What Are Microproteins and Why Are They So Important?

A class of tiny molecules known as microproteins is rewriting our understanding of the genome. For decades, these small proteins, composed of fewer than 100 amino acids, went largely unnoticed and were often dismissed as insignificant. Recent advancements in technology have brought these miniature molecules into the spotlight, revealing their involvement in a wide range of biological processes. Their discovery is compelling scientists to reconsider the definition of a gene and explore a previously hidden layer of biological regulation.

Defining Microproteins and Their Discovery

Microproteins are formally defined as proteins translated from small open reading frames (sORFs) and are composed of fewer than 100 amino acids. This small size is the primary reason they remained undiscovered for so long. For many years, methods used to identify genes and proteins were designed with a size filter, often set at a minimum of 100 amino acids, to avoid false positives. This arbitrary cutoff meant that the vast majority of microproteins were simply filtered out of genomic analyses. The turning point in their discovery came with new technologies that allowed for a more nuanced view of the genome, providing the evidence needed to establish microproteins as a legitimate class of biological molecules.

Genetic Origins of Microproteins

The genetic blueprints for microproteins are found in small open reading frames, or sORFs, which are short sequences of DNA that begin with a start codon and end with a stop codon. A surprising number of these sORFs are located in regions of the genome that were previously thought to be non-coding. This includes areas such as the untranslated regions (UTRs) of messenger RNAs (mRNAs) and within what were once classified as long non-coding RNAs (lncRNAs).

The existence of microprotein-encoding sORFs within these “non-coding” regions challenges the traditional view of a gene. It suggests that a single RNA molecule can have multiple functions, producing both a large protein and one or more smaller microproteins. The process of translating these microproteins is similar to that of larger proteins, involving the ribosome, but some microproteins are initiated from non-standard start codons, adding another layer of complexity.

Cellular Functions of Microproteins

Within the cell, microproteins perform a diverse array of functions. Many act as regulatory molecules, fine-tuning the activity of larger protein complexes by binding to them and either enhancing or inhibiting their function. This mode of action allows microproteins to have a significant impact on cellular processes despite their small size.

Well-characterized microproteins illustrate the breadth of their functional roles:

• Myoregulin is a microprotein that plays a part in muscle performance by regulating calcium uptake in muscle cells.
• Dwarf open reading frame (DWORF) microprotein stimulates the activity of a calcium pump in the heart.
• NoBody has been shown to be involved in the process of mRNA decay, a fundamental aspect of gene regulation.
• PIGBOS microprotein is located in the mitochondrial outer membrane and is involved in the protein quality control of the mitochondria.

These examples show they are a distinct class of molecules that can act as signaling molecules, structural components, or modulators of enzyme activity.

Microprotein Roles in Health and Disease

Because they are involved in a wide range of cellular processes, the dysregulation of microproteins has been linked to a variety of pathological conditions. Aberrant expression of specific microproteins has been observed in cancer, cardiovascular disease, and metabolic disorders.

In the context of cancer, some microproteins have been found to act as either tumor promoters or suppressors, making them potential targets for new cancer therapies. In cardiovascular disease, microproteins that regulate calcium handling in the heart, such as DWORF, are being investigated as potential therapeutic targets for heart failure. Mitochondria-derived peptides, a class of microproteins encoded by the mitochondrial genome, have shown protective effects in models of cardiovascular disease by improving mitochondrial function.

Furthermore, disruptions in microprotein function have been implicated in metabolic diseases like type 2 diabetes, where the microprotein mitoregulin has been shown to regulate lipid metabolism. These findings highlight the potential of microproteins as both biomarkers for disease diagnosis and as novel targets for therapeutic intervention.

Techniques for Studying Microproteins

The study of microproteins presents unique challenges due to their small size and often low abundance, requiring specialized techniques. Computational and bioinformatic tools are often the first step, used to scan genomes for potential sORFs that could encode microproteins, but these predictions must be experimentally validated.

One of the most powerful techniques for identifying translated sORFs is ribosome profiling, or Ribo-seq. This method allows researchers to sequence the small fragments of mRNA that are protected by ribosomes, providing a direct snapshot of which parts of the genome are being actively translated into protein.

Once a potential microprotein has been identified, advanced mass spectrometry techniques are used to confirm its existence and determine its sequence. These methods have been optimized to detect the small peptides that are characteristic of microproteins.