My colleagues and I are interested in eukaryotic RNA processing mechanisms. Specifically, we are investigating: (1) the very large and dynamic macromolecular assemblage (the spliceosome) that catalyzes the splicing of eukaryotic precursor-mRNA transcripts; and (2) the significant and dynamic structural changes that occur in the eukaryotic cell induced by its exposure to stress. In our studies we employ a diverse range of methods, including structural approaches (crystallography and electron microscopy), biochemistry, and studies in cell culture. See images of the laboratory, equipment and environment.
1. Splicing of precursor-mRNA transcripts
Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the 'spliceosome.' Integral to the spliceosome are five small nuclear ribonucleoprotein particles or U snRNPs (U1, U2, U4, U5, and U6) that assemble onto a precursor-mRNA transcript to catalyze its splicing, illustrated on your right (van der Feltz et al., Biochemistry 2012). The initial stage in precursor-mRNA splicing is the recognition of the junction between the 5' exon and intron (the 5' splice-site) by the U1 snRNP. This stage initiates assembly of the remaining U snRNPs and the subsequent catalytic reactions that result in the splicing of two exons and removal of the intron. I previously determined the crystal structure of the human U1 snRNP (see structure on your right), an 11-subunit complex, to understand the role of its various subunits, their participation in stabilizing the particle and in recognition of the 5' exon-intron junction (Pomeranz Krummel et al., Nature 2009). This work revealed the first detailed structure of a U snRNP and provided considerable insight into U snRNP architectural principles as well as the mechanism of 5' splice-site recognition by U1 snRNP. We are continuing studies along this line of investigation to provide insight into how U snRNPs cooperate to catalyze precursor-mRNA splicing.
2. Molecular basis for stress granule assembly
A eukaryotic cell possesses nuclear and cytoplasmic non-membranous subcellular organelles critical to processing and synthesis of RNA. The functions of these organelles include: neuronal mRNA transport (transport granules); RNA turnover and mRNA silencing by microRNAs (processing bodies); and assembly of telomerase and U snRNPs as well as other RNA-protein complexes (Cajal bodies). One of the most fascinating of these aggregates is the stress granule. A eukaryotic cell undergoes substantial biochemical and structural changes induced by stress, including formation in the cytoplasm of dynamic 100-200 nm diameter stress granules that appear to contribute to translational inhibition by sequestering mRNAs. We are investigating how this sub-cellular aggregate is formed, disassembled, and what is integral to its activity - including an RNA helicase called DDX3X shown above at left (Pugh et al., Nature 2012).
Hernandez H, Makarova OV, Makarov E, Morgner N, Muto Y, Pomeranz Krummel DA, & Robinson CV. Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP. PLoS One, 4: e7202 (2009).
Oubridge C, Pomeranz Krummel DA, Leung A, Li J, & Nagai K. Interpreting a low resolution map of human U1 snRNP using anomalous scatterers. Structure, 17: 930-938 (2009).
Pomeranz Krummel, DA, Oubridge C, Leung A, Li J, & Nagai, K. Crystal structure of human spliceosomal U1 snRNP at 5.5 Å resolution. Nature, 458: 475-480 (2009). (Comment in: Structural biology: Spliceosome subunit revealed. Nature, 458, 418-419 (2009); Protein structures: Structures of desire. Nature, 459, 24-27 (2009); see also Brandeis University Press Release).
Pomeranz Krummel DA, Nagai K, & Oubridge C. Structure of spliceosomal ribonucleoproteins. F1000 Biology Reports, 2: 39 (2010).
van der Feltz C, Anthony K, Brilot A, & Pomeranz Krummel DA. Architecture of the spliceosome. Biochemistry, 51: 3321-3333 (2012).
Pugh TJ, Weeraratne D, Archer TC, Pomeranz Krummel DA, et al. Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations. Nature, 488: 106-110 (2012). (see Press Release and Brandeis blog)
Anthony, K.C., and Pomeranz Krummel, D.A. (2013) Spliceosome, In eLS, John Wiley & Sons, Ltd.
Anthony, K.C., You, C., Piehler, J, and Pomeranz Krummel, D.A. (2014) High-affinity gold nanoparticle pin to label and localize histidine-tagged proteins in macromolecular assemblies. Structure.
Last update: March 11, 2014