32P] ATP . Transcripts were gel-purified. 2 pmol of RNA was then dephosphorylated using calf intestinal phosphatase (New England Biolabs) according to manufacturer's protocol. Dephosphorylated RNAs were 5′-end labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-32P] ATP . Gel-purified 5′-labeled RNAs were subsequently used for nuclease protection assays 2). After annealing, 1 µL of tRNA  was added, followed by the addition of increasing protein concentrations . To ensure protein binding to RNA, samples were incubated for 15 min at room temperature. Then, 0.005 units of RNase V1 (Ambion) were added and the reactions were incubated for additional 10 min at room temperature. Reactions were stopped by ethanol/salt precipitation. Samples were loaded together with alkaline hydrolysis ladder and denaturing RNase A (Ambion) and RNase T1 (Boehringer Mannheim) digests of RNAs on denaturing RNA gels.RNA was 2HPO4 pH 7.2, 7% SDS, 2× Denhardt solution, 50 mg/ml denaturated hering DNA at 50°C. Filters were washed twice with 3X SSC, 25 mM NaH2PO4 pH 7.5, 5% SDS at 50°C for 10 min, followed by one to two washes with 1× SSC, 1% SDS at 50°C. Signals were visualized using a phosphorimager  for quantification. The random-primed 32P-labelled probes used for the detection of the ARF3, ARF4 and SCL6-III mRNAs have been described previously 32P-labeled with T4 polynucleotide kinase (New England Biolabs). For U6 detection the following oligonucleotide was used: 5′-AGGGGCCATGCTAATCTTCTC-3′.Total RNA was extracted using inflorescences (stages 1–12), as described elsewhere Figure S1in vitro transcript of SB2 was subjected to protection by increasing concentrations of following dsRBPs: Dr2, HYL1, combination of Dr2 and HYL1, or DRB4. Regions protected by HYL1 are marked alongside short run gel (A), long run gel (B) and predicted folding pattern (C) by green bars. Three independent experiments gave similar results as the one presented. Nucleotides marked with asterisks seem to adopt more prominent helical structure upon protein binding and, therefore, become more prone to RNase V1 cleavage. (HL) represents a partially hydrolyzed RNA ladder. Denaturating RNase A and T1 digests give the position of pyrimidine and G residues respectively. The control lane shows untreated RNA samples and the (0) lane represent RNase V1 digestion without recombinant proteins added. A labeled 23-mer oligoribonucleotide was also loaded on the gel to help in band size determination. The binding of HYL1 to SB2 is weaker compared to SB1 or 11Dr2(7). In this case, both Dr2 and DRB4 are showing no effect on RNase V1 cleavage, suggesting their low in vitro binding affinity to SB2 RNA.Protection from RNase V1 digestion of SINE SB2 RNA by different dsRBPs. Prior to RNase V1 digestion, (4.82 MB TIF)Click here for additional data file.Figure S2mRNA levels of HYL1, DCL1 and HEN1 genes involved in miRNA production are unchanged in the Col-0-SB1(18) transgenic line. (A) HYL1 mRNAs accumulate to similar levels in wild-type and Col0-SB1.7(18) line as indicated by Northern blot analyses using total RNA from aerial tissues of 14 days-old plants. (B) Similar results were obtained for HYL1, DCL1 and HEN1 transcript levels by SemiQ-RTPCR using flower total RNA as samples and Actin2 (ACT2) and Ubiquitin1 (UBQ1) as internal references, suggesting that the inhibition of miRNA production in SB1.7-expressing lines is not attributable to misexpression of one of the main genes from the miRNA pathway. First strand cDNA synthesis was done by using 0.8 µg of flower total RNA following treatment with DNAse . PCRs were run for 25 cycles with the Actin2 (ACT2), Ubiquitin1 (UBQ1) and HYL1-specific primers and for 29 cycles for DCL1 and HEN1. For DCL1 the primers are positioned from each side of the miR162 target site, allowing amplification of the full-length DCL1 transcript only. Sequences of the primers are available upon request.(0.46 MB TIF)Click here for additional data file.


In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence  involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation Using a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores.In addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target. Retroviruses encapsidate two copies of the positive sense single-stranded genomic RNA. Encapsidation is very specific, as the virus has to select and package the full length genomic RNA over the vast excess of cellular and subgenomic RNA species. In human immunodeficiency virus type 1 (HIV-1), this mechanism is well understood. An RNA motif, downstream of the viral splice donor and upstream of the Gag start codon, interacts with the Gag structural protein and ensures specificity of encapsidation for full length rather than spliced RNAs .cis rather than a trans manner -labelled KS2ΨKE riboprobe was synthesized by in vitro transcription of linearised plasmid using T3 RNA polymerase (Promega) and gel purified prior to use in RPA, as described previously [6 cpm of viral RNA were used. For each experiment, a separate RPA was performed using the same RNA inputs but probing for viral plasmid DNA using a probe generated from plasmid KS2ΨEP. In addition, a probe for human GAPDH RNA was included in the reaction to control for variations in cytoplasmic RNA input. Any DNA contamination or variations in the GAPDH signal were accounted for when calculating encapsidation efficiencies, taken as the ratio of virion to cytoplasmic RNA of a mutant relative to the wild type.[α-eviously . RNA inpSupernatants of COS-1 cells transfected with envelope-deleted WT and DM deletion mutant proviruses were harvested as previously described . PelleteThe author(s) declare that they have no competing interests.AL, JSG and AML designed the study. AL carried out the experiments. RAC performed the EM experiment. AL, JSG and AML drafted the manuscript. All authors read and approved the final manuscript.


Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines.Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding  RNA and proteins have co-evolved to form the ribonucleoproteins (RNPs) that now carry out many of the fundamental steps of gene regulation, including mRNA processing and protein biogenesis The group I intron active site is composed of RNA. The catalytic core is formed at the interface of three RNA domains, termed the P1-P2, the P5-P4-P6, and the P9-P7-P3-P8 domains. These domains are held in a precise and catalytically active three-dimensional architecture by inter-domain tertiary interactions Protein cofactors use diverse strategies to stabilize group I intron RNA tertiary structure. Proteins such as CYT-18 bind multiple RNAs by recognizing conserved elements in the group I intron catalytic core The yeast mitochondrial bI3 group I intron is an instructive example of an RNA that has become dependent on proteins to fold and function correctly. bI3 RNA splicing requires specific binding by two proteins, the bI3 maturase and two dimers of the Mrs1 protein Azoarcus group I intron and the Bacillus subtilis ribonuclease P (RNase P) specificity domain RNA and at other sites in these non-cognate RNAs. These data indicate that Mrs1 has a general affinity for RNA coupled with selectivity for the GNRA tetraloop-receptor interaction, especially in its cognate bI3 RNA. Thus, Mrs1 has evolved from a DNA-binding resolvase to a protein capable of binding the GNRA tetraloop-receptor motifs found ubiquitously in large RNAs. This work also emphasizes the strong evolutionary pressure to usurp RNA-only structures with RNA-protein interactions. Every long-range RNA tertiary interaction in the bI3 ribonucleoprotein is stabilized by either the maturase or Mrs1 proteins and both proteins have been co-opted from earlier DNA-binding functions.In this work, we use high-throughput hydroxyl radical footprinting to identify the RNA binding sites for Mrs1. Mrs1 binds at each of two conserved GNRA tetraloop-receptor interactions in the bI3 RNA (where N is any loop nucleotide and R is a purine). The tetraloop-receptor motif was one of the first long-range tertiary interactions to be identified for RNA in situ from H2O2 in the presence of a Fe(II)-EDTA catalyst We identified RNA interaction sites for the Mrs1 protein using hydroxyl radical footprinting. Hydroxyl radicals are generated In the free bI3 RNA, only ∼20% of nucleotides are protected from cleavage prior to binding by the Mrs1 protein  with the P4 helix (position 60) and the stacking of RNase P nucleotide A130 in the P11 helix  the tetraloop itself, (ii) nucleotides in the receptor helix where the tetraloop interacts, and (iii) a region in the receptor helix extending towards the exterior of each RNA  each protein stabilizes a crucial long-range RNA tertiary interaction, (ii) no protein binds within ∼20 Å of the scissile phosphate and (iii) no protein binds within ∼50 Å of another protein.The catalytic active site of the intron is located roughly at the center of the RNP complex , 5 mM MgCl2, 2 mM spermidine, 10 mM DTT, 0.001% (v/v) Triton X-100, 0.166 µg of pyrophosphatase (Roche), 2 mM each nucleotide triphosphate, ∼10 µg double stranded DNA PCR template, 60 units SUPERNase-In (Ambion), 70 µg T7 polymerase], and purified by gel electrophoresis. The SL and B5 RNAs were synthesized from single stranded DNA templates with a double stranded promoter region. Mrs1 was expressed and purified as described The bI3 intron (with flanking exon sequences of 84 and 90 nucleotides) and the RNase P specificity domain (with flanking structure cassette sequences) RNAs were generated 2] Azoarcus RNA was incubated  at 52°C for 5 min, and 