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Citations
... We have previously described the naturally occurring lariat capping ribozyme (LCrz; formerly GIR1), that caps a homing endonuclease mRNA in several eukaryotic microorganisms, including myxomycetes and amoeboflagellates (Nielsen et al. 2008;Tang et al. 2011Tang et al. , 2014. LCrz cleaves in cis at an internal processing site (IPS) by branching, leaving the downstream fragment with a 5 ′ lariat in which the first and the third nucleotide are linked by a 2 ′ , 5 ′ phosphodiester bond (Nielsen et al. 2005;Meyer et al. 2014). ...
... Cleavage of the precursor transcript may occur during transcription or after completion of transcription. In its natural setting, the LCrz folds in an inactive conformation during transcription and is activated at a later stage by a conformational switching mechanism (Nielsen et al. 2008(Nielsen et al. , 2009). In the present construct, essential parts of the inactive conformation have been deleted to promote direct folding into the active conformation. ...
The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyse cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete Didymium to replace the mRNA m7G cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2', 5' phosphodiester bond. We show that the ribozyme is active in vivo in the budding yeast Saccharomyces cerevisiae and that lariat capping occurs co-transcriptionally. The lariat capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat capped mRNA with a templated polyA tail translates poorly underlining the critical importance of the m7G cap in translation. Finally, the lariat-capped RNA exhibits a 3-fold longer half-life compared to its m7G-capped counterpart, consistent with a key role for the m7G cap in mRNA turnover. Our study emphasizes important activities of the m7G cap and suggests new utilities of lariat capping as a molecular tool in vivo.
... The most complex nucleolar group I introns known are the twin-ribozyme introns [3,10]. These introns consist of a regular group I splicing ribozyme (GIR2) with an insertion in P2 or P6 that contains a HEG as well as a lariat capping ribozyme (LC ribozyme). ...
... Two natural variants of twin-ribozyme introns have been reported: the myxomycete Didymium iridis intron Dir.S956-1 [11,18], and introns (Nae.S516) in various species and isolates of the Naegleria amoeboflagellate [12,19]. Whereas both intron variants self-splice as naked RNA, express functional HEs and have a similar overall structural composition, a number of differences in distribution, inheritance and structural organization have been reported [10]. The Didymium LC ribozyme (DirLC; formerly DiGIR1) has been investigated in more detail and recent reports include studies of the catalytic reaction, RNA conformational changes, RNA:RNA interactions, 3D models and high-resolution X-ray crystal structures [10,16,[20][21][22]. ...
... Whereas both intron variants self-splice as naked RNA, express functional HEs and have a similar overall structural composition, a number of differences in distribution, inheritance and structural organization have been reported [10]. The Didymium LC ribozyme (DirLC; formerly DiGIR1) has been investigated in more detail and recent reports include studies of the catalytic reaction, RNA conformational changes, RNA:RNA interactions, 3D models and high-resolution X-ray crystal structures [10,16,[20][21][22]. Similarly, structural and functional properties of Naegleria LC ribozymes (NaeLC; formerly NaGIR1) have been studied [12,15,17,19,23]. ...
Background
Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Naegleria amoeboflagellates and the myxomycete Didymium iridis.
Results
We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents an efficient self-cleaving ribozyme that generates a small 2′,5′ lariat cap at the 5′ end of the homing endonuclease mRNA, and thus contributes to intron mobility.
Conclusions
The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a putative regulatory RNA element (AP2.1) in the Allovahlkampfia LC ribozyme that involves homing endonuclease mRNA coding sequences as an important structural component.
... An elaborate example of RNA-regulated splicing is the group I twin-ribozyme introns found in the small subunit (SSU) ribosomal precursor in several protists (Fig. 1A). These twin-ribozyme introns are composed of a conventional group I splicing ribozyme (GIR2), into which is inserted a cassette composed of a branching ribozyme upstream from a homing endonuclease (HE) gene (4). The branching activity results in cleavage and concomitant formation of a 3-nt lariat capping the 5′ end of the HE pre-mRNA ( Fig. 1B) (5), hence the name "lariat capping" (LC) ribozyme. ...
Significance
We report the crystal structures of precleavage and postcleavage forms of the lariat-capping (LC) ribozyme. The structures show how domains from an ancestral group I ribozyme have evolved due to loss of selection pressure for self-splicing. Instead, a branching activity has been selected, resulting in capping the downstream mRNA by a 3-nt lariat stabilized by the ribozyme core. The LC ribozyme constitutes an original ribozyme family with an unexpected 3D structure that departs significantly from that of group I introns. The structures also elucidate the regulatory domain’s role in transmitting a signal for cleavage to the ribozyme. The characterization of this natural evolutionary RNA speciation event is, to our knowledge, the first described at such an intricate level.
