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Untranslated Region of Mrnas As a Site for Ribozyme Cleavage-Dependent Processing and Control in Bacteria

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Untranslated Region of Mrnas As a Site for Ribozyme Cleavage-Dependent Processing and Control in Bacteria Erschienen in: RNA Biology ; 14 (2017), 11. - S. 1522-1533 https://dx.doi.org/10.1080/15476286.2016.1240141 RESEARCH PAPER The 30-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria Michele Fellettia,b, Anna Biebera, and Jorg€ S. Hartiga,b aDepartment of Chemistry, University of Konstanz, Konstanz, Germany; bKonstanz Research School Chemical Biology (Kors-CB), University of Konstanz, Konstanz, Germany ABSTRACT Besides its primary informational role, the sequence of the mRNA (mRNA) including its 50-and30- untranslated regions (UTRs), contains important features that are relevant for post-transcriptional and translational regulation of gene expression. In this work a number of bacterial twister motifs are characterized both in vitro and in vivo. The analysis of their genetic contexts shows that these motifs have the potential of being transcribed as part of KEYWORDS polycistronic mRNAs, thus we suggest the involvement of bacterial twister motifs in the processing of mRNA. 0 Aptazyme; bacteria; Our data show that the ribozyme-mediated cleavage of the bacterial 3 -UTR has major effects on gene hammerhead ribozyme; expression. While the observed effects correlate weakly with the kinetic parameters of the ribozymes, they polyadenylation; riboswitch; show dependence on motif-specific structural features and on mRNA stabilization properties of the secondary RNase; RNA decay; secondary structures that remain on the 30-UTR after ribozyme cleavage. Using these principles, novel artificial twister- structure; twister ribozyme based riboswitches are developed that exert their activity via ligand-dependent cleavage of the 30-UTR and the removal of the protective intrinsic terminator. Our results provide insights into possible biological functions of these recently discovered and widespread catalytic RNA motifs and offer new tools for applications in biotechnology, synthetic biology and metabolic engineering. Introduction UTRs, which was shown to favor the degradation of mRNAs, The function of the mRNA (mRNA) is primarily informa- seems to play an important role.10,11 tional, however, its protein coding sequence (open reading Protective RNA structures in the intergenic regions of poly- 0 0 frame, ORF) and its 5 -and3-untranslated regions (UTRs) cistronic transcripts that are processed by RNase-mediated present many features that can modulate gene expression at cleavage can play an important role in the stabilization of the the transcriptional, post-transcriptional and translational lev- secondary transcripts originating after mRNA processing. For 0 els.1,2 In bacteria the 3 -UTR contains the sequence for tran- example, Keasling and co-workers showed that the insertion of scription termination, which can be mediated by either rho- DNA cassettes containing an RNase E cleavage site and differ- dependent or rho-independent (intrinsic) terminators.3 The ent types of secondary structures could be used to tune the latter consists of a stable hairpin followed by U-rich regions.4 expression of the genes contained in an artificial polycistronic During rho-independent termination the presence of a weak mRNA by regulating the stability of the secondary transcripts U-rich RNA:DNA duplex in combination with the formation generated upon RNase E cleavage.12-14 of a stable RNA hairpin inside the RNA polymerase (RNAP) The differential stability of secondary transcripts might be a results in the dissociation of the enzyme from the DNA tem- common mechanism used by bacteria to differentially regulate plate and in the release of the mRNA.3,5,6 In addition to the the levels of expression of genes encoded by the same polycis- role in transcription termination, the presence of the intrin- tronic mRNA.15,16 Processing of mRNAs can also result in the sic terminator stem-loop structure is thought to protect the generation of regulatory small RNAs (sRNA). For example, it 0 mRNA from the attack of 3 -exonucleases RNase II, RNase R was shown that the 30-UTR regions of mRNA transcripts are a and polynucleotide phosphorylase (PNPase).7 Protective large reservoir for Hfq-dependent sRNAs in bacteria.17 In addi- intramolecular base pairing can also originate as the result of tion to RNases, small self-cleaving ribozymes were also sug- 0 exonucleolytic trimming from an unpaired 3 end.8 Interest- gested to be involved in mRNA processing in bacteria and 0 ingly, mRNA degradation initiation from the 3 end was pro- phages.18,19 In particular, in vitro and in vivo evidences of ham- posed to become relevant when the target mRNA does not merhead ribozyme (HHR) mediated cleavage of polycistronic present intrinsic RNase E cleavage sites or when mRNAs or mRNAs in bacteria were reported previously.18,20 0 3 -structured degradation intermediates accumulate abnor- A genetic context similar to the one observed for HHRs was 0 mally.7,9,10 In these situations, polyadenylation of the 3 - described for some of the reported bacterial twister ribozymes.21,22 CONTACT Jorg€ S. Hartig [email protected] Konstanz Research School Chemical Biology (Kors-CB), University of Konstanz, 78457 Konstanz, Germany. Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-174wr1gigkuyo6 1523 This widespread motif shows a high variability in terms of second- a theophylline-dependent HHR can be employed in the 30-UTR ary structure. It consists of a catalytic core composed of three stems of an E. coli expression system in order to control gene expres- (P1, P2, and P4) and two pseudoknots (Pk1 and Pk2). While the sion.24 Our experiments provide insights into possible biological length of the stems P2 and P4 are rather conserved, stem P1 shows functions of these interesting catalytically active motifs and offer certain variability in length of the stem and of the loop. Additional new possibilities for applications in biotechnology, synthetic non-conserved stem-loop structures (P0, P3, and P5) are present biology and metabolic engineering. in some twister motifs. Three circular permutated configurations are reported (Type P1, P3, and P5), with bacterial representatives in the type P1 and P5 categories. Type P3 contains only motifs Results found in metagenomes.21 In a previous work we used a type P3 twister motif for engineering synthetic ligand-dependent RNA The ribozyme motifs and their natural genetic contexts switches that regulate the accessibility of the ribosome-binding site In this study we employed six natural twister motifs fea- 0 23 (RBS) in the 5 -UTR. These twister-based switches were gener- tures that were identified recently by Breaker and coworkers ated by attaching aptamer sequences to the ribozyme core and by in bacterial genomes.21 The selected motifs originate from screening for optimized communication modules. six different bacterial species: five from Clostridia family Since it was reported that the twister ribozyme and similar and one from the Planctomycetes phylum. We chose three small self-cleaving motifs are frequently found in intergenic representatives of the type P1 format and three of the type region of polycistronic mRNAs, we decided to investigate the P5 format, each presenting different structural features. The influence of ribozyme cleavage on gene expression. In the pres- sequence, the predicted secondary structures, and the ent work we focus on the influence of ribozyme cleavage in the genetic contexts of the chosen motifs are shown in Fig. 1. 0 3 -UTR, an issue that has received very little attention so far. We Cbo-2-1 is a phage-associated type P1 twister motif that characterize several bacterial twister motifs for their potential to was found in the genome of Clostridium botulinum.Itcon- 0 influence gene expression in the 3 -UTR. We provide evidence tains a short P3 stem-loop structure and it is located in an of the possible involvement of these motifs in the processing of intergenic region between the fourth and the fifth gene of a 0 mRNAs, proposing the bacterial 3 -UTR as a possible target for prophage. Interestingly, the ribozyme is found at a point of cleavage-mediated processing and regulation events. Further- inversion of the orientation of the phage genes: the first more, we apply these findings for the development of novel arti- four genes (an integrase, a phage regulatory protein, a tran- ficial twister-based riboswitches that exert their switching scription regulator and a hypothetical protein) are encoded 0 activity via ligand-dependent cleavage of the 3 -UTR. To our on the (¡) strand, whereas Cbo-2-1 and all other phage knowledge there is only a single short report demonstrating that genes are encoded on the (C) strand. The ribozyme is Figure 1. Secondary structures and natural genetic contexts of the twister ribozyme motifs. (A) Pseudoknots Pk1 and Pk2 are highlighted in green and blue, respectively. The inactivating mutation is highlighted in yellow. The cleavage site is indicated with a red arrowhead. The constructs Gob-1-1_w/o_P0 and Sva-1-1_w/o_P0 were gener- ated removing the P0 stem at the position indicated by the gray arrow. The flanking sequences of the 3 type P5 motifs are shown in light gray and were employed in the in vivo assay. (B) Natural genetic context of the 6 investigated twister motifs. Genes of phage and bacterial origin are represented in orange and blue, respectively. Trans- posase, tRNA and hypothetical genes are represented in green, magenta and gray, respectively. Genes with the opposite orientation, whose coding sequence is present on the (-) strand, are indicated with an arrow. The putative promoters (scores between 0.95 and 1.00) are represented with a cornered arrow and putative intrinsic termi- nators are represented with a hairpin (for details see Table S1 and S2). The only promoter identified in proximity of the Gob motifs has a score of 0.88. 1524 presumably located in the 50-UTR of a polycistronic tran- The promoter and terminator search shows that it might be script where the first open reading frame (ORF) encodes transcribed as a part of a polycistronic mRNA, in the intergenic for the phage repressor protein cI, an important regulator region between two hypothetical genes.
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