37°C for 2 min 2] 4)Fe(SO4)2–7.5 mM EDTA (pH 8.0), 0.1% H2O2, and 50 mM sodium ascorbate [bI3 reactions were performed at one-half this Fe(II)-EDTA concentration]. Background was assessed using a reaction omitting Fe(II)-EDTA. Reactions were quenched with glycerol to a final concentration of 30% (v/v). Mrs1 was removed by proteinase K digestion  followed by phenol:chloroform:isoamyl alcohol (25∶24∶1) extraction. Reactions were recovered by precipitation with ethanol. Sites of cleavage were identified by primer extension using fluorescently labeled primers  in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 0.5 mM each dNTP, and 100 units of Superscript III reverse transcriptase (Invitrogen). Primer extension reactions corresponding to the plus and minus Fe(II)-EDTA and sequencing lanes were combined, precipitated with ethanol, dissolved in formamide and separated on an Applied Biosystems 3130 capillary electrophoresis instrument. Fluorescent cleavage data were analyzed using ShapeFinder Each RNA (3 pmol) was renatured under conditions previously shown to be optimal for folding of the specific RNA. The bI3 RNA was incubated in water at 95°C for 1 min, 4°C for 1 min, and 37°C for 10 min in reaction buffer  5′-end-labeled  or internally labeled  RNAs were folded in their respective buffers and incubated with Mrs1 for 30 min at 37°C before filtering. Wells were washed with 3 vol of reaction buffer before and after binding. Filters were quantified by phosphorimaging (Molecular Dynamics) and fit to an equation that accounts for cooperative binding by two dimers of Mrs1 A([P]n / [P]n + K1/2n), where A is the total fraction of RNA bound , [P] is the protein concentration, n is the apparent Hill coefficient, and K½ is the concentration of Mrs1 where one-half the RNA is bound. In all cases, RNA concentrations were at least 5-fold below the measured K1/2 values to ensure validity of the binding equation. For all binding curves, R2 was ≥0.9.Mrs1-RNA binding assays were performed by filter partitioning using preincubated nitrocellulose (Whatman) and Hybond (Amersham) filters The Mrs1 protein model was generated with I-TASSER


Phlebotomus ariasi sandfly. Of 60 human serum samples, 3 contained immunoglobulin G against SFSV. These data suggest SFSV is present in Algeria.To determine whether sandfly fever Sicilian virus (SFSV) is present in Algeria, we tested sandflies for phlebovirus RNA. A sequence closely related to that of SFSV was detected in a  Toscana virus  in countries surrounding the Mediterranean because the virus causes meningitis during summer. Sandfly fever Sicilian virus (SFSV) is a distinct arthropod-borne phlebovirus transmitted by sandflies, specifically by Phlebotomus papatasi , P. longicuspis (n = 61), P. sergenti (n = 21), P. ariasi (n = 6), P. perfiliewi (n = 3), P. papatasi (n = 1), and Sergentomyia minuta (n = 1). They were organized into 24 pools, each containing up to 30 sandflies. Each pool was ground in RNA NOW chaotropic solution . RNA purification was performed according to the manufacturer’s protocol. A total of 10 μL of RNA suspension was used for reverse transcription with random hexanucleotide primers with the Taqman Reverse Transcription Reagents  in a final volume of 50 μL, according to the manufacturer’s recommended protocol. To test these specimens for Toscana virus RNA and phlebovirus RNA, we used 10 μL of cDNA in the previously described assays  by 4%, which corresponds to 8 nt mutations; in addition, SFSV-Italy-Sabin has been manipulated after PCR amplification and sequencing of SFSV-Algeria to compare it genetically with the sequence obtained from Algerian sandflies.Over a 4-night period in July 2006, a total of 460 sandflies were trapped as described . CDC MinTogether with homologous sequences of selected phleboviruses, the 3 sequences determined in this study were used to perform genetic distance comparison and phylogenetic analysis. Nucleotide and amino acid distances are presented in the Detection of SFSV RNA in sandflies led us to test human serum for SFSV antibodies. We tested 60 samples from healthy persons for SFSV immunoglobulin (Ig) G by indirect immunofluorescence assay as described  indicated that the Algerian, Italian, and Cypriot strains of SFSV are closely related. Another study performed with M RNA sequences showed that Italian and Cypriot strains of SFSV are closely related as well (P. papatasi flies only. In this study, detection of SFSV RNA in 1 female P. ariasi sandfly must be interpreted with caution. In particular, this finding does not mean that P. ariasi is a vector of SFSV in the study region. The presence of SFSV RNA may result from mechanical transmission from a viremic vertebrate. Therefore, specific studies should be conducted to investigate vectors of SFSV in Algeria. Seroprevalence data demonstrate that SFSV or an SFSV-related virus can infect humans. Further studies are needed to determine whether the clinical picture is limited to self-resolving febrile illness, as previously reported in Italy.To our knowledge, SFSV has been previously isolated in


Sindbis virus (SV) is the prototype of alphaviruses which are a group of widely distributed human and animal pathogens. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. Our recent studies found that hnRNP A1 relocates from nucleus to cytoplasm in Sindbis virus (SV)-infected cells. hnRNP A1 binds to the 5' UTR of SV RNA and facilitates the viral RNA replication and translation.vitro system developed by our lab to assess the role of hnRNP A1 in SV positive strand RNA synthesis.Making use of standard molecular techniques, virology methods and an in in vitro.hnRNP A1 interacted with the genomic (G) and subgenomic (SG) RNA promoters. Knockdown of hnRNP A1 resulted in markedly decrease in the synthesis of G and SG RNA both in infected cells and Our study provides the first direct evidence that hnRNP A1 actively participates in viral RNA replication and is required for the synthesis of G and SG RNA. Alphaviruses are a group of widely distributed human and animal pathogens that includes almost 40 currently known members . In natuTogaviridae, genus alphavirus  ATP.24-mer and 45-mer oligoribonucleotides representing the negative strand sequence corresponding to nt 7579 to 7602 and nt 1-45 of the SV genome were synthesized by Dharmacon Research Inc. As already noted, these are the minimal sequences that have SG and G promoter activity respectively ,48. Olig32 P-labeled RNA probes (1×104 c.p.m). The reaction was carried out in binding buffer  and the final volume of the reaction mixture was 10 μl. Binding of hnRNP A1 to the G and SG promoters was recognized by a slower migration of the labeled RNA probes.An EMSA was carried out to determine the interaction between the promoters and hnRNP A1 as described previously . Briefly32 P-γ-ATP. After hybridization, the nylon membrane was washed twice with 2 × SSC and 0.1% SDS, exposed to a storage phosphor screen and scanned on a Molecular Dynamics Typhoon Phosphorimager.BSC40 cells were infected with SV at an moi of 40 pfu/cell and at 8 h post infection total RNA was extracted using RNeasy mini kit (Qiagen). Five μg of total RNA was denatured with formaldehyde, and electrophoresed through a 1.5% agarose gel. The RNAs were transferred to a nylon membrane and subjected to northern blotting. The negative-strand probe, SV7772(-), which contains the negative-strand sequence from nt 7772 to 7754 were end-labeled with BSC40 monolayers in T75 tissue culture flasks were infected with three different recombinant vaccinia virus vectors; these encode the SV nonstructural polyprotein P123 , SV nsP4, and T7 RNA polymerase, each at an moi of one pfu/cell. Twenty to twenty four hours after infection, the P15 fraction was prepared according to Lemm et al. . The P15in vitro synthesis of SV RNA (25 μl) contained 5 μl of 5 × reaction buffer , 10 mM dithithreitol, 40 units of RNase inhibitor (Promega), 2 μg negative-strand RNA which serves as promoter/template (P/T), and 12.2 μl (5.4 μg of protein/μl) of P15 extract prepared from BSC40 cells infected with recombinant vaccinia viruses expressing the T7 RNA polymerase, the SV polyprotein, P123, and the SV nsP4. Standard reaction mixtures contained 3 mM ATP, 2 mM UTP, 2 mM CTP, and 0.5 mM GTP. [32P]α-GTP  was included to label the transcripts. Incubation was at 37°C for 1 hour. RNA was extracted using phenol-chloroform and electrophoresed on a 1% denaturing agarose gel containing 2.2 M formaldehyde. The gel was transferred to a Genescreen Plus Hybridization Transfer Membrane (Perkin Elmer); the membrane was then exposed to a storage phosphor screen and scanned as described previously [The reaction mixture for the eviously . The negeviously .hnRNP A1 cDNA derived from SF268 cellular mRNA was cloned into pET30a and expressed as described . The recBSC40 cells were maintained in antibiotic-free media. 