Dimerization and RNA binding of paraspeckle proteins

[Truncated] Gene expression is regulated by numerous nucleic-acid-binding proteins. An important group of these proteins are members of the Drosophila behaviour and human splicing (DBHS) family. DBHS proteins are involved in gene regulation by binding to various DNA, RNA and protein partners, modulation of the cellular transcriptome and posttranscriptional processing of RNA. There are three mammalian DBHS proteins: paraspeckle protein component 1 (PSPC1), non-POU domain-containing octamerbinding protein (NONO) and the splicing factor proline/glutamine-rich protein (SFPQ). All three proteins share a characteristic domain ensemble, which is highly conserved and crucial for homo- and heterodimerization as well as nucleic acid binding.

Malfunction of DBHS protein-mediated regulation leads to various diseases: altered DBHS expression is linked to misbalanced transcription of genes involved in various types of cancer and neuronal disorders. As such, DBHS proteins may be useful therapeutic targets, but structural information is missing - a prerequisite for rational drug design.

This project aimed to solve DBHS protein structures in order to identify mechanisms of DBHS protein dimer formation and RNA binding capacity, which is imperative to understand DBHS function.

The first part of this study focuses on the structure of two DBHS protein dimers: the heterodimer of PSPC1/NONO and homodimer of PSPC1, which were solved by Xray crystallography. The PSPC1/NONO heterodimer structure is analyzed in detail, showing a unique arrangement of four RNA binding domains (RRMs), a novel proteinprotein interaction domain (NONA/paraspeckle domains (NOPS)) as well as a rare type of coiled-coil formation. Dimer formation and its influence on localization of the protein to subnuclear bodies is also addressed.

A second heterodimer construct is shown to have a strong pseudo-symmetric arrangement, which presented difficulties for structure solution and influenced the design of our expression constructs. This is presented as part of a case study in overcoming such problems in crystallography.

Finally, differences between the PSPC1 homodimer and PSPC1/NONO heterodimer structure are discussed, such as alternative conformations of conserved residues important for DBHS protein dimer formation.