DFBMC + IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales
Universidad de Buenos Aires, Argentina.

Regulation of alternative mRNA splicing

Regulation of Alternative Splicing

Alternative Splicing

Alternative splicing is a major contributor to protein diversity. Recent findings justify a renewed interest in alternative splicing. Estimated to affect nearly 90 percent of human genes, alternative splicing is more the rule than the exception, and mutations that affect alternative splicing–regulatory sequences are a widespread source of human disease. Indeed, many genetic disorders and cancers are caused by mutations that alter the function of alternative splicing–regulatory sequences. In addition, alternative splicing is particularly important in the development of the nervous system. Alternative splicing regulation not only depends on the interaction of splicing factors with their pre-mRNA target sequences but, like other pre-mRNA processing reactions, is coupled to RNA pol II transcription.

Research in the Kornblihtt lab focuses on the regulation of alternative pre-mRNA splicing, with particular emphasis on the mechanisms that couple the splicing and transcription machineries. The group studies how changes in the rate of transcriptional elongation and recruitment of splicing factors to the transcribing polymerase affect alternative splicing and contribute to the generation of multiple protein variants from a single gene.

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Coupling transcription with alternative Splicing

Elongation and factor recruitment may contribute independently or in a concerted way to the transcriptional control of alternative splicing. We are currently investigating different mechanisms that control alternative splicing in mammalian cells and plants.

Chromatin I

The chromatin context affects Pol II elongation rates and, in turn, alternative splicing. We discovered the mechanism by which membrane depolarization in nerve cells affects alternative splicing of the NCAM pre-mRNA by promoting intragenic histone acetylation that loosens the chromatin and elicits higher transcriptional elongation.

Chromatin II

We found that small interfering RNAs (siRNAs) targeted at the intron located downstream of an alternative exon affect alternative splicing through a mechanism known as transcriptional gene silencing (TGS). The intronic siRNAs trigger heterochromatinization on DNA target sequences by causing histone H3 Lys9 dimethylation and subsequent inhibition of transcriptional elongation which ijn turns affects alternative splicing. The effects of intronic siRNAs on alternative splicing are not related to conventional post-transcriptional gene silencing (PTGS). We found that the argonaute preotein AGO1 is necessary to observe these chromatin effects of siRNAs. We are currently performing genome wide studies aimed to identify AGO1 targets in the human genome acting in the control of alternative splicing by small RNAs.

Order of intron removal

We studied the relative order of removal of the introns flanking a model alternative cassette exon and found that there is a preferential removal of the intron downstream of the cassette exon before the upstream intron has been removed. Cis mutations and trans-acting factors that enhance exon inclusion by different pathways change the pattern of intron removal in ways that are consistent with their mechanism of action. However, reduction of transcriptional elongation, also causing higher inclusion of the cassette exon, does not change the order of intron removal. We propose that instead of promoting excision of the upstream intron, slow Pol II elongation favors commitment to splicing of this intron before the downstream intron is synthesized. To verify this hypothesis we are assessing the recruitment of the splicing factor U2AF to the 3´ splice site of the upstream intron under different conditions.

Night and day in plants

We use the model plant Arabidopsis thaliana to investigate the mechanism by which light/dark conditions affect alternative splicing. We found that the chlorpolast is the light sensing organelle involved in the generation of a retrograde signal acting in the nucleus in the regulation of alternative splicing of selected genes. Both H2O2 and sucrose mimic the effects of light on alternative splicing, which favors a role for sugar signaling but does not completely rule out the involvement of reactive oxygen species generated by the chloroplas. To get deeper insights into the nature of the signaling we are currently using Arabidopsis mutants and drugs that affect the redox-state of the plastoquinone pool. In a collaborative project with Marcelo Yanovsky’s group at the Leloir Institute of Buenos Aires, we have found that an enzyme that methylates splicesosomal proteins is essential for the proper functioning of the circadian rythms in Arabidopsis and Drosophila. This enzyme seems to control the alternative splicing of pre-mRNAs ofgenes involved in the controls of the cellular clock.

Techniques currently used in the lab

- Basic molecular biology (cloning, labeling, hyrbidizing, agarose gel and polyacrylamide electrophoresis, PCR, RT-PCR, Real-time PCR) / - Mammailian cell culture / - Cell transfections with DNA and RNA / - RNA interference / - Chromatin immunoprecipitation (ChIP) / - MspI chromatin accessibility assay / - ChIP-seq / - Western blotting / - Immunofluorescence / - RNase protection assay (RPA) / - Run-on analysis / - Gene expression using alpha amanitin resistant mutant RNA polymerases / - Arabidopsis basic manipulations