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 more than 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.

Coupling transcription with alternative Splicing

Transcriptional elongation and factor recruitment can contribute independently or in concert to the transcriptional control of alternative splicing. We have shown that for approximately 80% of the alternative splicing events that are affected by elongation in human cells, the higher the rate of elongation, the less inclusion of the alternative exon. We deciphered the mechanism by which the opposite effect is observed in the remaining 20% of elongation-sensitive events.

Chromatin and alternative splicing

The chromatin context affects the rate of elongation of Pol II, which in turn modifies alternative splicing decisions. We found that, by modifying post-translational modifications of histones, external signals regulate alternative splicing as a consequence of changes in the chromatin structure. A looser chromatin allows a higher rate of elongation of transcription and, therefore, affects alternative splicing events of various genes. Opposite effects are seen with a more compact chromatin structure.

Chromatin, alternative splicing and spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is a genetic disorder of the motor neurons that is the leading genetic cause of infant mortality and is caused by mutations of the SMN1 gene. In healthy individuals, SMN1 encodes the survival of motor neuron (SMN) protein, which plays a crucial role in the nervous system. Humans have a paralog of SMN1, called SMN2. Due to sequence differences, exon 7 of SMN2 (E7) is poorly included in its mRNA, which means that only 10-20% of SMN2 transcripts encode the full-length protein. Therefore, in the absence of SMN1 expression, SMN2 cannot compensate for the deficiency in the SMN protein. Dr. Adrian Krainer (CSHL, New York) developed a therapy for SMA approved in December 2016. Krainer designed a powerful antisense oligonucleotide (ASO), whose trade name is Spinraza, that promotes the inclusion of E7 in the SMN2 transcript, in cells, animals and patients. Spinraza interacts with a sequence located in the intron that follows E7 (intron 7), which is the binding site of the negative splicing factors hnRNPA1 and A2. When the hnRNPA1 / A2 binding site is blocked by the ASO, the inclusion of E7 in the mRNA increases and higher levels of full-length SMN protein are produced.

Epigenetics refers to changes in DNA methylation and / or post-translational modifications of histones that regulate gene expression patterns without altering DNA sequences. We have found, both in HEK293 cells and in fibroblasts from patients with SMA, that rapid transcriptional elongation, caused by chromatin relaxation due to histone acetylation, promotes the inclusion of SMN2 E7 and that the combined use of deacetylase inhibitors of histones (HDAC) such as trichostatin A (TSA) or valproic acid (VPA) and Spinraza-like ASOs synergistically regulate E7 inclusion. We also observed that the combined administration of Spinraza-like ASO and TSA has strong synergistic effects on the growth and survival of SMA mice.

This work is possible thanks to the support of Familias Atrofia Muscular Argentina (FAME) and CureSMA (EEUU).

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 chloroplast responds to light by generating a signal that acts in the nucleus to regulate the alternative splicing of certain genes. We show that the regulation of alternative splicing in plants involves an unexpected relationship between photosynthesis, which occurs in the chloroplast, and alternative splicing, which occurs in the nucleus of the plant cell, through what is known as retrograde signaling, initiated by the degree of oxidation of plastoquinones of photosynthetic electron transport.

We also verified that the regulation of alternative splicing by light action in plants responds to the coupling kinetic model, originally described in animal cells. We found that, in the presence of light, the rate of elongation of PolII increases, while in the dark the elongation is slower and this, in turn, regulates the alternative splicing of a set of genes. This shows that coupling is important for the response to an environmental stimulus at the level of a whole organism.

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