Title

Novel response to double stranded RNAs in mammalian cells

Date of Completion

January 2009

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

The Alu elements are conserved ∼300 nucleotide long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to hyper-editing by the ADAR enzymes, and at least 333 human genes contain such repeats in their 3'UTRs. In this thesis, I have shown that a pair of inverted Alus placed within the 3'-UTR of egfp reporter mRNA strongly represses EGFP expression, whereas a single Alu has little or no effect. Importantly, the observed silencing correlates with adenosine to inosine RNA editing, nuclear retention of the mRNA and its association with the protein p54nrb. Further, inverted Alu elements can act in a similar fashion in their natural chromosomal context to silence the adjoining gene. For example, the Nicolin 1 gene expresses multiple mRNA isoforms differing in the 3'-UTR. One isoform that contains the inverted repeat is retained in the nucleus, while another lacking these sequences is exported to the cytoplasm. Taken together, these results reveal a novel role for Alu elements in human gene regulation. ^ This Alu element mediated gene regulation exists in most cells, however, it is not operational in human embryonic stem cells (hESCs). In hESCs, the editing activity is robust and proteins involved in this pathway are all expressed, but the retention associated nuclear structures, called paraspeckles, are absent and only appear as cells differentiate. Paraspeckle assembly and function depends on the expression of a long nuclear-retained noncoding RNA, hNEAT1. This RNA is not expressed in hESCs, but is induced upon differentiation. Knockdown of hNEAT1 in HeLa cells results in both the loss of paraspeckles and the enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats in their 3'-UTRs. Taken together, these results assign a novel biological function to a large noncoding nuclear RNA and also demonstrate that hESCs allow the nucleocytoplasmic export of structured mRNAs that are retained in the nuclei of differentiated cells. ^