Discovery and biological function of self-cleaving ribozymes

Author(s): Jimenez, Randi Maria | Advisor(s): Luptak, Andrej | Abstract: RNA plays diverse biological roles, including in regulation and catalysis. Transcriptome analysis reveals transcription from throughout the genome, including many non-coding RNAs (ncRNA), distinct from housekeeping RNAs. The complexity and diversity of these ncRNAs presents an enormous task in relating RNA structure and function. The widespread distribution of self-cleaving RNAs (ribozymes) strongly suggests a biological significance. The ribozymes have been discovered in highly diverse genomic contexts throughout nature, from viroids to vertebrates. Their biological roles include self-scission during rolling-circle replication of RNA genomes, co-transcriptional processing of eukaryotic retrotransposons, and metabolite-dependent gene expression regulation in bacteria. Other examples, including highly conserved mammalian ribozymes, suggest that many new biological roles are yet to be discovered. The work presented here focuses on two families of ribozyme (hammerhead and HDV-like) which are widespread throughout all kingdoms of life, including in the human genome, but for which the biological roles remain unclear. Through the discovery of new ribozymes and characterization of their biochemical properties, we aim to elucidate the biological roles of self-cleaving ribozymes. Deep sequencing of viral or bacterial nucleic acids monitors the presence and diversity of microbes in select populations and locations. Metagenomic study of mammalian viromes can help trace paths of viral transmissions within or between species. High-throughput sequencing of patient and untreated sewage microbiomes showed many sequences with no similarity to genomic sequences of known function or origin. To estimate the distribution of functional RNAs in these microbiomes, we used the hammerhead ribozyme motif to search for sequences capable of assuming its three-way-junction fold. While only two of the three possible natural HHR topologies had been known, our analysis revealed highly active ribozymes that terminated in any of the three stems. Altogether, thirteen ribozymes were confirmed, the most abundant of these are type II HHRs, one of which is the fastest natural cis-acting HHR yet discovered. We demonstrate that a structure-based search for a known functional RNA is a powerful tool for analysis of metagenomic datasets, complementing sequence alignments. The hepatitis delta virus-like ribozyme (HDV) ribozyme was first discovered in the genome of the human pathogen, where self-scission by the ribozyme processes RNA concatamers during replication. The nested double-pseudoknot secondary structure of the ribozyme was used in bioinformatic searches to identify the widespread occurrence of this class of self-cleaving ribozyme throughout nature. Previous work using structure-based searches identified a HDV-like ribozyme located immediately upstream of the glmM gene, encoding glucosamine mutase, in the genome of the human gut bacterium Faecalibacterium prausnitzii. Recent searches identified an active HDV ribozyme located immediately downstream of the same glmM gene in the F. prausnitzii genome. Three additional ribozymes from three different bacterial genomes were also identified through bioinformatics approaches, however they do not map near glmM genes in the respective genomes. These five ribozymes are among a handful of HDV-like ribozymes mapping to bacterial genomes and their location suggests they play a role in regulating expression of a downstream gene. Our work demonstrates that in vitro catalysis by these ribozymes is sensitive to the presence of amine sugars such as glucosamine 6-phosphate, the substrate of the glmM enzyme. In vivo assays in E.coli demonstrate that expression of a downstream ORF is influenced by the ribozyme and its state of self-cleavage. The F. prausnitzii ribozymes represent a unique arrangement in which ribozyme self-cleavage may facilitate the regulation of the downstream gene.On the other hand, the occurrence of HDV-like ribozymes in eukaryotic genomes falls to diverse genomic loci, however in some cases their location hinted at potential biological functions. Many ribozymes mapped to the 5′ end of non-long terminal repeat retrotransposons and leads to a model where the ribozyme apparently plays several roles in the retrotransposition cycle. Of particular interest is the ribozyme mapping to the R2 retrotransposable element in Drosophila. The predicted coding sequence for the first three amino acids of the R2 protein maps within the ribozyme structure. Previous work showed that these HDV-like ribozymes promote translation initiation both in vitro and in vivo, but the precise location of the first translated codon remained unknown. Here we investigate the mechanism of translation initiation of ribozyme-terminated mRNAs. Our results indicate that the correct folding of the ribozyme core is important in promoting translation. Translation occurs in the absence of a cap, start codon, and poly-A tail. Taken together our data suggest that this translation is distinct from other cap-independent mechanisms. This work highlights the significance of self-cleaving ribozymes in both bacterial and eukaryotic genomes.