100 nmol of siRNA targeting hnRNP A1  was transfected along with 3 μl FuGENE HD transfection reagent (Roche) in 0.4 ml MEM supplemented with 10% FCS following manufacturer's directions. Three days after transfection cells were co-infected with recombinant vaccinia viruses expressing T7, SV-nsP123 and SV-nsP4 at an moi of one pfu/cell. P15 fraction was prepared as described . An aliqExpression of hnRNP A1 was examined by Western blotting using anti-hnRNP A1 antibody (Abcam). Briefly, cells were lysed in sample buffer and proteins were fractionated by SDS-PAGE in 12% polyacrylamide gels and transferred to PVDF membranes by wet transfer. Membranes were blocked with PBS containing 5% low-fat dry milk. Anti-hnRNP A1 mouse antibody was then added, and the membranes were washed with PBS containing 0.2% Tween 20. Goat anti-mouse horseradish peroxidase-conjugated antibody (BioRad) and the ECL kit (Pierce) were used to detect bound antibodies.Our previous study demonstrated that knockdown of hnRNP A1 in infected cells reduces the viral RNA synthesis . To furtWhile we have good evidence that hnRNP A1 binds to the G and SG promoters, the consequences of this interaction for viral RNA synthesis are not known. Our recent studies showed that hnRNP A1 re-localizes from nucleus to cytoplasm of SV-infected cells. Knockdown of hnRNP A1 inhibits the virus replication . These fin vitro system for the synthesis of G and SG RNA developed by our laboratory [32P-α-labeled GTP in the reaction mixture, along with the four unlabeled NTPs, the P/T, and the P15 fraction as described in Materials & Methods.We made use of an boratory ,49 to exin vitro, the reaction was set up as described [The level of endogenous hnRNP A1 in BSC40 cells was knocked down by siRNA targeting of hnRNP A1 as described in Materials & Methods. The knockdown efficiency was confirmed by Western blot using anti-hnRNP A1 antibody (data not shown). The hnRNP A1-depleted BSC40 cells were then co-infected with recombinant vaccinia viruses expressing T7 polymerase, SV P123 and SV nsP4 and the p15 were prepared as described . As showescribed  using eiHost factors together with viral proteins regulate virus RNA replication. The finding that mutations in the viral genome sometimes have different effects in vertebrate cells and mosquito cells has suggested that cellular factors play a role in viral RNA synthesis . Recent How does hnRNP A1 contribute to the replication of SV? As hnRNP A1 is an RNA-binding protein, we speculated that it might facilitate viral RNA synthesis via association with viral RNA. In our previous study we found that hnRNP A1 interacts with the 5'UTR of SV RNA and promotes the synthesis of negative strand RNA [, data noG and SG promoters are different in sequences. Our previous study showed that nsP4, the RDRP, has distinct sites for the binding of G and SG promoters ,48. We tHow does hnRNP A1 contribute to the viral RNA replication? hnRNP A1 is predominantly a nuclear protein. During SV infection nearly all of hnRNP A1 relocalizes to cytoplasm in association with viral RNA. Precisely what interactions lead to the recruitment of hnRNP A1 to the viral RNA replication complex is unknown. As hnRNP A1 is an RNA-binding protein, it may direct the viral RNA replication complex containing the viral RDRP, other viral and cellular factors to the sites where replication of viral RNA is initiated. Moreover, considering the reported physiological roles of hnRNP A1, we speculate that hnRNP A1 may interact with viral nonstructural proteins which comprise the viral RNA replication complex and facilitates the viral RNA replication. Further investigation is going on to address how hnRNP A1 contributes to viral RNA replication.To our knowledge, this is the first report to describe an RNA-binding protein (hnRNP A1) actively participating in SV RNA replication in molecular detail. These studies not only improve our understanding of the replication of SV, but also have the potential for use as the basis for developing antiviral agents that act by inhibiting the virus RNA replication.in vitro. Our study provides the first direct evidence that hnRNP A1 actively participates in viral RNA replication and is required for the synthesis of G and SG RNA.Our previous study and the present report demonstrate that hnRNP A1 is essential for the RNA replication of Sindbis virus. hnRNP A1 binds to the 5' UTR of SV RNA and facilitates the negative strand RNA synthesis. Moreover hnRNP A1 interacted with the genomic (G) and subgenomic (SG) RNA promoters and is required for the synthesis of G and SG RNA both in infected cells and The authors declare that they have no competing interests.HXG designed the experiments and carried out the in vitro RNA synthesis, virus titration and drafted the manuscript, CWL and SA carried out the Northern and Western blots, SA proof read the manuscript, VS and MLL designed and troubleshoot the experiments, and prepared the manuscript. All authors read and approved the final manuscript.


A new crystal structure of assembled subunits from the eukaryotic exosome complex gives insight into the interactions underpinning its various functions . Here, we focus on what the emerging structures tell us about the regulation of the exosome interactions with, and actions on, RNA. If a parallel may be drawn between the processes of life and of computation so that DNA is considered the imperfectly replicated template of inherited programs, then what do we make of RNA? Beyond its roles in translation as short-lived intermediary and component of the protein-synthesis machinery, RNA moonlights as a key regulator of gene expression. In its noncoding capacity, RNA boosts astronomically the information content of the DNA-encoded program. With so much RNA about that can impact the computational program, it is imperative that all RNAs—coding as well as noncoding—are maintained at appropriate levels, that the information they confer lasts only as long as needed and no longer, that their quality is controlled, and that they are efficiently scavenged for recycling at the end of their useful lifetimes.Cell, Perhaps unsurprisingly, several biological machines have evolved to meet the exacting demands for RNA turnover, surveillance, and precise maturation of structured precursor RNAs . One sucThe core of the exosome is a ring formed from six nearly identical subunits that are homologs of the eubacterial ribonuclease RNase PH . RNase POne puzzling finding is that the PIN endonuclease subdomain of Rrp44 is exposed to the solvent on the exosome surface, while the exonuclease domain is orientated to intercept the end of any RNA threaded through the central channel of the core. The 3′ exonuclease domain of Rrp44 is presumed to engulf the end of the substrate in the exosome  are added in the nucleus to target RNA for decay. It seems that the tail might aid channeling of RNA substrates into the nuclear exosome, and therefore the length of the tail is likely a key signature to mark RNA for destruction. The nuclease protection experiments suggest that the exosome can protect a length of 30 nucleotides in a single-stranded RNA. Degradation cannot be initiated near a stable stem structure, and polymerases can add a single-stranded tail that may act as a “landing pad” for the exosome. In yeast, a major cofactor for the nuclear exosome is the TRAMP polyadenylation complex that is involved in nuclear surveillance of RNAs and RNA-protein complexes . Its comThe question naturally arises why organisms with a nucleus have retained exosomes that look like bacterial PNPase, but have entirely lost the corresponding phosphorolytic activity? We suggest that this could have arisen to prevent the exosome from randomly adding tails to RNA. Polynucleotide phosphorylase and the archaeal exosome can work backward to add tails under conditions when phosphate is low but nucleoside diphosphates are abundant . In its The ring architecture of RNase PH, PNPase, and the exosome is truly ancient, and its conservation is a reflection of its important biological roles and just how deeply embedded it has become in the regulatory repertoire in all domains of life . The emb


MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5′-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo.Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of  Following mRNA synthesis, eukaryotic mRNAs undergo a series of critical modifications before being exported to the cytoplasm where they are translated into proteins. These processing events include the addition of a cap structure at the 5′ terminus, the splicing out of introns, the editing of specific nucleotides, and the acquisition of a poly(A) tail at the 3′ terminus. The cap structure found at the 5′ end of eukaryotic mRNAs is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation efficiency of mRNAs Nascent mRNA capping is a rapid, dynamic, and regulated co-transcriptional process that is subjected to quality control. Transcription initiation is associated with the RNA polymerase II (RNA Pol II) carboxy-terminal domain (CTD) Ser 5 phosphorylation, which recruits the capping apparatus The rate-limiting activity of the capping apparatus is the two-step ping-pong GTase activity in vitro studies have shown that foscarnet, an antiviral drug that targets the DNA polymerase of human cytomegalovirus, is a potent inhibitor of the GTase reaction de novo guanine nucleotide biosynthesis Very few inhibitors of the GTase activity have been identified. Recent Eupenicillium brefeldianumde novo pathway of guanine nucleotide synthesis almost exclusively. Guanosine nucleotide depletion impairs G protein coupled receptor transduction and cyclic GMP signaling, ultimately resulting in the interruption of the S phase of the cell cycle through specific inhibition of IMPDH Mizoribine monophosphate (MZP) is another compound which has been shown to specifically inhibit the cellular IMPDH Since ribavirin triphosphate and MZP share functional similarities, we investigated the attractive possibility that MZP could also inhibit the GTase activity of the human RNA capping enzyme (HCE). In the present study, we demonstrate that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. The biological implications of this inhibition are discussed.NheI and XhoI cloning sites of the pET28a plasmid (Novagen). In this context, the HCE protein is fused in frame with an N-terminal 6-His tag, and expression of the protein is driven by a T7 RNA polymerase promoter. Upon transformation of the pet-HCE plasmid into Escherichia coli BL21(DE3), cultures were grown at 37°C in Luria-Bertani medium containing 30 µg/mL kanamycine until the A600 reached 0.5. Induction was initiated with 400 µM isopropyl β-d-thiogalactopyranoside (IPTG) and 2% ethanol, protein expression was allowed for 20 h at 18°C. All subsequent procedures were performed at 4°C. The bacteria were harvested by centrifugation at 5000 rpm and resuspended in 50 mL of lysis buffer . 