Abstract:
Eukaryotic protein coding gene precursor-mRNA transcripts are processed or spliced to remove the non-coding sequences (introns) and connect or splice together the coding sequences (exons) for translation. The splicing process is catalyzed by a complex and dynamic macromolecular assembly called the spliceosome. Integral to the spliceosome are five uridine-rich small nuclear ribonucleoprotein particles, the U1, U2, U4, U5, and U6 snRNPs. The three-dimensional structures for most of the U snRNPs remain unclear. Recently, a low-resolution (~25 angstrom) electron microscopy negative-stain reconstruction of the S. cerevisiae 18-subunit U1 snRNP (~800kDa) as well as an improved resolution (~16 angstroms) electron microscopy reconstruction of the particle in vitreous ice (cryo) were obtained. However, at such resolutions interpretation of the experimental density map and thus inference into the function of individual subunits is a formidable challenge. Herein, I present strategies designed to aid model building into these low- resolution electron microscopy reconstructions. Specifically, I report on progress made to both label the single RNA subunit of the budding yeast U1 snRNP (U1 snRNA) and a protein subunit
with a monodisperse gold nanoparticle.
