Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

Illuminating Inner Life : A Comparison of Virginia Woolf's To the Lighthouse and Arthur Schnitzler's Fräulein Else

In the early 20th century, authors increasingly experimented with literary techniques striving towards two common aims: to illumine the inner life of their protagonists and to diverge from conventional forms of literary representations of reality. This shared endeavour was sparked by changes in society: industrialisation, developments in psychology, and the gradual decay of empires, such as the Victorian (1837–1901) and the Austro-Hungarian (1867–1918). Those developments yielded a sense of uncertainty and disorientation, which led to a so-called “turn [inwards]” in the arts (Micale 2). In this context, this essay examines Virginia Woolf’s (1882–1941) development of her literary technique by comparing To the Lighthouse (1927), written in free indirect discourse, with Arthur Schnitzler’s (1862–1932) Fräulein Else (1924), written in interior monologue. Instead of applying Freud’s theories of consciousness, I will demonstrate how empiricist psychology informed and partly helped shape the two narrative techniques by referring to Ernst Mach’s (1838–1916) idea of the unstable self, and William James’ (1842–1910) concept of the stream of consciousness. Furthermore, I will show that there is a continuous progression of literary ideas from Schnitzler’s Viennese fin-de-siècle connected to impressionism, towards Woolf’s Bloomsbury aesthetics connected to Paul Cézanne’s post-impressionist logic of sensations. In addition to that, I address how the women’s movement, starting in the end of the 19th century, inspired Woolf and Schnitzler to utilise their techniques as a means of revealing women’s restricted position in society. Methodologically, I will analyse the two novels’ narrative techniques applying close reading and by that point out their differences and similarities in connection to the above-mentioned theories as well as the two author’s literary approaches. I argue that this comparison demonstrates that modernist literary techniques of representing interiority evolved from interior monologue towards free indirect discourse. This progression also implicates that modernism can be seen as a continuum reaching back to the fin-de-siècle and culminating in the 1920s.