50 µg/mL lysozyme and 0.1% Triton were added prior to sonication. After removal of insoluble material by centrifugation at 13,000 rpm, the soluble extract was applied to a nickel−nitrilotriacetic acid−agarose (Qiagen) column. The protein was eluted stepwise with elution buffer  containing 50, 100, 200, 500, and 1000 mM imidazole. The polypeptide composition of the fractions was monitored by SDS-PAGE. The recombinant HCE protein was recovered in the 200 mM imidazole eluate. This fraction was dialyzed against buffer C  that was supplemented with 5 mM potassium pyrophosphate to ensure a homogeneous non-guanylylated enzyme. The protein concentration was determined by the Bio-Rad dye binding method and stored at −80°C. The amino acids 1–219 corresponding to HCE RTase domain (HCE-T1–219) and 229–597 corresponding to HCE GTase domain (HCE-G229–597) were also expressed separately using the same procedure.An expression plasmid containing the full-length HCE protein (597 amino acids) was generated by inserting the corresponding cDNA between the 5′-GGGCACACACAGTCGACCACACAAAACCACCC-3′) was synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. The 5′-triphosphate RNA substrate was purified on a denaturing 8% polyacrylamide gel and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantitated by spectrophotometry. Alternatively, radiolabeled RNA substrates were also synthesized by adding radiolabeled nucleotides to the transcription reaction.An RNA substrate of 32 nucleotides (Saccharomyces cerevisiae RTase (Cet1). The reaction was carried out in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 5 mM DTT, 1 µM of Cet1, and 5 nmoles of the 5′-triphosphate RNA for 30 min at 30°C. This 5′-diphosphate RNA was then purified on an 8% polyacrylamide gel, excised, and precipitated. The RNA substrate was quantified by spectrophotometry and stored at −20°C.A 5′-diphosphate RNA substrate was also synthesized by subjecting the 5′-triphosphate RNA to hydrolysis by the 32P]GTP, 5 µM of 5′-triphosphate RNA, 0.1 µM HCE, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 500 µM DTT, 0.5 ng/µl pyrophosphatase (Roche), and 1 U of RNAse Out (Invitrogen) were incubated for 15 min at 37°C. Reactions were stopped by the addition of 20 µl of phenol-chloroform. 10 µl of loading dye (97% formamide) was added to the aqueous phase and submitted to electrophoresis on a denaturing 10% polyacrylamide gel. Formation of the 32P-radiolabeled GpppRNA was quantified with a PhosphorImager.Reaction mixtures containing 5 µM GTP, 4 µM HCE, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 500 µM DTT, 0.5 ng/µl pyrophosphatase (Roche), and various concentration of MZP (as indicated). The reactions were stopped by the addition of 5 µl of loading dye  and analyzed on a 12% polyacrylamide gel containing 0.1% SDS. The formation of the radiolabeled HCE-[α-32P]GMP covalent complex was quantified with a PhosphorImager. Similar experiments were conducted using only the GTase domain of HCE (HCE-G229–597). Alternatively, equimolar concentrations of MgCl2 to MZP were added to the reaction in addition to the base 5 mM MgCl2 from the buffer (MZP✯Mg2+).The enzyme-GMP complex formation assays were carried out for 3 min at 37°C in a buffer containing 1 µM [α-32P]GTP, 1 µM HCE, 70 mM Tris-HCl, pH 7.5, 7.5 mM MgCl2, 70 µM DTT, 0.7 ng/µl pyrophosphatase (Roche), and 1 U of RNAse Out (Invitrogen). The transfer reaction was then initiated by adding a 5′-diphosphate RNA (32 nt) to a final concentration of 0.5 µM with various concentration of MZP (as indicated). After 30 sec at 37°C, the transfer reaction was stopped by the addition of 20 µl of phenol-chloroform. 10 µl of loading dye (97% formamide) was added to the aqueous phase and submitted to electrophoresis on a denaturing 10% polyacrylamide gel. The formation of the 32P-radiolabeled GpppRNA was quantified with a PhosphorImager.Formation of the enzyme-GMP complex was initially performed for 15 min at 37°C in a buffer containing 1.5 µM GTP, magnesium and increasing concentrations of MZP. As shown in 50 of 150 µM . A cold 5′-triphosphate RNA was incubated in the presence of HCE, GTP, magnesium, pyrophosphatase and various concentration of MZP were preincubated together, and the reaction was initiated by the addition of HCE. A concentration of 80 µM MZP resulted in a 50% decrease in the formation of the radiolabeled capped RNA (229–597) the activity was still inhibited by MZP, albeit with a 10-fold decrease in the potency . A 5′-diphosphate RNA, [α-pped RNA . Interes potency  and 4A.  potency . Consequ32P]GTP, magnesium and increasing concentrations MZP. Since this GTase reaction is known to be highly inhibited by its pyrophosphate product, pyrophosphatase was also added to the reaction in order to drive the reaction forward. The formation of the EpG covalent complex in single turnover condition was analyzed by SDS-PAGE and quantified by phosphorimaging. As seen in 2 to MZP did not influence this effect (data not shown). Nevertheless, MZP inhibition of the EpG complex formation is weak and could not be solely responsible for the global inhibitory potency of MZP.We next set out to identify which step (if any) of the GTase reaction is more susceptible to MZP inhibition. In order to investigate the inhibitory potency of MZP on HCE-GMP complex formation (first step), HCE was incubated with [α-32P]GTP, magnesium and pyrophosphatase. Next, various concentrations of MZP and a cold 5′-diphosphate RNA were added to initiate the transfer of the [α-32P]GMP moiety onto the RNA in single turnover condition. As seen in We next investigated if the second step of the GTase reaction could be impaired by MZP. EpG complex formation was allowed to initially form upon incubation of HCE with excess GTP, 0.1 µM of either HCE-WT or HCE-C126S and increasing concentrations of MZP. Radiolabeled RNA products were analyzed on denaturing polyacrylamide gel and quantified by autoradiography.(TIF)Click here for additional data file.Figure S2MZP as a potential cap donor substrate for HCE. A 5′-diphosphate acceptor RNA initiating with a [α-32P] guanosine was incubated in absence (lane 1) or in presence of MZP (lane 2) or GTP (lane 3) and HCE. The capping reaction was sequentially submitted to nuclease P1 then alkaline phosphatase prior to analysis by TLC on a polyethyleneimine-cellulose plate. An autoradiogram of the plate is shown. The locations of the origin and GpppG product are indicated by arrows (right).(TIF)Click here for additional data file.Figure S3HCE does not influence the firefly luciferase activity and stability. Cellular extract expressing the firefly luciferase protein was incubated alone (white) or supplemented with high concentration (1 µM) of recombinant HCE protein (gray). Alternatively, control cellular extract not expressing the firefly luciferase were incubated in presence of 1 µM of recombinant HCE (black). After the indicated time, the luciferase luminescence was quantified according to the manufacturer instruction (Promega). Luciferase intensity was normalized to the cellular extract expressing the firefly luciferase alone.(TIF)Click here for additional data file.Figure S4Molecular docking model for the binding of MZP to HCE. (A) Ribbon representation of HCE in open conformation (homology model) showing the orientation of the docked MZP.  Two close-up views of HCE active site are also shown, with emphasis on the residues coordinating MZP. (D) Uncluttered representation of HCE active site amino acids showing the orientation of the docked MZP (green) and GMP (blue). (E) Schematic representation of numerous amino acids that are predicted to interact with MZP. The homology model of HCE in open conformation was based on the 1P16-chain-A open conformation GTase using the ModWeb Modeller software. Docking calculations were carried out using the Docking Server software and the Dreiding force field was used for energy minimization of MZP using built-in Chemaxon tools in Docking Server (23). PM6 semi-empirical charges calculated by MOPAC2007 were added to the ligand atoms. Nonpolar hydrogen atoms were merged and rotatable bonds were defined (24). Docking calculations were carried out using the coordinated of the structural model of HCE. Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools (25). Affinity (grid) maps of 20×20×20 Å grid points and 0.375 Å spacing were generated using the Autogrid program (25). AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method (26). Initial position, orientation and torsions of the ligand molecules were set randomly. Each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 2,500,000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 Å were applied. The predicted distance are indicated in Angströms.(TIF)Click here for additional data file.Table S1Key interactions between MZP, GMP, and the active site residues of the HCE GTase domain as predicted by the molecular docking model.(TIFF)Click here for additional data file.