... Here, the secondary structure has been determined by structure probing and the biochemical reaction characterized in great detail. 6,9 Unfortunately, these studies predated the discovery of the branching activity of the ribozyme 5 and a length variant that predominantly cleaves by hydrolysis at the internal processing site (IPS), now considered to be an in vitro artefact, 9,16 was used. ...
... 20 At present, GIR1 has been found in 29 out of 78 Naegleria strains analyzed. 16,20 Characterization of several of these should help in refinement of the structure of GIR1, reveal new aspects of its regulation, and provide alternative variants for X-ray crystallography as well as in application of GIR1 for lariat capping of other RNA molecules. ...
The GIR1 branching ribozyme constitutes a separate class of naturally occurring ribozymes. Most studies have been performed with the single GIR1 known from the myxomycete Didymium iridis whereas the large number of GIR1s found in the amoeboflagellate Naegleria has remained largely uncharacterized. Here, we investigate ribozyme cleavage properties of a collection of Naegleria GIR1 ribozymes and define the variant from N. pringsheimi as a suitable model due to its superior activity in vitro. We identify the minimal ribozyme by deletion analysis applying a new RNase R based assay for the branching reaction, and by mutational analysis we demonstrate a surprising effect on the activity of structural elements J2/10 and L9 located outside the core of the ribozyme. These elements are located in regions that differ mostly from the Didymium ribozyme and illustrate the usefulness of comparative ribozyme studies.
... GIR1 is the only known naturally occurring catalytic RNA with structural resemblance to group I ribozymes, but with a biological function distinct from splicing (Cech and Golden, 1999;Johansen et al., 2002;Nielsen et al., 2005Nielsen et al., , 2008Beckert et al., 2008). GIR1 ribozymes are found as small domains (180-220 nt) within twin-ribozyme group I introns including the Dir.S956-1 intron from the myxomycete Didymium iridis and the Nae.S516 introns from several species of the amoebaflagellate Naegleria (reviewed in Nielsen et al., 2008;). ...
... GIR1 is the only known naturally occurring catalytic RNA with structural resemblance to group I ribozymes, but with a biological function distinct from splicing (Cech and Golden, 1999;Johansen et al., 2002;Nielsen et al., 2005Nielsen et al., , 2008Beckert et al., 2008). GIR1 ribozymes are found as small domains (180-220 nt) within twin-ribozyme group I introns including the Dir.S956-1 intron from the myxomycete Didymium iridis and the Nae.S516 introns from several species of the amoebaflagellate Naegleria (reviewed in Nielsen et al., 2008;). The GIR1s are inserted into peripheral domains of group I splicing ribozymes (see Figure 1A for the Didymium intron) and are always followed by homing endonuclease genes (HEGs) (Decatur et al., 1995;Einvik et al., 1997). ...
RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.
... The concept of self-induced conformational switching provides important clues to the function of the complex twin-ribozyme group I introns. These introns are composed of a conventional group I splicing ribozyme (GIR2) with an insertion into a peripheral loop of a cassette composed of the branching ribozyme GIR1 and a downstream homing endonuclease encoding sequence (for review, see Johansen et al. 2002Johansen et al. , 2007Nielsen et al. 2008). Examples of twin-ribozyme introns are currently limited to the extrachromosomal rDNA of the myxomycete Didymium iridis (Dir.S956-1) and several species and lineages of the amoebaflagellate Naegleria (Nae.S516) ). ...
DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5'-end of the 3'-cleavage product. The 3'-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5'- and 3'-deletions, we identify a hairpin in the 5'-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5'-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.
... Both GIR1 and the downstream HE mRNA are inserted into a peripheral domain of a regular splicing ribozyme (GIR2) making up the characteristic configuration of a twin-ribozyme intron. Such introns have so far only been found in the SSU rDNA genes of a unique isolate of Didymium iridis and in several Naegleria strains where it has been vertically inherited from a common ancestor (Johansen et al, 2002; Wikmark et al, 2006; Nielsen et al, 2008). The biological function of GIR1 appears to be in the formation of the 5′ end of the HE mRNA during processing from the spliced out intron and the resulting lariat cap seems to contribute by increasing the half-life of the HE mRNA (Vader et al, 1999; Nielsen et al, 2005), thus conferring an evolutionary advantage to the HE. ...
Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P.
