Our research centers on pre-mRNA processing, RNA quality control 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 and disassembly; (2) messenger RNP (mRNP) structure and function; (3) cellular fates of nonfunctional or damaged rRNAs and mRNAs, (4) RNP egress by nuclear envelope budding, and (5) development of novel therapeutics either based on RNA or targeting RNA-based processes. Our research spans the disciplines of neurobiology, 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 utilization, 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 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 single-molecule spectroscopy (CoSMoS), allows us to analyze the dynamic characteristics of individual spliceosomes in real time (Figure 1). This opens exciting new windows onto previously unaddressable questions regarding spliceosome assembly and its internal structural transitions. We are currently using CoSMoS to investigate the mechanisms of spliceosome disassembly in budding yeast as well as alternative splice site selection in mammals.
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 (Figure 2). We are currently investigating the structural nature and functional consequences of these interactions.
Cellular Fates of Damaged or Nonfunctional rRNAs and mRNAs
Cells contain numerous quality control mechanisms for ensuring the functional integrity of the transcriptome. One of these is No Go Decay (NGD), a mechanism for eliminating stalled translational complexes (i.e. mRNAs harboring stalled ribosomes). We recently showed that in addition to targeting defective mRNAs for degradation, NGD factors also target functionally defective 40S ribosomes for decay. We are currently investigating whether this same pathway rids cells of chemically damaged RNAs.
RNP Egress by Nuclear Envelope Budding
The canonical view of nucleocytoplasmic transport posited 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 were 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 and Anastasia Khvorova (University of Massachusetts Medical School) we are investigating new methods for the elimination of RNAs containing microsatellite expansions such as the CAG repeats in htt mRNA that lead to Huntington's disease, an incurable neurodegenerative disorder.
In a different collaboration with Anastasia Khvorova and 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 Cure Huntington's Disease Initiative (CHDI), and the Bill and Melinda Gates Foundation.
As of March 10, 2016