Mitochondrial tRNAs and their function in the nuclear genome

Transfer RNAs (tRNAs) belong to one of the evolutionary oldest nucleic acids (RNA molecules). Within the cell, tRNAs serve as adaptor molecules to bring amino acids - which they are charged with - to the ribosome, where amino acids become ligated to form proteins. Binding of tRNAs to the ribosome is carried out via so-called messenger RNAs (mRNAs), which also attach to ribosomes and which determine the sequence of the amino acids at the ribosome by base pairing to tRNAs (also designated as codon-anticodon interaction). Two different classes of tRNA genes exist: for once, tRNA genes are encoded with the nucleus of a cell, are transcribed from the nuclear DNA, exported into the cytoplasm and subsequently bind to the aforementioned ribosomes. Mitochondria which serve as a a source for energy production contain a separate mitochondrial genome which encodes for mitochondrial tRNA genes (mtRNA genes), different from the nuclear encoded ones, which are employed for mitochondrial protein synthesis. During evolution, parts of the mitochondrial genome became integrated into the nuclear genome; among these were also mitochondrial tRNA genes. For a long time it was unclear which functions these mitochondrial tRNA genes might have in the nucleus. Recently, our group could demonstrate that mtRNA genes which were located in so- called introns sequences of nuclear messenger RNAs were able to promote splicing of the respective intron sequences, resulting in an increased abundance of the respective mRNA and the corresponding encoded protein. However, mtRNA genes are not only present within intronic sequences but are also located in between nuclear genes, i.e. outside from intronic sequences. Already numerous examples exist that tRNAs in general have acquired new functions during evolution which are required for cell viability or function of specific tissues (such as brain). The pending questions which we would like to address in the grant proposal are : which functions do these intergenic mtRNAs exert within the nuclear genome and where are these functions located, i.e. in the nucleus or in the cytoplasm of a cell. By employing various molecular biology methods we want to solve these questions to decipher novel and yet unknown functions of these fascinating mitochondrial derived tRNA genes.

Our study investigates the potential expansion of the human nuclear genome through mitochondrial transfer RNA (tRNA) genes and explores their possible functions, as well as their integration and processing within the cell nucleus. The majority of genetic information in human cells is stored within the nuclear genome, which governs essential cellular processes. An exception to this are mitochondria, known as the "powerhouses of the cell," which are responsible for ATP production and possess their own genome. Mitochondria represent the sole source of extrachromosomal genetic material in human cells. The current form of mitochondria is derived from endosymbiotic bacteria that were engulfed and incorporated into ancestral eukaryotic cells during evolution. Over time, much of the mitochondrial DNA has been transferred to the nuclear genome, while essential genes remain retained within the mitochondrial genome and continue to be functionally active. The human genome is a dynamic entity. While critical genes are protected from mutations through various repair mechanisms, novel genetic elements continuously emerge. These include fragments of mitochondrial DNA that have been integrated into distinct regions of the nuclear genome in humans. Our research specifically focused on mitochondrial tRNA genes that have been incorporated into the nuclear genome and are located within intergenic regions, i.e., between coding sequences of other genes. We have termed these tRNAs inter-nmtRNAs. The primary function of tRNAs is to serve as adapter molecules facilitating the translation of genetic information from mRNA into protein. Through bioinformatic analyses of sequence data from various cell types, we confirmed the existence and expression of inter-nmtRNAs. Building on these findings, we conducted further experimental investigations to elucidate their potential functions. We observed that inter-nmtRNAs are transcribed and processed within the nucleus, despite the fact that the proteins involved in these processes differ in structure and recognition motifs from those found in mitochondria. Moreover, we demonstrated that inter-nmtRNAs are exported from the nucleus and remain stable outside the nuclear compartment. However, they are not aminoacylated and, therefore, do not participate directly in protein synthesis. Interaction analyses between RNA and proteins indicated that inter-nmtRNAs bind to the same set of proteins as their mitochondrial and nuclear counterparts. These findings suggest that inter-nmtRNAs may function as competitors for nuclear tRNAs and thereby might play a role in the regulation of tRNA-mediated processes.