The role of HRB87F inmRNA export and telomere biogenesis

This dissertation examines the role of the Drosophila hnRNP Al homologue, HRB87F, in mRNA export and telomere metabolism. It had been proposed that members of this hnRNP family play a role in mRNA export based on their ability to bind generally to mRNA and their ability to shuttle between the nucleus and cytoplasm via the M9 transport signal. To investigate this possibility, inducible transgenic stocks were created that express HRB87F with various mutations in the M9 signal. The wildtype and mutant proteins were localized in cells and on chromosomes, were tested for overexpression effects on fly viability, and were tested for the ability to rescue an mRNA export defect in flies with a mutation in the Drosophila orthologue of TAP/NXF1, small bristles. Excess HRB87F slightly enhanced the small bristles export defect, while overexpression of a mutant HRB87F with a manufactured NLS and NES suppressed this defect. The combined results indicate that HRB87F more likely functions in nuclear mRNA retention than export. Results also show that continuous overexpression of HRB87F is lethal to flies, perhaps due to nuclear retention of mRNA. Surprisingly, although the M9 signal was thought to be necessary for nuclear localization of the protein, results suggest that this requirement may be bypassed during heat shock.; In the course these experiments, it was noted that the HRB87F protein has a strong preference for binding to telomeres, and especially the 2L telomere where it induces a unusual puffed configuration in this heterochromatic region that we termed “flaring.” Using strains with telomere deletions, the sequence required for flaring was found to involve the subtelomeric TAS element rather the terminal retrotransposons. Flaring involves increased transcription at the telomere and a telomere reporter gene was activated by HRB87F overexpression, showing that HRB can modulate telomere position effect. Localization of various proteins and histone modifications by immunofluorescence suggest that flaring represents an initial relaxation of chromatin structure at the telomere, which is followed by re-establishment of the heterochromatic state. These results show for the first time a potential role for hnRNP proteins in modulating telomeric chromatin structure.