Mechanisms and biological functions of alternative splicing-induced first exon lengthening in developing neurons

Nerve cells, or neurons, are essential building blocks of the brain. They are responsible for acquiring, storing, processing, and transmitting information. Neurons develop from stem and progenitor cells through meticulously orchestrated changes in gene expression.

Gene expression typically involves (1) creating RNA copies of gene sequences in the cell nucleus, (2) modifying the RNAs using various nuclear factors, and (3) dispatching fully modified RNAs to the cytoplasm, where they serve as 'messengers' for protein synthesis. The second step of this process comprises several distinct reactions. One of them adds a chemical group called the 'cap' to the beginning of the RNA copy, while another appends the RNA end with a molecular 'tail.' In addition, there is a reaction known as splicing, which removes one or several internal portions of the original RNA sequence and joins the remaining segments together.

Previous studies have shown that the same RNA copy can be spliced in more than one way. The developing neurons often utilize such alternative splicing to regulate their gene expression. One particular type of alternative splicing affects the initial, cap-modified segments of RNA sequences. How they are regulated and what they do in neurons remains poorly understood.

Our work leading to this proposal revealed that cap-adjacent alternative splicing extends the initial sequence segments in hundreds of RNA messengers expressed in neurons. We found that some of these events are controlled by SRRT, a cap-associated protein abundant in stem cells but downregulated in neurons.

Here, we propose to test the hypothesis that this reduction in SRRT abundance helps neurons alter cap-adjacent splicing patterns and extend the initial sequence segments in many RNA messengers. We will examine whether such extended sequences determine the localization of RNA messengers inside the neuron, and control their engagement with the protein synthesis machinery.

We will develop our research program by pursuing the three objectives. Firstly, we will investigate the extent of cap-adjacent alternative splicing in the developing neurons, using both traditional and novel RNA sequencing approaches. Secondly, we will employ genetic, biochemical and computational techniques to dissect the mechanisms utilized by SRRT and other factors to control such splicing events.

Finally, we will discover the biological functions of the extended messenger RNA sequences. This will be achieved by determining if they enable specific RNA localization within the neuron, or modify their messenger function. We will also probe the functional outcomes of perturbing the natural splicing regulation by genetic engineering.

It is worth noting that, although these objectives are clearly related, each of them can be achieved independently.

The significance of our work is highlighted by the frequent deregulation of alternative splicing in the context of human diseases and aging. Defects in cap-adjacent RNA sequences have been linked to devastating neurological disorders, including Huntington's Disease, Fragile X Syndrome, Amyotrophic Lateral Sclerosis, and Frontotemporal Dementia. In our preliminary work, we have identified specific targets of cap-adjacent alternative splicing, including an Alzheimer's Disease biomarker and a gene mutated in Tuberous Sclerosis patients, who often develop epilepsy, autism spectrum disorders, and learning disabilities.

We are confident that the proposed study will uncover new fundamental mechanisms underlying normal brain development and function. This, in turn, will contribute to ongoing efforts to improve the diagnosis and treatment of medical conditions that cause significant suffering to patients and impose a substantial economic burden on society.