The brain is a plastic organ that undergoes frequent structural and functional modifications in response to input from the environment. Neuronal activity produces changes in the expression of proteins at synapses and in the connections between neurons. This plasticity relies on the transcription of different genes, which involves the production of messenger (mRNA) from DNA. The level of specific mRNA transcripts can be measured to determine when a gene is turned on or off.
In response to neuronal activity, two waves of mRNA are produced: immediate early genes, and activity-regulated transcripts. The first set of transcripts are made in approximately 30-60 minutes in response to neuronal activity. Some of these mRNAs encode transcription factors that activate another transcription cascade to produce proteins involved in growth, cell signaling, and synaptic function. Neuronal activity can also modify transcription through alternative splicing, whereby a single gene can encode multiple proteins. Each gene contains multiple introns and exons. Introns are excised before the final RNA sequence is translated to protein. Different combinations of exons can remain following intron removal, and each combination will produce a different protein.
Previous studies have shown that neuronal cells express a large number of long genes (>100 kilobases) that encode proteins important for synaptic function. Since genes of this length would require several hours to transcribe, this raised the question of how plastic changes occur so rapidly. This is the question Oriane Mauger and colleagues sought to answer in their recent study, “Targeted Intron Retention and Excision for Rapid Gene Regulation in Response to Neuronal Activity,” published in the journal Neuron.
The study, led by Peter Scheiffele from the Institut Pasteur in Paris, investigated whether alternative splicing is involved in rapid neuronal plasticity. Alternative splicing takes only seconds to a few minutes, and occurs during transcription. One type of alternative splicing is intron retention, where introns are retained in fully transcribed, mature transcripts. These transcripts are identified by the presence of a stretch of adenine bases, called the polyA+ tail, at the end of the mRNA sequence.
To investigate the role of intron retention in plasticity, the researchers first isolated and sequenced mature polyadenylated RNA sequences from brain cortex samples. The samples were isolated from mice on postnatal day 10 (juvenile) and day 50 (adult), and from 16.5-day-old mouse embryos that had been cultured for 2 weeks. They calculated the number of transcripts that were spliced and the number that retained introns. The intron-retaining (IR) transcripts made up 5-6% of all of the isolated transcripts.
They then treated cells with transcription inhibitors and found that the majority (84%) of intron retention events persisted. They theorized that these retained introns might be excised in response to neuronal activity in order to rapidly modify gene expression. They tested this hypothesis by treating cells with bicuculline, a drug that increases neuronal network activity. Some introns showed increased retention, while others were excised in response to neuronal stimulation. These results were validated for several specific transcripts, showing that the excision of Clk1, Fnbp11, and Tia1 occurred rapidly in response to bicuculline. Using drug treatments, the researchers were also able to show that activity-dependent intron excision is triggered by calcium signaling downstream of synaptic glutamate receptors, called NMDA receptors.
A gene ontology analysis was performed to see what categories of genes were modified by activity-dependent intron excision. In instances where an intron was excised in response to neuronal activity, these genes were shown to be involved in cell signaling and cellular architecture, including microtubule proteins, actin cytoskeleton proteins, phosphoisositide 3-kinase, protein kinase C, and Rho.
Cell fractionation experiments were performed to determine where in the cell the intron-containing transcripts were located. These experiments showed the presence of these transcripts in the cell nucleus. Neuronal stimulation caused an increase in the number of spliced transcripts in the cytosol, suggesting that they were exported. These cytosolic transcripts were found to be associated with ribosomes, indicating that they were being translated, or turned into protein.
Altogether, these experiments showed that a pool of mature DNA transcripts retain their introns. These introns are rapidly excised in response to neuronal activity, and the spliced RNA is transported to the cytoplasm for translation to protein. This mechanism allows new proteins to be rapidly generated in response to neuronal activity, modifying cellular architecture and supporting plasticity.
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Mauger, O., Lemoine, F., and Scheiffele, P. (2016) Targeted Intron Retention and Excision for Rapid Gene Regulation in Response to Neuronal Activity. Neuron 92(6), 1266–1278. doi:10.1016/j.neuron.2016.11.032