Leigh syndrome (LS) is a mitochondrial disease that causes irreversible developmental regression in children.
Currently, no treatment options are available, and most children do not live to see their second birthday. LS is
biochemically characterized by defective mitochondrial bioenergetics due to a variety of possibly mutated
mitochondrial proteins, which can be encoded in both mitochondrial and nuclear DNA. Here, we will focus on
two LS proteins: leucine rich pentacotripeptide repeat motif containing protein (LRPPRC), and transactivator of
COXI (TACO1). Loss of LRPPRC decreases stability of all mRNAs, and also leads to loss of mRNA
polyadenylation and the appearance of aberrant mitochondrial translation. Loss of TACO1 specifically prevents
translation of COX1 mRNA. Despite this knowledge, the molecular mechanisms involved remain unknown.
RNAs fold into complex structures that are integral to the diverse mechanisms underlying RNA regulation
of gene expression. Recent development of RNA structure profiling through the application of structure-probing
chemicals combined with high-throughput sequencing (such as DMS-MaPseq) has opened a new field that
enormously expands the amount of RNA structural information available. With this technology available, here
we propose to test the hypothesis that mutations in either LRPPRC or TACO1 lead to a downstream defect
in the folding of mitochondrial mRNAs and subsequently to aberrant mtDNA gene expression. To test
this hypothesis, we will follow two Aims and will use human cell lines knockout (KO) for each gene, which we
are generating by using innovative CRISPR-Cas9 gene-editing approaches. In Aim 1, the KO lines will be
reconstituted with wild-type or disease mutant gene variants and characterized physiologically in terms of cellular
and mitochondrial bioenergetics, mitochondrial RNA processing and stability, as well as mitochondrial translation.
Preliminary data on already available LRPPRC-KO cell lines have informed mitochondrial phenotypes
compatible with those of LS patients. This will follow with establishment of in-cello mRNA binding sites of these
proteins in wild-type cells using PAR-CLIP approaches. Finally, we will utilize ribosome-profiling assays to
determine whether LRPPRC and TACO1 are binding their target mRNAs during translation. In Aim 2 we will use
DMS-MaPseq to probe the native structures of the entire mitochondrial transcriptome in the KO and reconstituted
cell lines. To ensure success in this undertaking, we will capitalize on our collaboration with Dr. Silvia Rouskin,
the pioneer of DMS-MaPseq. We expect to answer basic questions regarding how these proteins interact with
mRNA, affect their folding, and ultimately their stability, modification and translation. To fully understand the
basic pathogenic mechanisms underlying the several LS forms is critical to the NICHD mission and a
fundamental pre-requisite to develop therapeutics to target this kind of disorders. With this perspective, the
proposed project is tailored to my training towards becoming a skilled physician scientist.