Journal: Nucleic Acids Research

Article Title: A versatile cis -acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

doi: 10.1093/nar/gkz1156

Figure Lengend Snippet: Characterization of circular pre-rRNA in H. volcanii and S. acidocaldarius . ( A ) Detection of circular pre-rRNA intermediates in S. acidocaldarius . The unique rDNA operon present in S. acidocaldarius (Saci_1299-1300 and flanking regions) is schematically represented ( , ). The double-stranded processing stems containing the bulge-helix-bulge motif, putative substrate of the archaeal tRNA processing machinery are indicated (upper panel). 5′extended linear 16S/23S pre-rRNA and circular pre-16S/23S pre-rRNA intermediates were amplified by RT-PCR and analyzed by agarose gel electrophoresis (lower panel). Reverse transcriptase was performed using reverse complement primers hybridizing at the 5′end of the mature 16S and 23S rRNA (depicted in light and dark blue, respectively). The 5′extended linear 16S/23S pre-rRNA were amplified using the indicated primer pairs (depicted in light and dark blue, light and dark green) respectively. Circular pre-16S/23S pre-rRNA intermediates were amplified using the indicated primer pairs (indicated in light and dark brown, and in light and dark blue, respectively). Expected sizes of the PCR products are as following: 5′extended linear 16S rRNA (primers Saci009/013) 98 bp; 5′extended linear 23S rRNA (primers Saci014/016) 168 bp; circular pre-16S rRNA (primers Saci009/010) 176 bp and Circular pre-23S (primers Saci014/015) 271 bp. ( B ) Detection of circular pre-rRNA intermediates in H. volcanii . Same as in (A), one (operon A: HVO_3038-HVO_3042 and flanking regions) of the two rDNA operons present in H. volcanii and characterized by the presence of an additional tRNA Cys at its 3′end is schematically depicted ( , ). Reverse transcriptase was performed using reverse complement primers hybridizing at the 5′end of the mature 16S and 23S rRNA (indicated in light and dark blue, respectively). The 5′extended linear 16S/23S pre-rRNA were amplified using the indicated primer pairs (depicted in light and dark blue, light and dark green) respectively. Circular pre-16S/23S pre-rRNA intermediates were amplified using the indicated primer pairs (depicted in light and dark brown, and in light and dark blue, respectively). Expected sizes of the PCR products are as following: 5′extended linear 16S rRNA (primers oHv200/40) 235 bp; 5′extended linear 23S rRNA (primers oHv201/42) 279 bp; Circular pre-16S rRNA (primers oHv39/40) 333 bp and Circular pre-23S 208 bp. ( C , D ) Determination of ligation extremities by DNA sequencing. Sanger sequencing chromatogram and the corresponding deduced nucleotide sequences of the region surrounding the RNA ligation position as determined by DNA sequencing of the PCR product obtained in (A, B) are provided for S. acidocaldarius (C) and H. volcanii (D), respectively. Nucleotides are color-coded as indicated in the figure (A: green; U: red; C: blue; G: black). ( E ) Schematic representation of rRNA processing stem. The bulge-helix-bulge motif and expected splicing endonuclease (endA) cleavage sites are indicated. ( F , G ) Schematic representation of S. acidocaldarius (F) and H. volcanii (G) rRNA processing stems. 2D structure prediction of the respective processing stems was determined using the ViennaRNA web services (RNAcofold and RNAfold servers - http://rna.tbi.univie.ac.at/ ) using default parameters. Nucleotides are color-coded as indicated above and in the figure. EndA cleavage sites within the respective bulge-helix-bulge motifs are indicated by scissors. The black lanes delineate the sequence boundaries shown in panel (C, D). ( H ) Relative abundance of circular-pre-rRNA after RNase R treatment. DNase-treated total RNA obtained from H. volcanii was treated with RNAse R to eliminate linear RNA prior to cDNA synthesis and quantitative PCR analysis (see Materials and Methods). The relative abundance of linear pre-rRNA versus circular pre-rRNA as determined by qPCR analysis was normalized to the non-RNAse R treated samples and arbitrarily set to one. The depicted results were obtained from analysis performed in biological duplicates and technical triplicates.

Article Snippet: PCR products within the 16S rDNA locus were digested with EcoRV-HF (NEB) for 4 h, PCR products within the 23S rDNA locus were digested with BssSIα (NEB) overnight.

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Ligation, DNA Sequencing, Sequencing, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: A versatile cis -acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

doi: 10.1093/nar/gkz1156

Figure Lengend Snippet: Plasmid-based engineered rDNA provides partial antibiotic resistance to H. volcanii cells. ( A ) Schematic representation of the plasmid-based rDNA cis -acting element reporter system used in H. volcanii . Indicated modifications expected to confer Pactamycin resistance (PCY R ) and Chloramphenicol resistance (CAM R ) were introduced in the rDNA sequence by molecular cloning (see text and Materials and Methods for details). Note that the individual changes provided different degrees of antibiotic resistance and are summarized in the respective boxes (data not shown). Changes in sequence generating new restriction enzyme digestion sites (RE) are also indicated. ( B ) Ribosomal RNA cis -acting element reporter provides partial resistance to Pactamycin. Cells transformed with a plasmid bearing unmodified rDNA operon A (level of gray) or the rDNA cis -acting reporter (level of green) were grown in Hv-Ca + lacking uracil and supplemented with the indicated amounts of Pactamycin. Growth was monitored as described above. Note that the presence of the cis -acting element reporter provides growth advantage in presence of Pactamycin in comparison to wildtype control. Representative results are provided. ( C ) Ribosomal RNA cis -acting element reporter provides partial resistance to Chloramphenicol. Same as is (B), except that cells were grown in presence of Chloramphenicol (level of orange). Note that the presence of the cis -acting element reporter provides growth advantage in presence of Chloramphenicol in comparison to wildtype control. Representative results are provided. ( D ) Partial antibiotic resistance depends on rRNA functional integrity. The cis -acting element reporter system was further modified by addition of a non-functional mutation affecting the SSU decoding center (16S G468U – H. volcanii numbering). Cells were transformed with the wildtype cis -acting element reporter and the 16S G468U mutated cis -reporter. Growth in presence of Pactamycin was monitored as described above. Note that the non-functional 16S G468U mutation abolishes the antibiotic resistance initially provided by the cis -acting element reporter plasmid.

Article Snippet: PCR products within the 16S rDNA locus were digested with EcoRV-HF (NEB) for 4 h, PCR products within the 23S rDNA locus were digested with BssSIα (NEB) overnight.

Techniques: Plasmid Preparation, Sequencing, Molecular Cloning, Transformation Assay, Functional Assay, Modification, Mutagenesis

Journal: Nucleic Acids Research

Article Title: A versatile cis -acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

doi: 10.1093/nar/gkz1156

Figure Lengend Snippet: Plasmid rDNA cis -acting element reporter is stably propagated in wildtype H26 cells. ( A ) Experimental strategy to assess genomic recombination events of the rDNA cis -acting element reporter. Properties of cells (antibiotic resistance/ restriction digest site) transformed with the rDNA cis -acting reporter plasmid were analyzed before and after curing the cis -reporter plasmid with 5-FOA. ( B , C ) Growth analysis of cells cured from the cis -acting reporter system. Cells transformed with either a plasmid containing the wildtype rDNA, or the cis -acting reporter were plated on 5-FOA containing plates to promote plasmid loss. Independent clones were selected and grown in presence of Pactamycin (B) or Chloramphenicol (C). Representative analysis is depicted. Similar experimental outcome was observed for all individual clones tested (data not shown). ( D ) Analysis of persistence of additional restriction digest site after plasmid-loss. Cells transformed with either a plasmid containing the wildtype rDNA, or the cis -acting reporter were plated on 5-FOA containing plates to promote plasmid loss. Independent clones were selected for PCR/restriction digest analysis using the 16S rDNA amplicon and analyzed by PAGE as described above. No significant recombination of the cis -acting reporter plasmid with genomic DNA could be detected as indicated by the absence of digested PCR product in the 5-FOA R clones. Representative analysis is provided. Similar experimental outcome was observed for all individual clones tested (data not shown).

Article Snippet: PCR products within the 16S rDNA locus were digested with EcoRV-HF (NEB) for 4 h, PCR products within the 23S rDNA locus were digested with BssSIα (NEB) overnight.

Techniques: Plasmid Preparation, Stable Transfection, Transformation Assay, Clone Assay, Amplification