Short, non-encoded oligo(A), oligo(U), or A/U tails can impact mRNA stability in kinetoplastid mitochondria. However, a comprehensive picture of the relative effects of these modifications in RNA stability is lacking. Furthermore, while the U-preferring exoribonuclease TbRND acts on U-tailed gRNAs, its role in decay of uridylated mRNAs has only been cursorily investigated. Here, we analyzed the roles of mRNA 3′ tail composition and TbRND in RNA decay using cells harbouring single or double knockdown of TbRND and the KPAP1 poly(A) polymerase. Analysis of mRNA abundance and tail composition reveals dramatic and transcript-specific effects of adenylation and uridylation on mitochondrial RNAs. Oligo(A) and A-rich tails can stabilize a proportion of edited and never-edited RNAs. However, non-tailed RNAs are not inherently unstable, implicating additional stability determinants and/or spatial segregation of sub-populations of a given RNA in regulation of RNA decay. Oligo(U) tails, which have been shown to contribute to decay of some never-edited RNAs, are not universally destabilizing. We also show that RNAs display very different susceptibility to uridylation in the absence of KPAP1, a factor that may contribute to regulation of decay. Finally, 3′ tail composition apparently impacts the ability of an RNA to be edited. Leishmania spp., Trypanosoma cruzi and T. brucei, which cause leishmaniasis, Chagas' disease, and African sleeping sickness, respectively. Kinetoplastid parasites are transmitted between mammalian hosts by insect vectors, and as such face very dramatic environmental changes during their life cycles. Thus, elaborate schemes of gene regulation, primarily effected at post-transcriptional levels, are invoked for both proliferative growth in insect and mammalian hosts, and to instigate the developmental changes required to complete the life cycles of these parasites. One dramatic example of regulation during the life cycle is in the mitochondria of T. brucei, which undergoes extensive changes in gene expression, physiology, and morphology as the organism cycles between its insect and mammalian hosts The kinetoplastid parasites include several human pathogens such as T. brucei, 12 of the 18 mitochondrially-encoded mRNAs undergo some degree of editing (the other six mRNAs are termed “never-edited”). Differences in the degrees of editing of specific RNAs in different life cycle stages of T. brucei suggest that regulation of the editing process may contribute to changes in the abundance of mature mitochondrial RNAs A hallmark of kinetoplastid mitochondria is the process of uridine (U) insertion/deletion RNA editing. RNA editing, directed by small guide RNAs (gRNAs) and catalyzed by a multiprotein complex called the editosome or RNA editing core complex (RECC), is required to generate the mature, translatable form of many mRNAs in vitro degradation assays in partially purified mitochondrial extracts, untailed and oligo(A) tailed RNA substrates are differentially susceptible to decay, in a way that is consistent with results of the in vivo analysis in KPAP1 depleted cells In addition to editing, regulation of RNA levels in trypanosome mitochondria also appears to occur via RNA turnover. For example, the abundance of mature monocistronic mRNAs, including those that do not undergo editing, often varies dramatically between human bloodstream form (BF) and insect procyclic form (PF) life cycle stages T. brucei mitochondria, but functional studies are not consistent with a role for these enzymes in general mRNA decay. Two of the four mitochondrial proteins with known exoribonuclease domains, KREX1 and KREX2, are U-specific components of the editosome whose activities are apparently devoted to U deletion RNA editing Several exoribonucleases have been described in cis-acting sequences on mRNA stability are more complicated that previously appreciated.In this study, we sought to better clarify the roles of uridylation and adenylation in mRNA stability and determine whether TbRND could impact mRNA stability under conditions where RNAs are preferentially uridylated. We analyzed RNA levels and 3′ tail composition in cells depleted of KPAP1, TbRND, or both enzymes simultaneously. Our results indicate that mitochondrial RNAs in wild type cells often harbor a mixed population of 3′ tails, and the depletion of KPAP1 leaves transcripts differentially susceptible to uridylation. Furthermore, although the appearance of oligo(U) tails on mRNAs was common upon KPAP1 depletion, these uridylated transcripts do not appear to be direct targets of TbRND. TbRND depletion in the KPAP1 RNAi background did result in both positive and negative indirect effects on transcript abundance and 3′ tail characteristics that were transcript specific. Finally, our results suggest that 3′ tail composition can impact the ability of an RNA to enter the RNA editing pathway. Overall, these studies reveal that both the composition of mitochondrial mRNA 3′ tails and the impacts of these 5′-GCGGATCCATGAGAAAGTTTTCAGCTTTTCG-3′ and 5′-GCGGAT CCTGGAAGACGCAAAGGGATGTC-3′, were cloned into the p2T7-177 plasmid For expression of tetracycline (tet)-inducible RNAi, nucleotides 1 to 687 nt of the KPAP1 gene (Tb11.02.5820) amplified using primers T. brucei strain 29-13 , which contains integrated genes for the T7 RNA polymerase and the tet repressor, were grown in SDM-79 media supplemented with 10% fetal bovine serum (FBS) as indicated previously 6 cells/ml, with cells harvested at day 3 for RNA collection. RNA was collected in the same way for the TbRND RNAi cell line 6 cells/ml and diluted as necessary every 24–48 hours. Values from three or four independent growth experiments were averaged to generate growth curves. To isolate mitochondria for subsequent RNA extraction, cells from 1 L of culture grown from each RNAi cell line, both with and without tet induction of RNAi, were harvested and the isolation was performed as described PF To collect RNA from mitochondria from uninduced and induced TbRND RNAi and KPAP1 RNAi, and KPAP1/TbRND RNAi cells, the acid guanidinium thiocyanate-phenol-chloroform method of extraction was used on the isolated mitochondria For qRT-PCR, four µg of RNA was treated with a DNase kit (Ambion) to remove any residual DNA. RNA was reverse transcribed and amplified using a MyiQ single-color real-time PCR detection system as described 2O and 1 µg was reverse transcribed using SuperScript III (Invitrogen) and one of the following gene-specific oligonucleotides: 5′-CCCATAAAAAATACAAATCATAGACTG-3′ for ND4; 5′-TTATTCAAAAGAAGCTCTCCGTCG-3′ for pre-edited RPS12; 5′-CAAAACGTAAACAACAACCATA-3′ for edited RPS12; and 5′-ATCAAACCATCACAATATAAAATCATATG-3′ for edited MURF2, according to the manufacturer's instructions. For PCR, weighted dNTPs were used . For amplification of ND4 RNA 3′ tails, primers 5′-GTATTTATGTCAATATCAATATCAACTATAG-3′ and 5′-GTAACAATTAACAATATAAAATTTATAC-3′ at 0.2 µM final concentration were used in a 100 µl final volume with 20% of the gene-specific RT reaction and Taq polymerase in a 40 cycle reaction. For amplification of pre-edited RPS12 RNA 3′ tails, primers 5′-GAAACATCGTTTAGAAGAGATTTTAGA-3′ and 5′-CCACTCAAAAAATCCTCGCC-3′ at 0.2 µM final concentration were used in a 100 µl final volume with 12.5% of the gene-specific RT reaction and Taq polymerase in a 40 cycle reaction. Partially-edited RPS12 was amplified with the same antisense primer as pre-edited RPS12, but using the sense primer 5′-ATTATACACGTATTGTAAGTTAGATTTAGA-3′ and 12.5 percent of both RPS12 RT reactions. Finally, for amplification of edited MURF2 RNA 3′ tails, primers 5′-TCAGTTTTGTTTAACACAGTTATTATC-3′ and 5′-CAAAGCACAAAAATAAAACTAAATTAAAA-3′ at 0.1 µM final concentration were used in a 100 µl final volume with 12.5% of the gene-specific RT reaction and Taq polymerase in a 40 cycle reaction. Products of the entire PCR reaction were precipitated with ethanol and used in a 40 cycle nested PCR reaction with 0.1 µM fresh addition of the same sense primer and 0.2 µM concentration of the antisense primer 5′-TAAAACTAAATTAAAACAACCAAAC-3′. In all cases, PCR products were purified with the Illustra GFX PCR purification kit  or phenol/chloroform/isoamyl alcohol (25∶24∶1) extraction, precipitated, and resuspended in 6 µl H2O. A third of this yield was ligated into TOPO pCR2.1, and used to transform Top10 E. coli cells.Mitochondrial RNA was DNase treated with a DNase kit (Ambion), followed by extraction with phenol/chloroform/isoamyl alcohol (25∶24∶1) and precipitation. Ten µg of the DNase treated RNA was circularized in a 400 µl reaction volume with 80 units of T4 RNA ligase (Epicentre) with the included