A biochemical challenge

According to recent studies, 95-100% of human pre-mRNAs are subject to regulation by alternative splicing. “The consequences of this type of splicing are still rarely studied, several scientific publications have recently been released and they underline the role of alternative splicing in oncogene regulation. Some oncogenes are indeed more powerful according to the way they are spliced”, indicates the researcher Pierre Close. Gene regulation by alternative splicing has up to the present mainly been studied in relation to cancers and neurodegenerative diseases.

Thanks to a grant from EMBO (European Molecular Biology Organization), Pierre Close joined the laboratory of Dr Jesper Svejstrup at Cancer Research UK, London Research Institute, Clare Hall Laboratories, for a post-doctoral internship “The laboratory of Dr Svejstrup studies the biochemical mechanisms of gene transcription in the wider sense. That was the starting point of the present study. The objective of the project was to establish new connections between newly –synthesized RNA and transcription by RNA polymerase II by purifying complexes associated with nascent RNA”. In order to do this, the researchers rose to a huge technical challenge in biochemical terms: “We purified protein complexes associated with nascent RNA and therefore still connected to RNA polymerase II, at the chromatin level”, explains Pierre Close.

Among the purified complex proteins, the scientists identified one which was particularly interesting because it was linked to the formation of RNA and to transcription. “We named it DBIRD. This protein complex is composed of two proteins: DBC1 (Deleted in Breast Cancer protein 1) and another protein that was hitherto unknown, which we called ZIRD (ZNF-protein interacting with nuclear RNPs and DBC1)”, continues Pierre Close.

DBIRD speeds things up

Once DBIRD was identified and named, the scientists applied themselves to describing the role that this protein complex could play in transcription. “We carried out studies of DNA-microarray (or DNA chips) which allowed us to measure the abundance of each exon in the cells at a given moment”, explains Pierre Close. “We used control cells and deficient cells for the DBIRD complex and we analyzed the abundance of each exon in the two cell- types”, he continues. In this way the researchers obtained a list of about 3000 exons whose abundance varied according to the presence or the absence of DBIRD in the cell. “These exons are therefore spliced differently according to whether DBIRD is functional or not”, specifies Pierre Close.

To understand how DBIRD influences the splicing of these exons, the scientists carried out a bioinformatics study of these sequences in collaboration with a team from the Munich Gene Center (Germany). “We found a quite powerful correlation between the abundance of pairs of nucleic acid bases adenine-thymine (A/T) and the exons which were differently spliced”, the researcher reveals. Thus the sequences rich in A/T sequences would be particularly sensitive to the DBIRD complex because they are more or less included in the final messenger RNA according to the absence or presence of this complex. 

It remains to be understood how DBIRD regulates the inclusion or exclusion of these exons. “In a normal cell, the RNA polymerase II moves along the single strand DNA corresponding to the gene and transcribes it. When this enzyme complex meets an exon rich in A/T this poses a problem because these are sequences that are difficult to transcribe”, explains Pierre Close. “We think that the DBIRD complex somehow helps the RNA polymerase II to transcribe these sequences rich in A/T, by a mechanism we have yet to understand”, continues the post-doctoral researcher. This is what the results of the measurement of the elongation speed of RNA polymerase II showed in the genes containing these exons.  The speed of transcription is in fact reduced when DBIRD is absent. “So the speed of RNA polymerase II directly influences the inclusion or exclusion of exons during the splicing process”, continues Pierre Close.