Alternative splicing may not be the key to proteome complexity

Alternative splicing is commonly believed to be a major source of cellular protein diversity. However, although many thousands of alternatively spliced transcripts are routinely detected in RNAseq studies, reliable large-scale mass spectrometry-based proteomics studies identify only a small fraction of annotated alternative isoforms. The clearest finding from proteomics experiments is that most human genes have a single main protein isoform, while those alternative isoforms that are identified tend to be the most biologically plausible: those with the most cross-species conservation and those that do not compromise functional domains. Indeed, most alternative exons do not seem to be under selective pressure, suggesting that a large majority of predicted alternative transcripts may not even be translated into proteins.

One gene, one protein or one gene, many proteins?

Alternative splicing of messenger RNA produces a wide variety of differently spliced RNA transcripts that may be translated into diverse protein products. The presence of alternatively spliced transcripts is unequivocally supported by EST and cDNA sequence evidence [], by microarray [] and RNAseq data [,]. It has been estimated that most multi-exon human genes can undergo alternative splicing [].

The breadth of alternative splicing detectable at the transcript level has lead to claims that alternative protein isoforms could be the key to mammalian complexity []. How much of this alternative splicing is functional at the protein level is a long-standing open question of great importance for understanding eukaryotic biology

Proteomics experiments find little evidence of alternatively spliced proteins

The low number of protein splice isoforms is in stark contrast to the abundance of alternative transcripts in microarray and RNAseq experiments and is especially surprising in light of the fact that the eight large-scale experiments interrogated more than 100 different tissues, cell lines and developmental stages [].

Detected splice events have comparatively subtle effects on the protein

Most alternative exons are not under selective pressure

Alternative splicing is well documented at the transcript level, and microarray and RNAseq experiments routinely detect evidence for many thousands of splice variants. However, large-scale proteomics experiments identify few alternative isoforms. The gap between the numbers of alternative variants detected in large-scale transcriptomics experiments and proteomics analyses is real and is difficult to explain away as a purely technical phenomenon. While alternative splicing clearly does contribute to the cellular proteome, the proteomics evidence indicates that it is not as widespread a phenomenon as suggested by transcript data. In particular the popular view that alternative splicing can somehow compensate for the perceived lack of complexity in the human proteome [,] is manifestly wrong.

What happens to alternative transcripts that are not translated in detectable quantities is not clear. Some may be expressed in small quantities, in limited tissues or under special circumstances, some may be regulated by cellular quality control pathways [,], ensuring that isoforms with damaged domains are not present in the cell, and some may have functions other than generating a protein product []. Resolving the fate of these missing isoforms will be of great importance to help understand the cellular machinery.

dominant cellular isoforms can be predicted for any well annotated genome.

These results show that most alternative exons are evolving neutrally, suggesting that most alternative splice events are not evolutionary innovations. Of course, this also suggests that many alternative transcripts will not be translated into functional proteins.

Gene expression levels, not alternative splicing, seem to be the key to tissue specificity []. While a small number of alternative isoforms are conserved across species, have strong tissue dependence and are translated in detectable quantities, most have variable tissue specificities and appear to be evolving neutrally. This suggests that most annotated alternative variants are unlikely to have a functional cellular role as proteins.

Landscape of ribosome-engaged transcript isoforms reveals extensive neuronal cell class-specific alternative splicing programs

Nervous system function relies on complex assemblies of distinct neuronal cell types that have unique anatomical and functional properties instructed by molecular programs. Alternative splicing is a key mechanism for the expansion of molecular repertoires, and protein splice isoforms shape neuronal cell surface recognition and function. However, the logic of how alternative splicing programs are arrayed across neuronal cells types is poorly understood. We systematically mapped ribosome-associated transcript isoforms in genetically defined neuron types of the mouse forebrain. Our dataset provides an extensive resource of transcript diversity across major neuron classes. We find that neuronal transcript isoform profiles reliably distinguish even closely related classes of pyramidal cells and inhibitory interneurons in the mouse hippocampus and neocortex. These highly specific alternative splicing programs selectively control synaptic proteins and intrinsic neuronal properties. Thus, transcript diversification via alternative splicing is a central mechanism for the functional specification of neuronal cell types and circuits.

Regulation of neuronal differentiation, function, and plasticity by alternative splicing

Post-transcriptional mechanisms provide powerful means to expand the coding power of genomes. In nervous systems, alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatio-temporal control of transcripts. Thus, alternative splicing programs play instructive roles in the development of neuronal cell type-specific properties, growth, self-recognition, synapse specification, and neuronal network function. Here we discuss the most recent genome-wide efforts on mapping RNA codes and RNA binding proteins for neuronal alternative splicing regulation. We illustrate how alternative splicing shapes key steps of neuronal development, maturation, and synaptic properties. Finally, we highlight efforts on dissecting spatio-temporal dynamics of alternative splicing and their potential contribution to neuronal plasticity and the mature nervous system.

Nervous systems have an astounding complexity and exhibit a remarkable level of functional and structural organization. This organization arises during embryonic and early postnatal development. Neuronal precursors give rise to specific neuronal cell types, characterized by their neurotransmitter phenotypes, routes of migration, morphology, as well as physiological properties. The molecular repertoires driving these differentiation steps are spatially and temporally regulated by transcription factors, with terminal selector genes ultimately driving the gene battery unique to each cell type

However, many aspects of the specification of neuronal and synaptic properties remain to be understood and recent work points to an important role for post-transcriptional mechanisms in this process.

RNA Motifs, RNA-Binding Proteins, and Dissection of the “Splice Code”

From Tissue to Cell Type-Specific Splicing Dissection

Neuronal Cell Type-Specific Splicing Regulators

Alternative Splicing-Dependent Neuronal Recognition and Synapse Specification

Control of Neuronal Transcript Dynamics by Neuronal Signaling and mRNA Processing

Over the past decade, there have been breathtaking advances in genome-wide transcriptomics and the systematic identification of RNA-binding proteins, their binding specificities, and RNA targets. These advances have allowed for testing hypotheses about the importance of alternative splicing in specific steps of neuronal development, maturation, and function. Recent work revealed that alternative splicing programs do not simply provide fine-tuning or “tweaking” of fine-grained details of neuronal properties. Much rather, splicing programs have emerged as instructive signals for key steps of neuronal growth, recognition, synapse specification and plasticity. New genetic tools for cell type-specific experimentation will further accelerate the progress towards the fascinating question of how genetic programs and neuronal activity-driven mechanisms synergize to achieve the functional organization of a complex tissue like the nervous system.