In genetics, an isoform is a member of a set of similar products that originate from the same gene; such a set can consist of mRNAs or their resultant proteins. The best-studied isoforms are the protein isoforms, or "protein variants", resulting from differences in the coding DNA sequences (CDS) of their precursor mRNAs. While many perform the same or similar biological roles, some protein isoforms have unique functions. A set of protein isoforms may be formed from alternative splicings, variable promoter/transcription start site (TSS) usage, or other post-transcriptional modifications of a single gene; post-translational modifications are generally not considered. (For that, see Proteoforms.) Through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein. The discovery of isoforms could explain the discrepancy between the small number of protein coding regions of genes revealed by the Human Genome Project and the large diversity of proteins seen in an organism: different proteins encoded by the same gene could increase the diversity of the proteome. Isoforms at the protein level can manifest in the deletion of whole domains or shorter loops, usually located on the surface of the protein. Isoforms at the RNA level are readily characterized by cDNA transcript and RNA-Seq studies. Many human genes possess confirmed alternative splicing isoforms. It has been estimated that ~100,000 expressed sequence tags (ESTs) can be identified in humans. Isoforms harboring changes in the CDS have been the most thoroughly characterized because they commonly give rise to proteins with different functional properties. RNA isoforms can differ not only in the CDS (leading to the well-known differences in resultant proteins), but also in their untranslated regions (UTRs), which regulate the levels of primary transcript in numerous ways: transcript stability, folding and turnover, as well as translation efficiency. UTRs are often the target of miRNA, which typically downregulate transcript expression by triggering degradation or halting translation. As a result, the UTR can determine how much of a transcript is translated. In outdated usage, isoforms is also used as a vague synonym for paralogs, the products of different genes from the same organism that are related by an ancient gene duplication. For example, PRKAA1 and PRKAA2 are paralogs, but many sources simply refer to them as "isoforms".
Definition One single gene has the ability to produce multiple RNAs (and thus proteins) that differ both in structure and composition; this process is regulated by the alternative splicing of mRNA, though it is not clear to what extent such a process affects the diversity of the human proteome, as the abundance of mRNA transcript isoforms does not necessarily correlate with the abundance of protein isoforms. Three-dimensional protein structure comparisons can be used to help determine which, if any, isoforms represent functional protein products, and the structure of most isoforms in the human proteome has been predicted by AlphaFold and publicly released at isoform.io. The specificity of translated isoforms is derived by the protein's structure/function, as well as the cell type and developmental stage during which they are produced. Determining specificity becomes more complicated when a protein has multiple subunits and each subunit has multiple isoforms.
Mechanism The primary mechanisms that produce protein isoforms are alternative splicing and variable promoter usage, though modifications due to genetic changes, such as mutations and polymorphisms are sometimes also considered distinct isoforms.
Alternative splicing
Alternative splicing is the main post-transcriptional modification process that produces mRNA transcript isoforms, and is a major molecular mechanism that may contribute to protein diversity. The spliceosome, a large ribonucleoprotein, is the molecular machine inside the nucleus responsible for RNA cleavage and ligation, removing non-protein coding segments (introns). Because splicing is a process that occurs between transcription and translation, its primary effects have mainly been studied through genomics techniques—for example, microarray analyses and RNA sequencing have been used to identify alternatively spliced transcripts and measure their abundances. Transcript abundance is often used as a proxy for the abundance of protein isoforms, though proteomics experiments using gel electrophoresis and mass spectrometry have demonstrated that the correlation between transcript and protein counts is often low, and that one protein isoform is usually dominant. One 2015 study states that the cause of this discrepancy likely occurs after translation, though the mechanism is essentially unknown. Consequently, although alternative splicing has been implicated as an important link between variation and disease, there is no conclusive evidence that it acts primarily by producing novel protein isoforms. Alternative splicing generally describes a tightly regulated process in which alternative transcripts are intentionally generated by the splicing machinery. However, such transcripts are also produced by splicing errors in a process called "noisy splicing," and are also potentially translated into protein isoforms. Although ~95% of multi-exonic genes are thought to be alternatively spliced, one study on noisy splicing observed that most of the different low-abundance transcripts are noise, and predicts that most alternative transcript and protein isoforms present in a cell are not functionally relevant.
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