Brahma regulates a specific trans-splicing event at themod(mdg4)locus ofDrosophila melanogaster

Feb 15, 2014RNA biology

Brahma controls a specific RNA splicing event at the mod(mdg4) gene in fruit flies

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Abstract

A specific trans-spliced mod(mdg4) mRNA isoform is influenced by the levels of Brahma (BRM) in Drosophila cells and larvae.

The mod(mdg4) locus in Drosophila contains multiple transcription units that undergo alternative splicing and significant trans-splicing.
Anti-sense mod(mdg4) transcripts were characterized in S2 cells, revealing specific transcription start and cleavage sites.
Brahma (BRM) was shown to regulate the abundance of a specific trans-spliced mod(mdg4) mRNA isoform.
This regulation occurs alongside increases in RNA polymerase II density and changes in the phosphorylation state of its C-terminal domain.
The effect of BRM on mod(mdg4) splicing does not rely on its ATPase activity, indicating an alternative mechanism of action.

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The mod(mdg4) locus of Drosophila melanogaster contains several transcription units encoded on both DNA strands. The mod(mdg4) pre-mRNAs are alternatively spliced, and a very significant fraction of the mature mod(mdg4) mRNAs are formed by trans-splicing. We have studied the transcripts derived from one of the anti-sense regions within the mod(mdg4) locus in order to shed light on the expression of this complex locus. We have characterized the expression of anti-sense mod(mdg4) transcripts in S2 cells, mapped their transcription start sites and cleavage sites, identified and quantified alternatively spliced transcripts, and obtained insight into the regulation of the mod(mdg4) trans-splicing. In a previous study, we had shown that the alternative splicing of some mod(mdg4) transcripts was regulated by Brahma (BRM), the ATPase subunit of the SWI/SNF chromatin-remodeling complex. Here we show, using RNA interference and overexpression of recombinant BRM proteins, that the levels of BRM affect specifically the abundance of a trans-spliced mod(mdg4) mRNA isoform in both S2 cells and larvae. This specific effect on trans-splicing is accompanied by a local increase in the density of RNA polymerase II and by a change in the phosphorylation state of the C-terminal domain of the large subunit of RNA polymerase II. Interestingly, the regulation of the mod(mdg4) splicing by BRM is independent of the ATPase activity of BRM, which suggests that the mechanism by which BRM modulates trans-splicing is independent of its chromatin-remodeling activity.

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Brahma regulates a specific trans-splicing event at themod(mdg4)locus ofDrosophila melanogaster

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