Our research centers on pre-mRNA processing and large RNA-protein (RNP) complexes. We study the basic structures and functions of these complexes as well as their contributions to human disease. Current areas of investigation include (1) single-molecule analysis of spliceosome assembly, (2) messenger RNP (mRNP) structure and function, (3) RNP egress by nuclear envelope budding, and (4) development of novel therapeutic approaches targeting RNA-based processes. Our research spans the disciplines of cell and molecular biology, biochemistry, chemical biology, biophysics, and bioinformatics.
Single-Molecule Analysis of Spliceosome Assembly
The majority of protein-coding genes in multicellular organisms contain introns, noncoding regions that must be excised from newly made transcripts (pre-mRNAs) to generate messenger RNAs (mRNAs). Intron excision is mediated by the spliceosome, a highly dynamic macromolecular machine containing five stable small nuclear RNAs (snRNAs) and scores of proteins. By altering the splice sites it utilizes, the spliceosome can produce many different mRNAs from a single gene. Such alternative splicing greatly enhances the number of different proteins that can be encoded within a single genome and is crucial for development, maintenance, and function of diverse tissue types.
A central goal of our research is to elucidate the basic mechanisms by which spliceosomes accurately identify pre-mRNA splice sites and then catalyze intron excision. Recently we have developed methodologies for fluorescently labeling individual proteins in crude cell lysates. In collaboration with Jeff Gelles (Brandeis University), we use these lysates to monitor spliceosome assembly and intron excision on individual pre-mRNA molecules. The use of a multiwavelength fluorescence method, colocalization of single-molecule spectroscopy (CoSMoS), allows us to analyze the dynamic characteristics of individual spliceosomes in real time. This opens exciting new windows onto previously unaddressable questions regarding spliceosome assembly and internal its structural transitions.
mRNP Structure and Function
In addition to removing introns, the process of pre-mRNA splicing loads the mRNA with proteins that fine-tune later steps in gene expression. One example is the exon junction complex (EJC), a set of proteins stably deposited by the spliceosome ~24 nucleotides upstream of mRNA exon-exon junctions. By combining RNA immunoprecipitation with next-generation deep-sequencing techniques, we recently mapped EJC deposition sites genome-wide. We find that EJCs reside at ~80 percent of exon-exon junctions, as well as at sites that contain sequence motifs targeted by SR proteins, another set of mRNP proteins linked to pre-mRNA splicing. Detailed proteomics reveals that endogenous EJCs interact stably with numerous SR proteins to form multimegadalton complexes that protect long stretches of spliced mRNAs from nuclease digestion. We are investigating the structural nature and functional consequences of these interactions.
RNP Egress by Nuclear Envelope Budding
The canonical view of nucleocytoplasmic transport posits that the sole gateway into and out of the nucleus is the nuclear pore complex (NPC). Thus all RNPs synthesized and assembled in the nucleus have been thought to enter the cytoplasm by transiting the NPC. However, in a collaboration with Vivian Budnik (University of Massachusetts Medical School), we recently reported that a subset of endogenous RNPs are able to escape the nucleus by budding through the inner and outer nuclear membranes in a mechanism akin to that used by herpes viruses. Studies currently under way are designed to identify the complete set of RNAs that utilize the budding pathway and their associated proteins and to determine the relationship of nuclear envelope budding to known NPC-dependent export pathways.
RNA and Disease
Aberrant RNAs and RNA-binding proteins are important contributors to human disease. Several of our current projects are aimed at developing therapeutics targeting aberrant RNAs. In collaboration with Neil Aronin (University of Massachusetts Medical School) and Matthew Disney (Scripps Florida), we are investigating new methods for the elimination of RNAs containing microsatellite expansions. One example is htt mRNA, in which expanded CAG repeats lead to Huntington's disease, an incurable neurodegenerative disorder. Another is C9ORF72, in which a GGGGCC expansion leads to amyotrophic lateral sclerosis (ALS).
In collaboration with Ananth Karumanchi (HHMI, Beth Israel Deaconess Medical Center), we are working to develop novel treatment modalities for preeclampsia. Preeclampsia, characterized by hypertension, edema, and proteinuria after the 20th week of pregnancy, is a leading cause of premature birth. It complicates up to 10 percent of pregnancies worldwide and is conservatively estimated to cause >75,000 maternal and 500,000 infant deaths globally each year (www.preeclampsia.org). The causative agent is soluble Flt1protein, a truncated tyrosine kinase receptor generated by intronic polyadenylation of placental Flt1 pre-mRNA. We are working to develop RNA-silencing approaches to limit placental production of soluble Flt1.
Our work is also supported by grants from the National Institutes of Health, the ALS Therapy Alliance (ATA), the Cure Huntington's Disease Initiative (CHDI), and the Bill and Melinda Gates Foundation.
As of July 13, 2012