Spliceosomal snRNA Modification
The major spliceosomal snRNAs U1, U2, U4, U5, and U6 are present in eukaryotic cells as small nuclear ribonucleoprotein particles (snRNPs) that are essential for the splicing of the major class of introns. Notably, these snRNAs share common features of post-transcriptional modification. Aside from trimethylation of the 5' guanosine cap (the exception is U6 that possesses a γ-methyl guanosine cap), numerous internal nucleotides are also modified by pseudouridylation, 2'-O-methylation and, in some cases, base methylation.
Importantly, the modified nucleotides in spliceosomal snRNAs are remarkably conserved from species to species, strongly suggesting that they are biologically significant. Yet, detailed investigation of this process has not been possible, due at least in part to the lack of effective assays and experimental systems. Recently, our lab and others developed several highly sensitive assays for detecting modified nucleotides in RNA. Using Xenopus oocytes and cultures of yeast and mammalian cells, we also developed powerful experimental systems for performing extremely efficient modifications in spliceosomal snRNAs. For the first time, these newly developed assays, coupled with the effective experimental systems, provide us with tools to address the fundamental questions regarding spliceosomal snRNA modification.
Using combined approaches of molecular biology and cell biology, we are currently focusing on three important areas of spliceosomal snRNA modification:
Which modified nucleotides are required for spliceosomal snRNA function and how do these modifications contribute to function?
Where do spliceosomal snRNA modifications occur within the cell?
What cellular machinery facilitates the modification of spliceosomal snRNAs, and what biochemical mechanisms are involved?
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