buffer, a final concentration of 20 µM ATP, and 80 units of RNase inhibitor at 4°C overnight. After another phenol/chloroform/isoamyl alcohol (25∶24∶1) extraction and precipitation, the RNA was resuspended in H15 clones for each uninduced RNAi cell line, 15 clones from the induced TbRND RNAi cells, and 30 clones from the induced KPAP1 and KPAP1/TbRND RNAi cell lines were selected for sequencing. Sequences from all uninduced cultures were combined to generate the sequences of the “Control” population. Random C or G nucleotides interspersed in the sequences  were eliminated from the subsequent analysis, as they occurred too seldom to determine whether they were indeed part of the tail sequence rather than a PCR or sequencing artifact. General characteristics of tails were calculated in an Excel spreadsheet, and dot plots were generated with a dot plot generator created by Tatsuki Koyama at Vanderbilt University. Tails were considered homopolymeric if from the second nucleotide to the end of the tail, they consisted of only one nucleotide. A paired, one-tailed Student's t-test was used to verify the shortening of U tails on pre-edited RPS12.in vivo, and test whether alterations in the percent of U residues in 3′ tails impact the ability of TbRND to degrade mRNAs. We reasoned that depleting the major mitochondrial poly(A) polymerase (KPAP1), while leaving the mRNA TUTase, RET1, intact would lead to increased U content in mRNA 3′ tails. This change in average mRNA tail U content might permit us to detect an effect of TbRND on mRNA decay patterns. Additionally, these experiments have the potential to provide insight into the interplay between mRNA 3′ adenylation and uridylation and to reveal whether different mRNAs are differentially sensitive to these modifications.We previously reported that depletion of the TbRND exoribonuclease to approximately 40% of normal levels lead to increased gRNA oligo(U) tail length, but did not have significant effects on levels of mitochondrial mRNAs as determined by qRT-PCR To answer these questions, we generated procyclic form cells lines harboring inducible dual KPAP1/TbRND RNAi, as well as single TbRND and KPAP1 RNAi lines for comparison. Upon tet induction, TbRND mRNA was depleted to 30–35% of normal levels in both the single and double RNAi lines . KPAP1 met al.We next asked whether altered mRNA levels could be a factor contributing to the slow growth of the KPAP1/TbRND RNAi cell line by comparing the levels of mitochondrial RNAs in cells depleted of KPAP1 or TbRND to those simultaneously depleted of both enzymes  tail. If this were the case, we would expect edited RNAs to increase in abundance in the dual knockdown compared to the KPAP1 RNAi line. We do observe this trend for edited A6, RPS12, COII, and to a lesser extent CYb . LikewisIn an effort to identify characteristics of 3′ tails on mRNAs whose abundance is altered by TbRND depletion in the KPAP1 background, we directly investigated 3′ tail compositions in the differing cell lines. We began by examining the never-edited ND4 RNA, which increased in abundance upon dual KPAP1/TbRND depletion. Specifically, ND4 RNA levels were unchanged upon TbRND depletion, decreased 30% upon KPAP1 depletion, and increased 3-fold over uninduced cells when TbRND was depleted in the KPAP1 RNAi background . The 3′ Upon depletion of TbRND alone, we observed no obvious changes in 3′ tail composition or length in any RNA examined . When KPin vitro decay assays In contrast to the never-edited ND4 RNA, RPS12 RNA undergoes extensive editing along almost the entire length of the RNA. The pre-edited version of RPS12 RNA was 1.8-fold stabilized by KPAP1 depletion, and thus differs from ND4 RNA in this regard. However, pre-edited RPS12 RNA is similar to ND4 RNA in that it is significantly stabilized (>2-fold) by dual KPAP1/TbRND depletion compared to KPAP1 depletion alone . To deteUpon co-depletion of KPAP1 and TbRND, the distribution of RNAs with different classes of tails remains relatively unchanged compared to the single KPAP1 knockdown. However, we noticed that the oligo(U) tails in the double knockdown line appeared generally shorter than those in the KPAP1 single knockdown, prompting us to sequence a larger number of clones to generate statistically significant data. These analyses revealed that 3′ tails on RPS12 pre-edited RNA in the double knockdown cell line averaged 6.7 nt in length compared to 12.2 nt in length for the tails from KPAP1 RNAi cells, and this difference was statistically significant (p<0.01) , while oTo this point, we have shown that pre-edited RPS12 RNA is stabilized upon KPAP1 depletion  and thati.e., U addition and deletion patterns that did not match the fully edited sequence et al.Lastly, we examined the 3′ tail composition of a transcript for which TbRND and KPAP1 co-depletion resulted a decrease rather than an increase in abundance. Edited MURF2 RNA abundance as a percentage of the total was essentially unchanged by TbRND RNAi, decreased 30% by KPAP1 RNAi, and decreased 65% by TbRND/KPAP1 co-depletion . To corrUpon depletion of KPAP1, the percentage of U-tailed edited MURF2 RNAs increased dramatically . This isT. brucei, and examines a potential role for the exoribonuclease, TbRND, in mRNA decay. Our results reveal that the effects of 3′ tails on mitochondrial mRNA stability in this organism are not as simple as previously anticipated. Oligo(A) tails have been implicated as a stabilizing element for never-edited RNAs and for edited RNAs that have begun the editing process in vitroThe present study provides insights into the relationship between 3′ non-encoded tails and mRNA turnover in the mitochondria of Given the substantial and transcript-specific impacts of 3′ tails on mRNA stability, it stands to reason that RNAs may be differentially susceptible to 3′ end modification. We were able to demonstrate this, as KPAP1 depletion lead to a predominance of non-tailed ND4 RNAs, but caused a very dramatic increase in the proportion of oligo(U) tailed pre-edited RPS12 and edited MURF2 RNAs. Thus, the latter two RNAs appear much more susceptible to U tail addition. Furthermore, a previous study showed that pre-edited COIII RNA appears to be primarily uridylated even in the presence of KPAP1 d for mRNAs similar to that for oligo(U)-tailed gRNAs that are known to be substrates (results not shown). However, TbRND binding to mRNA does not appear to be influenced by the presence or composition of a 3′ tail, so in this scenario, the specificity of recruitment to a particular RNA would be due to factors other than the 3′ tail. Alternately, TbRND in the KPAP1 RNAi line could be directly degrading an RNA we have not selected for further study here. Upon TbRND co-depletion, this transcript would accumulate and might then sequester the majority of the ribonuclease that was previously degrading transcripts such as ND4 and pre-edited RPS12  ed RPS12 . Indeed,ed RPS12 . Indeed,Our ability to modulate the composition of mRNA 3′ tails also allowed us to address whether such extensions on pre-edited RNAs can affect the ability of these RNAs to enter the editing pathway . This isThis study reveals the complicated interplay between mRNA 3′ end modifying enzymes, their target RNAs, and the ribonucleases that mediate RNA decay. Although TbRND does not appear to degrade mRNAs or their tails, the dual-depletion system utilized here remains a way to modulate both mRNA stability and tails to examine the relationship between the two. However, our complex results compellingly demonstrate the limits of our understanding imposed by the fact that we have not identified the major players in the mRNA decay pathway: the ribonucleases. While the depletion of enzymes that end-modify RNA can be a component of RNA stability research, a crucial next step will be the identification of these ribonucleases.Figure S1Circular RT-PCR primer locations. Relevant portions of sequences from ND4, RPS12, and MURF2 mRNAs are shown with primer locations indicated by arrows. Red arrows indicate reverse transcription primers, blue arrows indicate PCR primers, and the turquoise arrow indicates a nested PCR primer.(PDF)Click here for additional data file.Dataset S1Spreadsheet containing 3′ non-encoded tails from the various cell lines used in this study. Tails acquired from the four transcripts examined are categorized in separate sheets of the Excel file, and categorized by different cell lines within each sheet. The dataset for the MURF2 transcript also includes a designation of whether or not the tail was acquired from a completely edited MURF2 transcript.(XLS)Click here for additional data file.


Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize  Northern hybridization, although less sensitive than other methods for RNA analysis, is the only technique providing information about the concentration of specific transcripts among a complex, overlapping RNA population. This information is required in the study of important events of gene expression regulation, such as RNA transcription termination, processing, and degradation. The quality of a northern hybridization protocol depends on three main points: protection of RNA integrity, unbiased recovery of the entire transcripts population, and its efficient and even transfer to the filter.Streptomyces RNA purification protocols include an initial incubation step during which some RNA degradation pathways active in vivo may keep on going in vitro. This often results in RNA degradation. To overcome these artifacts, an efficient lysis must be promptly achieved and quickly followed by addition of a fully denaturing agent which protects RNA integrity.Streptomycetes are largely studied antibiotic-producing soil bacteria, which undergo a complex life cycle characterized by the differentiation of a vegetative and an aerial, spore-producing mycelium. Lysis of these and of other gram-positive organisms is difficult to achieve. Most A second point that must be considered is the loss of low M.W. transcripts occurring when the RNA is purified by column chromatography.Finally, blotting in the presence of a neutral buffer results in a non-efficient transfer of high M.W. transcripts to the filter.Streptomyces coelicolor dnaK operon, indicating that early published data Mo7O24.4H2O.R5 Agar: 103 g/l sucrose, 0.25 g/l KCellophane (Bio-Rad)2O.Phenol (USB) saturated with HChloroformThe transfer apparatus consist in filter paper, Whatman 3MM filter paper, and nylon membrane ZetaProbe (BioRad).RNeasy Kit (Qiagen)Dnase I (Invitrogen)Chemicals were from Sigma, where not otherwise specified.8 spores ml-1) of S. coelicolor M145 was plated directly onto sterile cellophane covered R5 plates [2 and ground in Mikrodismembrator II (Sartorius) at 2,000 rpm for 45 s .Dense spore suspension  was carried out accordingly to the manufacturer's instructions.RNA was glyoxylated using 10 μl glyoxal mixture/2 μl RNA . The sam254nm fixed for 15 s, and hybridized to a 32P-labeled riboprobe (spec. act. 7 × 108 cpm/μg) in PerfectHyb Plus Hybridization Buffer (Sigma), at 73°C overnight. After hybridization, the membrane was washed twice in 0.4× SSPE at 73°C and twice in 0.1× SSPE at 67°C.After electrophoresis the gel was soaked in 30 mM NaOH for 5 min with gentle agitation, then put onto downward transfer apparatus, using 20 mM NaOH as transfer solution. After 2 h, the gel was removed, the filter air dried for 1 min, neutralized in BTPE/50 mM Tris-acetate pH 7, air dried, UV2, by means of Mikrodismembrator II (MD) reduces it, maintained in a frozen state, to a fine and homogeneous powder in a very short time (45 s) thus preventing all degradative processes. MD is a small apparatus where a small steel ball is moved in a bilateral direction inside a small grinding chamber, successfully used for nucleic acids extraction from various biological samples ranging from tumoral tissues [Bacillus subtilis cells [2. This precaution allows fast, efficient, and highly reproducible grinding of Streptomyces mycelium. The RNA is then purified according to [260/280 of purified RNA appears to be equivalent to that obtained by column procedures. Moreover, the use of an alkaline medium for transfer of the RNA to a filter [The first crucial step in the present protocol is the grinding of the frozen mycelium. Grinding of the mycelium, previously frozen in liquid N tissues  to Bacilis cells . To mainrding to . The usert time 4 s thus prt time 4 s thus pdnaK operon of S. coelicolor. Figure dnaK operon. This probe hybridizes with transcripts ranging from 4.3 Kb (corresponding to the entire operon [dnaK operon during the heat shock response [We applied our protocol to the analysis of RNA complementary to the e operon  to aboute operon . Their ee operon ,6. Comparesponse  it clearAll this stresses the quality of our protocol for studying dynamic events of RNA metabolism where the relative concentration of high and low M.W. transcripts must be compared.


Experimental substrates were used to assess rates of calcification in sclerobionts  across key reef microhabitats: cryptic (low-light), exposed  and vertical topographic settings. Sedimentation negatively impacted calcification by photosynthesising crustose coralline algae in exposed microhabitats and encrusting foram cover (%) in exposed and cryptic substrates. Heterotrophs were not affected by sedimentation. Fore-reef, turbid water encruster assemblages calcified at a mean rate of 757 (SD ±317) g m−2 y−1. Different microhabitats were characterised by distinct calcareous encruster assemblages with different rates of calcification. Taxa with rapid lateral growth dominated areal cover but were not responsible for the majority of CaCO3 production. Cryptobiont assemblages were composed of a suite of calcifying taxa which included sciaphilic cheilostome bryozoans and suspension feeding barnacles. These calcified at mean rates of 20.1 (SD ±27) and 4.0 (SD ±3.6) g m−2 y−1 respectively. Encruster cover (%) on exposed and vertical substrates was dominated by crustose coralline algae which calcified at rates of 105.3 (SD ±67.7) g m−2 y−1 and 56.3 (SD ±8.3) g m−2 y−1 respectively. Globally, encrusting organisms contribute significant amounts of carbonate to the reef framework. These results provide experimental evidence that calcification rates, and the importance of different encrusting organisms, vary significantly according to topography and sediment impacts. These findings also highlight the need for caution when modelling reef framework accretion and interpreting results which extrapolate information from limited data.It is widely accepted that deteriorating water quality associated with increased sediment stress has reduced calcification rates on coral reefs. However, there is limited information regarding the growth and development of reef building organisms, aside from the corals themselves. This study investigated encruster calcification on five fore-reefs in Tobago subjected to a range of sedimentation rates (1.2 to 15.9 mg cm These include primary reef building corals and secondary reef builders, e.g. crustose coralline algae, bryozoans, foraminifera and serpulid worms −2 y−1 on degraded reefs to 14.3 kg m−2 y−1 on healthy, clear water reef sites −2 y−1 for CCA Worldwide, reef-building organisms are under increasing sediment stress from natural (e.g. storm and catchment runoff) and anthropogenic disturbances  which have resulted in the subsequent decline in near-shore water quality Globally, increased catchment runoff (e.g. sediment and nutrients) threatens reefs by elevating turbidity and sedimentation and causing eutrophication The rate of calcification associated with reef-building organisms is a key indicator of reef health. However, due to a paucity of information, relatively little is known about rates of calcification for different encrusting, carbonate-secreting sclerobionts, sensu Taylor −1) −2 y−1) is the combined result of both linear growth (extension mm y−1) and skeletal density (g cm−3). Focusing on only one growth parameter can be misleading when assessing reef accretion, as high extension rates do not always correspond with high calcification rates Most encruster studies have focused on ecology and community development, relying on visual estimates of encruster cover (%), morphological assessments and linear growth rate measurements  substrates, downwards facing, cryptic-shaded (c) substrates, and vertical, light-exposed substrates (v). This study assessed: 1) which encruster groups distinguished communities in different microhabitats; 2) encruster calcification on fore-reefs; 3) if sedimentation impacted encruster cover (%); and 4) if calcification rates for CCA, encrusting bryozoans, barnacles (episkeletozoans) and ‘other’ encrusters varied with a) substrate orientation, or b) sediment impacts.No specific permits were required for the described field studies. The activities for this study were conducted in collaboration with the Tobago House of Assembly.−1, but following runoff and high energy events can exceed 200 mg L−1 , Culloden (C), Kariwak (K), Little Englishman’s Bay , and Mt Irvine (M) with a control water quality site at Sisters Rocks (S) . All sit−1) were attached to the reef framework 50 cm above the substrate at 10 m water depth and remained in place from February to October 2007. Traps were positioned adjacent to experiments and collected routinely every month or when logistically possible. Sedimentation (mg cm−2 d−1) rates were estimated for: total sediment, clastic sediment and carbonate sediment. The carbonate and clastic content of each sediment trap was determined using hydrochloric acid digestion to remove calcium carbonate grains. Sedimentation rates are used here as an indicator of sediment stress at each site.Sediment impacts differed between sites with mean sedimentation rates ranging from low to moderate Benthic communities were generally dominated by scleractinians and gorgonians . The excCeramic, unglazed, artificial settlement plates (10 cm x 10 cm) were deployed at the five fore-reef locations. Settlement plates were attached to gently sloping reef framework at a depth of 10 m, ca. 30 cm above the reef substrate. Tiles were attached to PVC poles using cable ties and the poles anchored to reef framework using nails and concrete blocks. At each location 66 plates were positioned horizontally in pairs, with half facing upwards and half facing downwards mimicking exposed (e) and shaded/cryptic (c) reef substrates. An additional 66 plates were positioned in pairs hanging vertically (v) mimicking vertical reef topography. Settlement plates were attached to anchored PVC poles using cable ties. A random selection of 6 plates per orientation were retrieved from each of the 5 locations after 12 months of emersion (May 2007 - May 2008). On collection, the 90 plates were individually bagged underwater and subsequently oven dried at 30°C and air-cooled.In order to assess the contribution of different calcifying organisms to reef accretion across all sites, community composition was first analysed, using the 30 randomly collected