Effect of Dicer on Expression of Sense/Antisense Overlapping Transcripts 

Jon Kralik
Marquette University
Milwaukee, WI
Mentor: Dr. Stephen Munroe

The transcriptional complexity of mammalian genomes appears to be significantly greater than originally thought. Investigation into this complexity has exposed many unexpected and fascinating features of the transcriptome’s composition and regulatory mechanisms. Although there are only approximately 22,500 novel protein-coding genes, the total number of transcripts that comprise mammalian transcriptomes is in excess of 100,000. The significant difference between the number of protein coding genes and the number of transcripts that comprise the transcriptome is due to the alternative processing of coding transcripts and the presence of non-coding RNAs, which are thought to play a regulatory role. Another unexpected characteristic of mammalian genomes is their organization into complex loci. Traditionally, genes were thought to be the sole occupant of a distinct region. However, further exploration of the transcriptome is uncovering that many genes share transcribed regions by utilizing opposite strands of the DNA to assume an antisense orientation and form sense/antisense pairs (SAPs) of transcripts to produce complex loci.

Transcripts in the Fantom2 Consortium’s full-length mouse cDNA can be divided into 4 transcriptional classes and 10 corresponding pair classes. Two of the most abundant SAP pair classes comprise a spliced mRNA and a non-coding RNA. These pair classes occur more frequently than expected by random pairing. This observation is consistent with a functional role for non-coding antisense RNAs in gene regulation. To investigate the question of whether or not SAPs form double-stranded RNA and function in gene regulation, a custom antisense microarray was designed to measure the expression levels of 4862 transcripts constituting 2431 SAPs in mouse embryonic stem cell lines. From the 2431 SAPs, 24 SAPs which showed the greatest difference in expression between the wild type and Dicer null ES cell lines were selected for validation and further quantification of expression levels using Real Time PCR. 

The results obtained from Real Time PCR confirm that the selected 24 SAPs are in fact differentially expressed in the two ES cell lines.  There is a general increase in expression in the Dicer null cell line that is seen in controls as well as the selected transcripts. The increase in transcript expression in the Dicer null cells suggests that SAPs regulate gene expression by forming double-stranded RNA to activate Dicer and initiate the RNA interference pathway. However, this will have to be further investigated as such changes may reflect secondary effects caused by the deletion of Dicer which is known to play a role in a variety of pathways, including the biogenesis of microRNAs. 
 
 
 

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