Multilevel regulation of the glass locus during Drosophila eye development

Background

For an animal to develop and function normally it is important for genes to be expressed in the correct tissues at the right time. This requires multiple levels of regulation that act on the transcription as well as translation of any given gene. Gene expression relies on the presence of regulatory sequences such as enhancers, which promote transcription in a context specific manner. Translation is controlled by interaction between mRNA and regulatory proteins or the ribosome. Both these processes are hugely complex and only partially understood. In this study, Fritsch et al. characterise features relating to the transcriptional and translational control of the glass transcription factor in Drosophila eye development. Of particular interest is the discovery of a small open reading frame (smORF) peptide in the glass locus which regulates the translation of the gene.

 

Key findings

A smORF in the glass locus regulates expression of a GFP reporter.

Placing the sequence of an upstream region of a gene in front of a fluorescent protein is a common way to make a visible reporter for gene expression (i.e. the fluorescent protein is usually observed in the same cells as the endogenous protein would be). Upon investigating the glass locus, Fritsch et al found that a GFP reporter constructed in this manner did not result in visible fluorescence in the expected post-mitotic cells of the developing Drosophila eye. After further examination the authors saw that a smORF was present upstream of, and overlapping with, the start codon of glass. Genetic manipulation of the locus showed that when the smORF was deleted the expected expression patter became visible. Interestingly, when GFP was brought into the same frame as the smORF, GFP was observed in the nucleus, indicating that the smORF encodes a functional nuclear localisation sequence.

The glass locus contains multiple context dependent enhancer sequences.

The authors go on to investigate the expression of their reporter construct and the influence of cis-regulatory sequences in the glass upstream region. By making various deletions the authors were able to dissect the regions of the locus that contain putative enhancers and their activity. The glass locus is shown to be complex, containing individual elements that were responsible for the expression of glass in the various cell types of the eye. The action of multiple enhancers together was required for the proper localisation of the reporter.

Brainy smurf tunes glass translation levels.

Having extensively characterised the function of glass enhancer sequences, the authors turn their attention back to the enigmatic smORF. Examination of other Drosophila species, as well as more distantly related flies, showed that the smORF is well conserved, indicating that it performs a similar role in regulating glass in other flies. Fritsch et al. then set about trying to ascertain the role of the smORF by creating mutations that would interfere with the potential peptides’ activity. These alleles were nicknamed ‘brainy-smurf’ (see Fig 1.).  Despite this strong conservation of the smORF itself, when mutations were made that introduced frameshifts into the smORF no phenotypes were detected, indicating that the peptide itself does not have a significant role in eye development or function.

Overexpression of the glass coding sequence is usually lethal. However, when glass was overexpressed with the upstream smORF present, the phenotype was much less severe, indicating that the presence of the smORF was sufficient to reduce the levels of the functional glass protein. In contrast, expression of the smORF alone, yielded no detectable phenotypes. These results point towards an intriguing evolutionarily conserved mechanism for the upstream smORF that acts to buffer levels of glass translation to ensure that appropriate levels of this important transcription factor are produced during development.

 

Why I like this preprint

This study takes a multi-level approach to broadly characterise various aspects of gene regulation. In doing so, the authors have provided an illuminating insight into how complex the regulation of a single locus can be, from sequence level enhancer function, through to production of a functional protein.

Despite being highly prevalent in metazoan genome, and having been first characterised over a decade ago, the function of most smORFs is still largely unknown. Whilst some have been shown to encode peptides with important biological roles, examples of smORFs with proven functions in translational regulation are uncommon. This preprint highlights a fascinating role for smORFs in the regulation of a developmentally important and evolutionarily conserved gene.

 

Questions for the authors

Do you think that the smORF actually produces a peptide, or could it be that the smORF sequence is somehow recognised by the ribosome without being translated?

Brs mutations demonstrate that any putative peptide encoded by the smORF has no discernible function. However, it seems unusual that the sequence of the peptide would be so well conserved, to the extent that the nls sequence is still functional. Can these observations be reconciled?

Do you have any idea about how the difference in expression of the GFP reporter and glass can be explained when expressed from the same regulatory sequences? Is it likely that the context of the glass sequence itself is required to interact with the peptide / translation machinery in the correct manner?

Do you think there would be a difference in phenotypes if glass mutants were rescued, either with coding sequence alone or Brs alleles?

 

doi: https://doi.org/10.1242/prelights.8668

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