bi-directional transcription experiment

Question

We suspect a bi-directional transcription event is happening at a locus in our organism where two genes are directly adjacent to each other. The annotation data is not well established. The intergenic distance is probably less than 200 base pairs.

The two genes are expressed in opposite directions towards each other. Base on the preliminary transcriptomics data, it seems like one gene is over transcribing (3' UTR perhaps?) into the adjacent gene, possibly resulting in some kind of transcriptional regulation of the adjacent gene.

Here is a rough diagram of what we think might be happening:

------------------------==========gene A================>----------------------

----------------------------------------<====gene B=====-----------------------

Of course we need to first confirm this by designing primers to see if this over transcription is actually happening.

If this is happening, we intend to do some knock down experiments. We have no transgenesis available in our organism, only RNAi by dsRNA. It is possible to specifically knock down geneA by introducing dsRNA to the 5' region of geneA that does not overlap with geneB. Perhaps this will lead to ectopic/over expression of geneB.

Is there anyway to knock down geneB specifically without knocking down geneA? It looks like designing dsRNA for geneB would knock down both A and B.

Answer 1

How well is gene A annotated? Do you have the gene A sequence (after post-transcriptional modifications)? If you do, you can order it from a company like DNA2.0 and they will synthesize it for you for like $0.35/base. Then you can transform the cells with this plasmid and do a knockout of gene B by inserting some sequence at the ~200bases overlap.

Also, I guess you can transform the immediate transcript (without modifications). It will also be modified once it is in the cell. But it will be longer and more expensive to synthesize, but there are a few methods you can use to do that.

Let me know if this idea appeals to you. I can give you more info if you are interested.

Answer 2

If gene B makes a protein product, you can try designing a morpholino against the 5'-UTR of gene B. This can prevent translation initiation at the ribosome as the morpholino occludes mRNA entry into the ribosome. You can detect this by western blotting.

If gene B makes some sort of regulator RNA, you will need to target expression of gene B at the DNA level. If gene B has well established splice sites, a morpholino can interfere with splicing enhancers and silencers, and therefore, should produce a non-functional or impaired RNA product. You can detect splice variants by amplifying gene B from cDNA.

Theoretically, the advantage of using a morpholino is due to it's single stranded nature. Morpholinos designed against gene B should have no effect on gene A due to non–complemenetarity.