Reduction of Drosophila Mitochondrial RNase P in Skeletal and Heart Muscle Causes Muscle Degeneration, Cardiomyopathy, and Heart Arrhythmia.

In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo. Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.

Loss of Individual Mitochondrial Ribonuclease P Complex Proteins Differentially Affects Mitochondrial tRNA Processing In Vivo.

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.

Structural Divergence of the Group I Intron Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases That Function in RNA Splicing.

The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.

Sequence-dependent structural changes in a self-assembling DNA oligonucleotide.

DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.

Probing the role of sequence in the assembly of three-dimensional DNA crystals.

DNA is a widely used biopolymer for the construction of nanometer-scale objects due to its programmability and structural predictability. One long-standing goal of the DNA nanotechnology field has been the construction of three-dimensional DNA crystals. We previously determined the X-ray crystal structure of a DNA 13-mer that forms a continuously hydrogen bonded three-dimensional lattice through Watson-Crick and non-canonical base pairs. Our current study sets out to understand how the sequence of the Watson-Crick duplex region influences crystallization of this 13-mer. We screened all possible self-complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson-Crick pair influenced the crystallization propensity and the speed of crystal self-assembly. We determined X-ray crystal structures for 13 of these oligonucleotides and found sequence-specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self-assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self-assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self-assembly is nucleated by the formation of the Watson-Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures.