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Multilevel regulation of the glass locus during Drosophila eye development

Cornelia Fritsch, F. Javier Bernardo-Garcia, Tim Humberg, Sara Miellet, Silvia Almeida, Armin Huber, Simon Sprecher

Posted on: 19 February 2019

Preprint posted on 31 January 2019

Article now published in PLOS Genetics at http://dx.doi.org/10.1371/journal.pgen.1008269

Through the looking-glass: small open reading frames regulate the translation of the glass gene in eye development.

Selected by Gabriel Aughey

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.

Figure 1:
A) Blue and Bespectacled – Brainy smurf (Brs) I.e. a smu(o)rf with glass(es)!
B) Eye phenotypes resulting from expression of glass (E+F), Brs (G), and glass with upstream Brs smORF (H). [Figure 3E-H in preprint].

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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Author's response

Cornelia Fritsch & Simon Sprecher shared

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?

The results of our GFP-reporter construct showing increased GFP expression after removal of the smORF and of our UAS-constructs where the smORF interferes with overexpression of Glass indicate that in those constructs translation efficiently starts from the upstream AUG resulting in the production of the small peptide. This is also supported by the bona fide Kozak sequence at the upstream AUG, the nuclear localization of the smORF-GFP fusion protein and the conservation of the peptide sequence in higher Diptera. As a consequence, the translation of Glass should be strongly inhibited by the translation of the smORF. However, under wildtype conditions, Glass levels are sufficiently high to ensure normal eye development, while our CRISPR mutant that puts the Glass coding sequence in frame with the upstream AUG, did not show any observable defects in eye development or function suggesting that there may be other mechanisms tightly regulating Glass protein levels. It may also be that the defects are too subtle to be seen with the assays that are currently available.

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?

Given the high sequence conservation we also expected to see an eye-phenotype. Therefore, after introducing frameshifts in the peptide sequence by CRISPR, we crossed the G0 flies to deficiency lines that take out the glass locus, and screened for eye phenotypes in the F1 generation. We found F1 flies with very mild rough eye phenotypes not only over the deficiency chromosome, but also with the TM6b balancer. We used mainly those flies for further crosses to establish stocks and screening the glass locus for the introduced single nucleotide deletion as well as for other sequence alterations. However, it turned out that not all F1 flies that had irregular eyes, carried a mutation in the smORF sequence and even those that had such a mutation did not show a rough eye phenotype under homozygous conditions. When we cross these flies back over the glass deficiency, we sometimes see flies with slightly rough eyes, but most offspring have normal eyes. So, if the Brs peptide has a function in eye development, it might only become evident if eye development is already compromised.

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?

There are no stop-codons in the third reading frame of the GFP sequence we used in our reporter construct. Therefore, translation from the upstream AUG results in the production of a 36.4 kDa protein that is actually bigger than GFP itself (26.9 kDa). Thus, the production of this non-fluorescent protein is probably highly preferred by the ribosome than translation starting from the actual GFP start codon. In the case of the endogenous Glass translation only a small peptide is produced when translation starts from the upstream AUG. While there are no mechanisms known that would allow the ribosome to jump back to the Glass AUG once the translation of Brs is finished, translation might be re-initiated at an AUG codon in the next exon of glass producing a shorter version of the Glass protein that is lacking the first 25 amino acids. In the mosquitoes A darlingi and C quiquefasciatus translation is predicted to start from this place, and in Tribolium Glass the first 40 amino acids are missing compared to Drosophila Glass suggesting that the N-terminal region of Glass might be dispensable. This would suggest a scenario in which in higher Diptera an exon containing several AUGs was inserted upstream of the original Glass coding sequence that would extend the length of the Glass protein by 25 amino acids at its N-terminus, but at the same time would interfere with translation of this extended version by the presence of the smORF and instead support translation from the original start codon by re-initiation of translation after Brs production.

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

This could be an interesting experiment. Taken all our results together, it looks like Glass protein levels are well buffered by different mechanisms so that we only see effects of the upstream AUG when we strongly over-express Glass. So, the prediction would be that under the control of the endogenous regulatory sequences both constructs should rescue glass mutants. It would also be interesting to test a rescue construct lacking the upstream the smORF and the first 25 amino acids of Glass to see if such a truncated version of the Glass protein is functional in Drosophila. Since we have defined different glass enhancer elements in our reporter gene assays, one could also try to rescue glass mutants specifically in photoreceptor or non-photoreceptor cells and see if the presence of Brs makes a difference in such a case when eye development is impaired.

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