plates per orientation . Encruster community composition was analysed by placing a 100 point grid over each tile and identifying the organism underneath each intersection to taxonomic group . Prior work has demonstrated that this approach mimics findings on natural reef substrates −2 y−1) were then estimated for: 1) each plate and 2) three taxa: CCA; barnacles; and cheilostome bryozoans. All other encrusters were grouped together as ‘other’. Calcification rates for each settlement plate were determined following methods detailed in −2 y−1) were then determined for each tile as m1–m2.Total calcification rates (g m2) removed carefully from each tile using a scalpel blade. A pre-cut template was used to remove a known surface area for CCA and cheilostomes. The calcareous section was then weighed, and the tile reweighed. CCA and cheilostome sections were typically 1 cm2, whilst entire barnacles were removed and the mass and surface area calculated for each individual. Bulk calcification by other organisms was then determined using the method outlined above.After 12 months, calcification rates were determined for three calcifying groups: CCA; barnacles; and cheilostomes. Plates were weighed, and then a known surface area of each organism . Relationships between sedimentation rates and calcifiers were assessed using linear regression analysis (R2). Data transformation was not required to meet the assumptions of these tests.Data were analysed using SPSS 19 statistical software. Data distributions were determined using the Levene statistic for homogeneity of variance and Kolmogorov-Smirnov to test for data distribution. Normally distributed data were compared using one-way ANOVA with Multivariate analyses were carried out using the PRIMER 6 p>0.05; −2 d−1) were significantly different between sites for all three parameters: total sedimentation, carbonate sedimentation and clastic sedimentation (p<0.001) (−2 d−1) ranged from 0.3 at Sisters Rocks control site to 14.6 at Kariwak. Terrestrial (clastic) sedimentation (mg cm−2 d−1) was also highly variable ranging from 6.9 at Mt Irvine to <0.01 at Sisters Rocks.IWCAM p<0.001) . Total sp  = 0.035, and Mann-Whitney U test: exposed median  = 99%, vertical median  = 81%, U  = 310.5, p  = 0.038). No difference in total calcareous cover was observed between the cryptic and vertical plates (p  = 0.8).The mean (±SD) total cover (%) of encrusters on all plates was 79 (±21.7), with CCA dominating cover (53.8±36.7), followed by cheilostomes (10.6±19.1), serpulid worms (16.9±17.9), barnacles (6.7±5.6) and forams 2.0±3.3; . Total cH  = 48.0, df  = 2, p<0.001). Exposed and vertical plates were dominated by CCA (median 98% and 67% respectively). Cryptic plates were characterised by serpulid worms (median 21.5%) and cheilostomes (median 8.8%) and had lower levels of CCA (median 9%).There was significant variation in the cover (%) of key calcifying organisms between substrate orientations . Signifip<0.001). Pairwise tests between microhabitats showed significant differences: exposed ‘v’ cryptic: R  = 1 (p<0.01), exposed ‘v’ vertical: R  = 0.976 (p<0.01), cryptic ‘v’ vertical: R  = 0.936 (p<0.01). MDS ordinations  (p>0.5) but was significantly elevated (p<0.001) on exposed substrates (1039.3±300.9). Across all settlement plates, calcification rates (g m−2 y−1) were significantly different between CCA, cheilostomes and barnacles . CCA produced the most CaCO3 compared to cheilostomes and barnacles (−2 y−1).Total encruster calcification rates (g mp<0.001) . A Bonfearnacles . Other (−2 y−1) differed significantly between calcifying organisms . Cheilostomes produced more skeletal carbonate framework (median ±IQR: 13.5±35.2) than CCA (median ±IQR: 0±2.45) or barnacles  with CCA calcification rates (median ±IQR: 105.4±92.1) exceeding cheilostome (median: 0) and barnacle rates . Vertical calcification by CCA (median ±IQR: 47.9±63.8) was greater than cheilostomes (median: 0) and barnacles . Pairwise tests between microhabitats gave: exposed ‘v’ cryptic: R  = 0.95 (p<0.01), exposed ‘v’ vertical: R  = 0.564 (p<0.05), cryptic ‘v’ vertical: R  = 0.664 (p<0.01). SIMPER demonstrated the importance of coralline algae and ‘other’ calcifiers  in distinguishing between the microhabitats. In all cases, these two categories contributed to >65 cumulative % of the dissimilarity between groups.MDS ordination, coded for microhabitat, reveals excellent two dimensional representation for encruster calcification  were significantly different between the five sites  and the cover of encrusting foraminifera on cryptic  and exposed tiles  when compared to estimates of global reefal carbonate production  This study demonstrates the important contribution of encrusters to benthic cover and reefal carbonate accretion  organism was considered in these analyses. These findings highlight the important CaCO3 contribution of solitary organisms which may not dominate areal cover. The potential importance of underlying (overgrown) encrusters to CaCO3 production clearly warrants further attention.Organisms that dominated encruster cover (substrate area) did not necessarily produce the most skeletal carbonate in early encruster assemblages. Whilst CCA dominated benthic cover in exposed and vertical microhabitats it did not contribute the most CaCOFree space on reefs  is typically rare, often only becoming available in patches following predation and physical disturbances. The results presented here focus on early encruster development (≤12 months) on freely available space (settlement plates). No attempt was made to account for competition for space. Longer experiments on deep (40 m) cryptic Jamaican reef environments have noted how areal cover of solitary organisms doubled in the first 7 to 14 months and then dropped sharply by 60% over the subsequent 12 months −2 y−1) on exposed plates in Tobago were almost an order of magnitude greater than those reported for similarly sediment impacted (<10 mg cm−2 d−1) Jamaican fore-reef sites , as were cryptic calcification rates  Results from Tobago are consistent with other observations of coralline algae dominated reefs from the Pleistocene and Holocene, for a review see −1 and skeletal density g cm−3) from other non-related studies. In the absence of contemporaneous, in-situ growth parameters, this approach is perhaps justified. However, results should be used with caution as findings are based on the assumption that such growth rate parameters are constant across all reefs and the major variable in calcification is benthic cover (%). A growing number of studies Previous studies have estimated coralline algae calcification using census-based methods to estimate percentage cover and then extrapolated growth parameter data  were found to range from 0 to 821 g m−2 y−1−3Porites blocks in the shallows (1–2 m) in French Polynesia after a 24 month experiment ranged from 180 to 1130 g m−2 y−1Census based estimates, which utilise non-site specific growth rates, of encrusting CCA carbonate production vary widely. By combining Stearn −2 y−1. Results confirm that bryozoans are still important secondary contributors to reef construction as they accrete and overgrow cryptic reef substrates. Cheilostomes typically colonised and accreted on cryptic substrates with a preference for sediment free, dimly-illuminated surfaces Colonial bryozoans, in particular cheilostomes, are capable of overgrowing extensive areas of reef substrate Filter feeding barnacles made a noticeable contribution to encruster communities on vertical and cryptic substrates in Tobago. Whilst barnacles occur in many coral reef locations, they have not formed an important component of prior reef accretion or encruster studies. As a result little is known about their contribution to the carbonate budget of tropical coral reefs. It is hypothesised here that barnacles and other heterotrophs may play a more important role in reefal carbonate production in locations that are characterised by increased sediment disturbance.3. This is an area which future research should focus on.Settlement plate studies from Jamaica and Barbados note that forams and serpulid worm assemblages made a clear contribution to encruster cover at 10 m, particularly on cryptic plates which did not accumulate sediment in-situ calcification process on natural reefs as it relied on the use of experimental substrates deployed on coral reefs. However, there is reason to believe that it still provides useful data. Earlier comparisons of coralline algae recruitment and growth rates between natural versus experimental substrates found that results were comparable and that artificial substrates could be used to calculate carbonate production 3 production need to be identified. Future studies should focus on functionally important carbonate secreting organisms (e.g. forams and serpulid worms) in a range of optimal to suboptimal reef-building environments.This study might not fully represent Discorbis bertheloti bioerodes carbonate substrates Future research over longer timeframes is required to understand temporal shifts and environmental drivers of encruster calcification and the implications for reefal carbonate budgets. This study spanned 12 months and no attempt was made to account for succession or seasonal variation. The rate of occupation attained in the first 12 months to a bare substrate was rapid. However, calcification rates might increase in subsequent years as colonies mature. Early life stages are likely to be sensitive to water quality and settlement plates were deployed during Tobago’s dry season when sediment runoff (and sedimentation) was minimal. Encruster recruitment might have been less successful if substrates were deployed during the rainy season as sediment cover of available substrates can limit recruitment. Previous researchers have noted how solitary organisms often become dominated or overgrown by colonial species resulting in reduced diversity Ensuring the continued growth, structural integrity and topographic complexity of coral reefs is essential if they are to adapt to changing oceanic conditions. The distribution and growth of calcareous reef building organisms is heavily influenced by both abiotic factors  and biotic factors (e.g. predation and competition) Table S1−2 y−1) by site (n = 5) and orientation (n = 3) for coralline algae, cheilostomes, barnacles and all groups combined.Calcification rates (g m(DOC)Click here for additional data file.