Tuesday, March 15, 2016

Guide RNAs: A Uniquely Kinetoplastid Way of Editing Mitochondrial mRNA

by Paul Hoffman

            Mitochondria are found nearly all eukaryotic cells on Earth, generating most of the energy for eukaryotic life. These organelles have their own genome, independent from the nuclear genome that controls a majority of a cell’s functions. Much like nuclear DNA, mitochondrial DNA, or mtDNA, follows the central dogma of molecular biology: DNA gets transcribed to messenger RNA, or mRNA, which gets translated to protein or other products. The roles of the mitochondrial genome vary by organism, with some species utilizing their mtDNA more than others. As such, expression of mtDNA varies wildly across eukaryotic life. The kinetoplastids, for example, are a class of protozoans known for having large mitochondria with a massive network of mtDNA, containing multiple duplicates of the entire mitochondrial genome. Kinteoplasts are organized into circular fragments, which vary in size and function. Unlike most mtDNA, or nearly all DNA for that matter, kinetoplast DNA, or kDNA, is encrypted and mRNA produced from it needs to be decoded before it can be translated into useable products. A unique editing process is required for the mRNA to be functional, and this is what makes the kinetoplastids so unique.
Guide RNAs, or gRNAs, are encoded by certain fragments of the kDNA. Kinetoplast fragments can be organized into two classes: maxicircles, approximately twenty thousand to forty thousand base-pairs in length, and minicircles, approximately five hundred to one thousand base pairs in length. Maxicircles encode protein products, and are transcribed into mRNA transcripts. These transcripts are called cryptogenes; the message that is normally readable in regular mRNA is hidden due to highly conserved mutations within kDNA. In order for the message to be read by the cellular machinery, the mRNA transcripts need to be decoded into mRNA, and that is where gRNAs come into play. gRNAs come from the minicircles, vary from species to species (1), and range in number and class between species (2).
There are two major subgroups within the kinetoplastids, the trypanosomatids and the bononids, with major differences to gRNAs and kinetoplast structure between, and even within, these groups (3). Leishmania species fall under the former group, and form a clade, or close relationship, with Crithidia species within the trypanosomatids, with the Trypanosoma species forming another (4). This clade only has one gRNA coded per minicircle. Some trypanosomes contain multiple genes per minicircle (5), with three gRNAs per minicircle being coded for in some species of Trypanosoma (6). RNA editing in the Trypanosoma clade is performed at both ends of each editing domain while the  Leishmania-Crithidia clade is at the 5’ prime end of a domain (3). The bononids are often seen as more primitive than their cousins. Unlike the trypanosomatids, the bononids have unconcatenated ktDNA, meaning that it is not tightly bound, but rather loose and free-floating. Furthermore, the bononids have two gRNAs encoded per minicircle, unlike the Leishmania-Crithidia clade’s one or the Trypanosoma’s three to four (3). These differences make the kinetoplasts and gRNAs unique between the subdivisions of the kinetoplastids and incompatible between one another, despite sharing a common lineage. Of these groups, the trypanosomatids are far better studied, and seen as the model for studying gRNAs.
            Of the two clades that exist in the trypanosomatids, the Leishmania-Crithidia clade is often less studied, but just as important as these are the organisms with the most amount of classification to gRNAs. In some species of Leishmania, there can be upwards of sixty different classes of minicircles, each of which can encode multiple gRNAs. Each class is a unique minicircle sequence that codes for a group of gRNAs that act on a certain mRNA sequence (7). Minicircles all share a conserved sequence and differentiate based on variable sequences (8). In Leishmania species, the coding region for gRNAs is around one hundred fifty base-pairs from the conserved region; each minicircle codes for a single specific gRNA (9). Certain Leishmania species contain gRNAs with duplicate functions, with some having upwards of nineteen redundant gRNAs (10). These redundant gRNAs vary in expression level, with some of the redundant gRNAs being completely non-functional (5). Some of this redundancy may allow for minicircles to be lost and gained without any major change in function to the cell. Leishmania species have a wide range of minicircle numbers, even between strains of the same species, yet are able to function normally and have all the required gRNAs needed for survival (3).
            gRNA function can be boiled down to adding or removing uracil-residues from mRNA transcripts (11). All genetic material is coded for with nucleotides: DNA uses adenine, guanine, cytosine, and thymine; while RNAs swap out thymine for uracil. gRNAs decode mRNA transcripts by shifting the coding region of these transcripts by the addition and deletion of uracils in the transcript. The gRNAs have precise points within the transcripts where they perform their editing: gRNAs bind to a block of bases, ranging from one to ten nucleotides, within the transcript and signal a cleavage reaction in these blocks (12). Multiple blocks are strung together to create an editing domain. It is within these domains that the vast majority of mRNA transcript editing occurs. Different combinations of gRNA blocks can be formed on the same transcript to create different domains, yielding different mRNA products from the same transcript. Should a transcript be misedited by the wrong gRNA, other gRNAs can re-edit the transcript to form a correct sequence (12).
            A big question is how gRNAs, and the kinetoplast structure as a whole, arose from the standard mitochondrion. Evidence for early divergence of  the kinetoplastids from the euglenoids comes from ribosomal RNA sequences (13). The kinetoplastids are highly divergent from other euglenoids and, unlike many other groups, are monophyletic (14). The first models on the origin of the kinetoplast and gRNAs is a three step process: the ability to edit mRNA is acquired from enzymatic activity, then mutations occur at certain sites within the mRNA, finally editing mRNA becomes essential for growth (15). Expansions to this model propose that cryptogenes could then code for multiple proteins with different editing procedures unlocking different protein products from the mRNA. Another model suggests that RNA editing with gRNAs arose to combat mutations. kDNA, like other mtDNA, is highly unstable when not in use. Leishmania species have complex lifecycles, and different genes within the kDNA are needed at different times. When selective pressure is relived from kDNA and mtDNA, mutation rates tend to skyrocket within the sequences, leading to a vastly different kinetoplast genome than before. RNA editing with gRNAs could combat these mutations, restoring lost functionality by returning the mRNA transcripts to a working state. Furthermore, the kinetoplastids is that they do not use the universal genetic code (3). In most organisms, the codon UGA signals and end to reading an mRNA sequence when translating from nucleic acids to protein. Kinetoplastids, however, use UGA to code for tryptophan. However, the kinetoplasts themselves do not code for tRNA, or transfer RNA, that carries tryptophan to the mRNA sequence in the process of translating to protein. Because these tRNAs are imported into the kinetoplast, they rely on the genetic code of the cell rather than that of the kinetoplast. The differences between the cell’s code and the kinetoplast’s code may warrant RNA editing to be preserved and selected for in the kinetoplastids.
            Kinetoplasts and gRNAs make up the unique mitochondria system in kinetoplastids. Unlike nearly every other eukaryote, the kinetoplastids have the unique capability to change the expression of their mitochondrial genes by changing their mRNA transcripts using other RNAs as the editor. While the origin of gRNAs are still unclear, their function is well classified and incredibly interesting as no other organism uses this mechanism of RNA editing.


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