Identifying the RNA targets of DBHS proteins to give insights into their role in gene regulation and paraspeckle formation

Ellen Fortini

    Research output: ThesisDoctoral Thesis

    350 Downloads (Pure)


    [Truncated] RNA binding proteins are important in many aspects of cellular function. In particular, RNA binding proteins regulate gene expression at multiple levels, a process that is de-­regulated in many diseases. One family of RNA binding proteins, called DBHS (Drosophila behaviour and human splicing) proteins are of interest as they regulate gene expression at the transcriptional and post-­transcriptional levels. In addition, the three members of the DBHS protein family in mammals, Non-­POU domain containing octamer binding protein (NONO), splicing factor proline/glutamine-­rich protein (SFPQ) and paraspeckle protein component 1 (PSPC1), along with the long non-­coding RNA (lncRNA) NEAT1 (Nuclear Paraspeckle Assembly Transcript 1) are involved in the formation of sub-­nuclear structures called paraspeckles.

    Paraspeckles are RNA:Protein complexes located in mammalian cell nuclei, and are unusual in that their formation and maintenance relies on the specific interaction between the paraspeckle proteins and NEAT1. Paraspeckles are thought to play a role in the regulation of gene expression via the sub-­nuclear sequestration of specific proteins, including the DBHS proteins, to attenuate their function. In addition, paraspeckles are involved in binding and retaining specific RNAs in the nucleus as a means of post-­transcriptional regulation.

    At present there is a lack of knowledge about the molecular interactions occurring within paraspeckles between the RNA binding proteins and the RNAs they interact with. These interactions are the key to both the formation and function of paraspeckles, as well as the component proteins and RNAs.

    In this project I optimized and applied PAR-­CLIP (Photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation) to isolate RNAs bound by the DBHS proteins NONO and SFPQ. The isolated RNA was sequenced and a bioinformatics analysis pipeline implemented with the program PARalyzer to identify, to single nucleotide resolution, the NONO and SFPQ binding sites in RNA.

    Original languageEnglish
    QualificationDoctor of Philosophy
    Publication statusUnpublished - Feb 2014


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