Molecular analysis of wheat snRNAs

Pre-mRNA splicing is a complex event in which introns are removed and exons spliced together. This process is mediated by small ribonucleoprotein particles (snRNPs), formed from small nuclear RNAs (snRNAs) and their associated nuclear proteins.To determine if the snRNAs within the snRNP particles might be responsible for the observed splicing deficiencies, the snRNA populations of selected monocots and dicots have been examined. The individual U1, U2, U4, U5 and U6 snRNAs involved in splicing have been identified on Northern blots. Plant nuclei contain a large, heterogeneous population of U1, U2, U4 and U5 snRNAs. U6 snRNA is expressed as a single, abundant species.To clarify the differences between the spliceosomal snRNAs expressed in pea (dicot) and wheat (monocot) systems, the U1 and U2 snRNA variants found in wheat have been sequenced and compared with the pea U1 and U2 snRNAs. Sequence differences were found to occur in regions not involved in direct snRNA:pre-mRNA interactions but rather in stem and loop structures important for binding U1 and U2 snRNA-specific proteins. Variations in these regions suggest that monocot and dicot U1 and U2 snRNPs may have different protein variants associated with them and that these differences may mediate the splicing differences observed between plant species. RNase protection assays have demonstrated that all of the U1 and U2 snRNA variants characterized as part of this thesis are expressed at some level at each stage of development.To further characterize protein components associated with snRNAs, methods have been developed for the partial purification of snRNPs from pea, tobacco and wheat nuclei. Using RNase protection and a monoclonal antibody directed against the mammalian U2B$\prime\prime$ protein has demonstrated that wheat U2 snRNPs purified by these protocols are intact and still associated with this U2-specific protein.To determine whether sequence differences in the wheat U2.1 snRNA variant affect the binding of snRNP proteins in a heterologous dicot species, this variant was expressed in tobacco using an Agrobacterium/geminivirus vector system and heterologous dicot U2 snRNA promoter and terminator sequences. Wheat U2.1 snRNA variant is accurately transcribed and processed in tobacco nuclei. The presence of the characteristic trimethylguanosine cap of snRNAs indicates that snRNA variants can be expressed and processed in plant cells using heterologous 5$\prime$ and 3$\prime$ flanking sequences. (Abstract shortened by UMI.)U of I OnlyETDs are only available to UIUC Users without author permissio