Research Interests – Nucleic Acid-Protein Complexes, Gene Expression, Transposons, RNA Splicing and Disease Connections

The general interest of our laboratory is the specificity of nucleic acid transactions, and how these become altered in disease states. Specifically, we study a type of DNA recombination, called transposition, where mobile genetic DNA elements can move around then genome and can be used for gene transfer. We are also interested in the process why which functional messenger RNAs are processed from pre-mRNA that encoded by split genes. The process of RNA splicing leads to extensive protein diversity from a fixed set of human protein-coding genes and occurs extensively in the germline and in the nervous system. RNA splicing is known to be altered by mutations in RNA binding proteins, both in cancer and in neurodegenerative diseases. Our intertest is in understanding how RNA binding proteins control patterns of alternative pre-mRNA splicing.

About half the human genome is composed of transposons and transposable DNA insertions have been linked to human disease gene mutations and are thought to be important for genome and organismal evolution. Our research focuses on the P element family of transposable elements first found in the fruit fly, Drosophila melanogaster and more recently vertebrate homologs of the P element transposase called THAP9 have been found. P element transposition is related to retroviral DNA integration, such as HIV virus, and to the process by which immunoglobulin and T-cell receptor genes are rearranged in the vertebrate immune system [V(D)J recombination]. The P element system offers the ability to effectively combine the use of biochemical, structural, genetic, molecular biological and proteomic approaches to study fundamental aspects of transposition mechanisms. We also use these approaches to study how nuclear RNA binding proteins set up patterns of alternative splicing of pre-mRNA in Drosophila and human cells. Splicing patterns occur in a cell-type and tissue-specific manner and splicing is an important mechanism for the regulation of gene expression, the evolution of organismal complexity in metazoans and leads to significant proteomic diversification. Understanding how pre-mRNA splicing is controlled is important since because human disease gene mutations in RNA binding proteins affect the splicing process. RNA binding proteins, like transcription factors, contain low complexity sequence domains that undergo associations via liquid-liquid phase separation (LLPS), which is a process that allows concentration of RNA processing components in the nucleus. Our studies deal with the interaction and assembly of proteins with RNA and DNA as well as the composition, structure, function and biochemical activities of these complex nucleoprotein machines.

Current Projects

Biochemistry of P element transposase, the mechanism of transposition and P element-related THAP 9 genes in humans and zebrafish.

The 87kD P element-encoded transposase protein is required to catalyze P element transposition and belongs to a large polynucleotidyl transferase superfamily, that includes RNaseH, RuvC, retroviral integrases, transposases and the argonaute proteins.  Biochemical studies using the purified protein revealed that guanosine triphosphate  (GTP) is an essential cofactor for the reaction.  Current studies involve the use of biochemical and cell-based assays, along with cryo-electron microscopy (cryo-EM) to investigate to the role(s) that GTP plays in transposition and to dissect the detailed assembly and reaction pathway of transposase on DNA.  Interestingly, the THAP9 gene in humans and zebrafish shares extensive sequence homology to the Drosophila P element transposase and the human and zebrafish THAP9 proteins are active to mobilize P elements in human and Drosophila cells.  We are interested in engineering P element transposase or human THAP9 to target specific sites for genome-editing applications.

Figure 1. Cryo-EM structural model of the P element strand transfer complex.  A) overall structure showing protein and DNA. B) Unusal A-form DNA structure and refolded DNA at the transposon ends.  Taken from Ghanim et al. 2019.

RNA binding proteins and the control of alternative pre-mRNA splicing in Drosophila and humans.

The Drosophila P element transposon pre-mRNA undergoes tissue-specific splicing and we showed that regulation of the third P element intron (IVS3) involves RNA binding proteins (PSI, hrp48, hrp36 and hrp38) that recognize an exonic splicing silencer (ESS) regulatory RNA element in the 5′ exon adjacent to IVS3, resulting in splicing inhibition. This RNA silencer element binds the splicing repressor protein PSI (P element somatic inhibitor) protein which directly interacts with the spliceosomal U1 snRNP and modulates U1 snRNP binding to specific sites on the pre-mRNA. We are using RNA-seq and bioinformatic analysis with the recently developed Junction Usage Model (JUM) software to identify changes in alternative splicing patterns and to identify and characterize new splicing silencers in cellular genes. We are also interested in analyzing the splicing pattern changes in human cells in disease states using JUM. This project involves genome editing mutations in the human splicing repressor hnRNPA1 that is mutated in ALS (Amyotrophic Lateral Sclerosis), a neurodegenerative disease and analyzing splicing pattern changes caused by the disease mutations. We are also analyzing RNA-seq data from human patient samples for splicing pattern changes in ALS and Parkinson’s disease to identify changes in gene expression correlated with disease state to find potential therapeutic targets and biomarkers. This project is part of the Aligning Science Against Parkinson’s (ASAP) Collaborative Research Network.

Figure 2. P element exonic splicing silencer complex and sequence-specific RNA binding proteins.  A)  The P element exonic splicing silencer complex (ESS) is assembled containing U1 snRNP and the RNA binding proteins PSI, hrp48, hrp36, hrp38 and PABP-C.  Assembly of this complex blocks U1 snRNP binding to the accurate IVS3 5’ splice site.  B)  The RNA binding proteins PSI and hrp48 bind specifically to the P element ESS.  PSI has 4 N-terminal KH-type RNA binding domains and a C-terminal region that interacts with U1-70K protein.  hrp48 contains two N-terminal RNP-CS type RNA binding domains and a C-terminal RGG low complexity domain.