Highly motif- and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression

<p>Recent bioinformatics studies have demonstrated a wide-spread occurrence of the hammerhead ribozyme (HHR) and similar small endonucleolytic RNA motifs in all domains of life. It is becoming increasingly evident that such ribozyme motifs participate in important genetic processes in diverse organisms. Although the HHR motif has been studied for more than three decades, only little is known about the consequences of ribozyme activity on gene expression. In the present study we analysed eight different naturally occurring HHR sequences in diverse genetic and organismal contexts. We investigated the influence of active ribozymes incorporated into mRNAs in mammalian, yeast and bacterial expression systems. The experiments show an unexpectedly high degree of organism-specific variability of ribozyme-mediated effects on gene expression. The presented findings demonstrate that ribozyme cleavage profoundly affect gene expression. However, the extent of this effect varies and depends strongly on the respective genetic context. The fast-cleaving type 3 HHRs [CChMVd(-) and sLTSV(-)] generally tended to cause the strongest effects on intracellular gene expression. The presented results are important in order to address potential functions of naturally occurring ribozymes in RNA processing and post-transcriptional regulation of gene expression. Additionally, our results are of interest for biotechnology and synthetic biology approaches that aim at the utilisation of self-cleaving ribozymes as widely applicable tools for controlling genetic processes.</p

A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans

The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3'-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington's disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.publishe

Twister ribozymes as highly versatile expression platforms for artificial riboswitches

The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.publishe

Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.publishe

Distances of repeats to neighboring ORFs.

<p>(A-C) Analysis of the distance of repeat sequences relative to adjacent ORFs for <i>Xcc</i> (A), <i>Xac</i> (B) and <i>Ana</i> (C). Repeats can be either located upstream or downstream of the next neighboring ORF. Repeats were grouped into three categories according to increasing distance from the respective: distance of > 100 bp (gray), 50–100 bp (light blue) and repeats overlapping with ORFs or located in a distance of up to 50 bp from the respective start or stop codon are grouped together (dark blue). (D) Schematic of a repeat being located in close proximity, upstream of the neighboring ORF on the non-coding strand. (E) Schematic of a repeat being located in close proximity, downstream of the neighboring ORF on the non-coding strand.</p

Sequence comparison between repeat containing regions in <i>Xcc</i> and <i>Xac</i>.

<p>(A): Presence of repeats patterns in <i>Xac</i> for repeat containing sequences from <i>Xcc</i>. Homologous repeats are depicted in blue, absent or mutated repeats depicted in gray, non-homologous alignments in white. (B) Analysis of changes of the identity of the neighboring genes for intergenic repeats from <i>Xcc</i> in comparison to <i>Xac</i>. Perfect alignments are grouped as “no change” (blue). Deletions (dark gray) or insertions (light gray) into intergenic regions were detected. Alignments showing only homology for one neighboring ORFs were grouped as “flanking region changes” (green). Non-homologous alignments are shown in white. (C) Orientation of neighboring genes relative to intergenic regions are shown for the repeat-containing intergenic regions from <i>Xcc</i> and the randomly chosen control sets 1–5. Sequences of control 5 were chosen to reflect the orientation of genes as found for the repeat containing intergenic regions in <i>Xcc</i>. ORFs can be either aligned (gray), convergent (white) or divergent (blue).</p