PARQUE DE BARRIO EXT DIA

I identified a tissue where Symplekin accumulates at the septate junction, together with CPSF73 and YPS, but not the general polyadenylation factor CstF64. This has the hallmarks of a cytoplasmic polyadenylation complex. The genetic interaction between Symplekin and YPS suggests that detection of Symplekin at the junction is a consequence of mRNA trafficking. YPS has been implicated in translational repression of Osk mRNA and is part of the mRNP that traffics the mRNA along microtubules (Mansfield et al., 2002; Wilhelm et al., 2000). Preventing translation until the mRNA reaches its destination ensures localized expression. This is mediated through extension of the poly(A) tail through

cytoplasmic polyadenylation, which can modulate translation.

The absence of YPS results in diffuse, but detectable Symplekin staining in the cytoplasm of stage 10B follicle cells. This extensive accumulation outside of the nucleus contrasts with all other Drosophila cells characterized thus far, where Symplekin staining appears exclusively nuclear. That is not to say that cytoplasmic polyadenylation, or participation of Symplekin in that processes, does not occur, but if it is spread throughout the cytoplasm, it is likely below our threshold of detection. Our ovarian sample preparation conditions do not result in Symplekin penetrance into nurse cells or the developing oocyte, where we might detect additional cells with cytoplasmic or strongly positioned enrichment of Symplekin protein.

If Symplekin and YPS localization to the septate junction indicates localized

regulation of mRNA translation, there is likely a highly abundant transcript(s) localized to this region. Stage 12-14 ovarian follicle cells produce the chorion proteins for the egg shell and during stage 10B of oogenesis, these cells produce Yolk and Vitellin, so these transcripts could be localized to the cell periphery. Microarray analysis of follicle cells during the late stages of oogenesis has provided a complete identification of follicle cell specific mRNAs, which are candidates for localized mRNAs (Tootle et al., 2011). Performing in situ

122

hybridization to a candidate list of mRNA might identified a localized transcript(s). Since YPS was implicated in translational repression preceding Osk protein synthesis, it is

possible that this transcript(s) might be stored at the septate junction and then not translated until a later stage of development.

YPS was identified through a BLAST search for homologues to ZONAB. This transcription factor binds to the tight junction protein ZO-1 (Balda and Matter, 2000). A recent study showed that ZO-1 mRNA is regulated by cytoplasmic polyadenylation. ZO-1 requires CPEB (Orb in Drosophila) binding sites in the 3’ UTR for localized translation and consequently tight junction integrity and polarization of the cells (Nagaoka et al., 2012). It is therefore possible that a structural protein is locally translated at in this cell type.

Identification of a localized mRNA will confirm our hypothesis that Symplekin localizes to this region as part of the cytoplasmic polyadenylation machinery. This hypothesis can now also be tested in cells where Symplekin localization to the tight junction has been observed.

Overall, our characterization of Symplekin during development indicates that it is an essential gene with multiple in vivo roles. Our results also imply that the role of Symplekin at the tight junction is possibly related to RNA biology. Expression of Symplekin mutant

proteins and characterization of developmental, mRNA and localization phenotypes will help dissect its various roles in gene expression. This work provides a foundation for such

studies.

ACKNOWLEDGMENTS

I thank James Wilhelm for kindly sending the YPS antibody and mutant flies. From the Marzluff lab, I thank Xiao Yang for the GST-CstF-64 and GST protein, Mindy Steiniger for the CPSF-30 depleted RNA sample and Brandon Burch for H2a total and H2a readthrough primers.

123

CHAPTER 5: OVERALL CONCLUSIONS AND SIGNIFICANCE

Every eukaryotic cell division requires synthesis of new histone protein to package the newly replicated genome. The unique 3’ end of metazoan histone mRNA mediates precise

expression of this special class of genes. How the cell coordinates biosynthesis of these distinct transcripts for all five classes of histone proteins is still unknown. In this thesis, I approached this question from two directions. One goal was to determine the contribution of the HLB to histone mRNA expression. My other aim was to understand how general 3’ end formation factors assemble into the distinct histone pre-mRNA processing machinery. Using Drosophila as a model system, I tested the contribution of the histone locus in HLB

assembly and expression (Chapter 2), asked if localizing a histone pre-mRNA processing factor to the HLB influenced 3’ end formation (Chapter 3) and established a system to test Symplekin function in histone pre-mRNA processing (Chapter 4). From these studies, I demonstrate a role for the histone locus in HLB maturation and consequently show that formation of the nuclear body is tied to histone mRNA expression. I also show that raising the local concentration of FLASH at the site of histone mRNA synthesis increases the rate of histone pre-mRNA processing. These observations, and my characterization of Symplekin during development, advance our understanding of how the cell coordinates gene

124