Journal: Nucleic Acids Research

Article Title: A versatile cis -acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

doi: 10.1093/nar/gkz1156

Figure Lengend Snippet: Role of the bulge-helix-bulge motif for the synthesis of circular-pre-rRNA and total rRNA. ( A ) Schematic representation of the rDNA cis -acting reporter system. The 16S and 23S rRNA processing stems containing the bulge-helix-bulge motif are depicted in yellow and red, respectively. A close-up representation (dashed-line box) indicates the structural elements that were subjected to site-directed mutagenesis (see below). The tRNA splicing endonuclease cleavage sites are indicated by scissors. ( B ) Structure predictions of processing stem mutants analyzed in this work. The indicated processing stem mutants collection was generated by molecular cloning and transformed into wildtype H. volcanii cells as described in Material and Methods. The predicted structural consequences (ViennaRNA webservers) of the respective mutations introduced in the rDNA cis- acting reporter are depicted. ( C ) Bulge-helix-bulge motif/processing stem integrity is required for efficient circular-pre-rRNA and total rRNA formation. Cells transformed with the cis -reporter carrying the additional mutations depicted in (B) were analyzed using the schematically depicted work flow (left panel). In brief, relative total DNA amounts (plasmid-based/endogenous rDNA) were analyzed by PCR/restriction digest analysis (upper left panel). Relative amounts of circular-pre-rRNA were determined by RT-PCR/restriction digest analysis using a reverse transcription primer (blue arrow) positioned at the 5′end of the respective 16S or 23S rRNAs and extending through the rRNA region subjected to PCR amplification only when circularized (middle left panel). Relative total rRNA amounts (essentially corresponding to mature rRNA) were obtained by RT-PCR/restriction digest analysis using the indicated primers (blue arrow indicates the reverse transcription primer used). PCR fragments were digested (16S: EcoRV ; 23S: BssSI ) and separated by PAGE. Fluorescent signals were acquired on a Li-COR Odyssey system. Exemplary PAGE analysis of wildtype, 16S 5′bulge deletion and 23S 3′bulge deletion is provided. Note that the relative rDNA amounts are similar, independent of the mutations examined. ( D ) Early steps of rRNA maturation are functionally coordinated in H. volcanii . Results of semi-quantitative relative expression analysis of 16S/23S circular-pre-rRNAs (upper panel) and total 16S/23S rRNAs (lower panel) obtained from the processing stem mutants analysis depicted in (B) are summarized. Relative ribosomal RNA expression was normalized to the relative amounts of the respective rDNA template (plasmid-based versus endogenous) and is expressed in comparison to the wildtype situation (arbitrarily set to one) (see Materials and Methods for details). Each rDNA variant analysis was performed at least in biological duplicates (two independent transformants) and technical quadruplets (two fluorescent channels and two independent quantifications)

Article Snippet: PCR products within the 16S rDNA locus were digested with EcoRV-HF (NEB) for 4 h, PCR products within the 23S rDNA locus were digested with BssSIα (NEB) overnight.

Techniques: Mutagenesis, Generated, Molecular Cloning, Transformation Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, RNA Expression, Variant Assay

Journal: Nucleic Acids Research

Article Title: A versatile cis -acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

doi: 10.1093/nar/gkz1156

Figure Lengend Snippet: A putative model accounting for functional coordination of early rRNA maturation steps in archaea. ( A ) Ribosomal DNA organization and rRNA bulge-helix-bulge motif conservation across archaea. A selected survey of archaeal rRNA operon organizations suggests 2 predominant classes of linked rRNA organization found in representative organisms of the Euryarchaeota and TACK Superphylum ( T haumarchaeota– A igarchaeota– C renarcheota– K orarchaeota) and one minor class of unlinked organization (e.g. Thermoplasmata genus order/ Nanoarchaeum equitans ). 16S and 23S rRNAs processing stem secondary structures were predicted using the ViennaRNA Web servers. Presence of predicted bulge-helix-bulge is indicated in black. Presence of heterogeneous rRNA operons with heterogeneous presence of bulge-helix-bulge motif within the processing stem is depicted by a brown circle ( Haloarcula genus). Absence of predictable bulge-helix-bulge motifs is depicted by a red circle (e.g. Thermoplasmata genus/ Nanoarchaeum equitans ). ( B ) Proposed models for early coordination of rRNA maturation in H. volcanii . Upon processing of the 16S rRNA processing stem presumably performed by the tRNA splicing machinery, ligation of the processed 16S rRNA flanking region generates circular-pre-16S rRNA intermediates, whereas the remaining processed 5′leader and downstream internal spacer sequences are ligated to the nascent pre-23S rRNA. This ligation event may potentially generate a molecular feature facilitating the subsequent maturation (circularization) of the pre-23S rRNA. Alternatively, rRNA elements upstream of the 23S rRNA processing stem may have an inhibitory effect on the 23S processing stem folding/processing. This inhibitory effect is only alleviated when rRNA elements upstream of the 23S rRNA processing stem are partly or completely removed during early rRNA maturation events (see Discussion for additional details).

Article Snippet: PCR products within the 16S rDNA locus were digested with EcoRV-HF (NEB) for 4 h, PCR products within the 23S rDNA locus were digested with BssSIα (NEB) overnight.

Techniques: Functional Assay, Ligation