Mitochondrial Sequencing Identifies Long Non-Coding RNA Features that Promote Binding to PNPase.

Extranuclear localization of long non-coding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where Polynucleotide Phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and crosslinked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. LncRNA sequence and structure were evaluated through supervised (Classification and Regression Trees (CART) and Support Vector Machines, (SVM)) machine learning algorithms. In HL-1 cells, qPCR of PNPase CLIP knockout mutants (KH and S1) were performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. 112 (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most highly expressed. Most non-coding RNAs binding PNPase were lncRNAs, including Malat1. LncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. Binding of lncRNAs to PNPase was decreased through knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs to bind to PNPase and undergo import into the mitochondrion.

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Structure of a 10-23 deoxyribozyme exhibiting a homodimer conformation.

Deoxyribozymes (DNAzymes) are in vitro evolved DNA sequences capable of catalyzing chemical reactions. The RNA-cleaving 10-23 DNAzyme was the first DNAzyme to be evolved and possesses clinical and biotechnical applications as a biosensor and a knockdown agent. DNAzymes do not require the recruitment of other components to cleave RNA and can turnover, thus they have a distinct advantage over other knockdown methods (siRNA, CRISPR, morpholinos). Despite this, a lack of structural and mechanistic information has hindered the optimization and application of the 10-23 DNAzyme. Here, we report a 2.7 Å crystal structure of the RNA-cleaving 10-23 DNAzyme in a homodimer conformation. Although proper coordination of the DNAzyme to substrate is observed along with intriguing patterns of bound magnesium ions, the dimer conformation likely does not capture the true catalytic form of the 10-23 DNAzyme.