Mbl is a RNA binding protein that was initially described in Drosophila. It is conserved in mammals and regulates alternative splicing of pre-mRNAs. The Mbl gene locus is complex and gives rise to 13 protein coding transcripts as well as 30 circRNAs in Drosophila melanogaster (circBase as of 15 December 2015, NCBI Gene as of 9 May 2019) [108], [736]. Eight of these circRNAs harbor the potential to code for a circRNA-specific protein, as they contain ORFs that span the head-to-tail junction [1]. One of these ORFs is an infinite ORF, meaning that the circRNA does contain a start but no stop codon in none of the three reading frames, which would generate a “Möbius protein” [1].
In order to investigate, if circMbl can indeed be translated into peptides, collaborators from the Kadener Lab in Jerusalem performed ribosome-profiling in fly heads. They identified a number of circRNAs with ribosome-footprinting (RFP) reads that span the circular backsplice
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junction [1]. The most abundant among them was circMbl that also exhibited a high stop codon score, strongly suggesting that the stop codon is used during active circRNA translation.
Furthermore, the translation of circMbl was supported by minigene assays in S2 cells and transgenic flies. The minigene experiments revealed that circMbl produces protein and that protein production was abrogated by mutation of the 5’ss and enhanced when cap-dependent translation was repressed by starvation, 4E-BP or FOXO overexpression [1]. Additionally, mass spectrometry of fly heads identified a peptide that can only be synthesized from one of the circMbl isoforms and not from a linear Mbl RNA isoform [1].
However, transcription of the minigene constructs generated also linear concatemers that could potentially have contributed to peptide expression. Further, it was not possible to validate the peptide encoded by the most abundant ribo-circMbl by MS, as it could not be ionized sufficiently. Thus, we performed in vitro translation assays to collect additional evidence for cap-independent translation of circMbl and to determine the characteristics of circMbl translation initiation in greater detail.
First, we determined the capacity of circMbl to mediate translation initiation from linear m7Gppp-capped, Appp-capped or m7Gppp-capped bicistronic RNA reporters. Comparing translational rates of all three reporters allows to make distinctions between the circMbl efficiency in mediating cap-dependent, cap-independent and 5’-end independent/internal translation initiation. In vitro translation assays in a cell-free Drosophila system revealed that the m7 Gppp-capped circMbl reporter was ~8-fold more efficiently translated than the Appp-Gppp-capped circMbl reporter and ~10-fold more efficiently translated than the bicistronic circMbl reporter (Fig. 13A).
These results suggest that circMbl translation is less powerful than linear Mbl translation and that the prevailing mechanism of protein expression is cap-dependent. However, as circRNAs are in general more stable than mRNAs and cap-independent translation can be predominant under certain cellular conditions, a circMbl translation efficiency of 10 % could still be meaningful.
Moreover, circMbl encoded peptides are unique in their domain composition and could have specific regulatory function in RNA metabolism or Mbl protein production through regulatory feedback loops.
For that reason, we continued to investigate the translational efficiency of circMbl in comparison to other cap-dependent and cap-independent reporters as well as in the context of cap-dependent translation inhibition. Therefore, we generated circularized reporters containing either full-length circMbl (circMbl-RLuc), a deletion of most of the circMbl sequence (del-circMbl-RLuc) or full-length circMbl in reverse orientation (rev-circMBL-RLuc) (Fig. 13B). In the in vitro translation system, the translational efficiency of circMbl was ~25 % of the translation efficiency of a linear cap-dependent reporter (m7Gppp-RLuc) (Fig. 13C). In comparison, CrPV
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IRES displayed ~75 % of the translation efficiency of a linear cap-dependent reporter. Deletion of most of the circMbl sequence completely abrogated translation and reverse orientation of circMbl decreased translation by a third.
Addition of m7Gppp-cap in trans inhibited cap-dependent translation remarkably so that cap-dependent control reporter expression was reduced by ~85 % (Fig. 13C). In contrast, translation efficiencies of circMbl and CrPV IRES were elevated more than 2-fold. Also, circMbl in reverse orientation displayed a more than 2-fold increase in translation efficiency.
Taken together, these results demonstrate that circMbl like CrPV IRES translation is indeed cap-independent and can further be stimulated under conditions of cap-dependent translation repression. In this context circMbl can be translated as efficiently as a cap-dependent reporter under normal conditions. Thus cap-independent translation of circMbl has the potential to make a meaningful contribution to Mbl protein production when cap-dependent translation is reduced. Surprisingly, reversed circMbl was also able to mediate circRNA translation even though the sequence contains multiple uORFs. Some of them even display a strong AUG context (3 out of 5 uORFs with AUG codons matching in -3 and/or +4 position the Kozak sequence [17]) which is associated with reduced translation of the downstream ORF [84], [319]. This suggests that either the effect of translational repression by uORFs in 5’UTRs cannot be transferred to uORFs within circRNAs or that circMbl is functionally active through its secondary structure and not through its primary nucleotide sequence.
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Fig. 13: CircMbl can be cap-independently translated in vitro. (A) Drosophila cell-free in vitro translation assay comparing a linear monocistronic circMbl reporter carrying a m7Gppp-cap to a linear monocistronic circMbl reporter carrying an Appp-cap analogue and a linear bicistronic circMbl reporter carrying a m7Gppp-cap. Renilla luciferase activity (RLuc) was normalized to Firefly luciferase activity (FLuc). Error bars represent standard deviation of three biological replicates. (B) Linear and circular mbl-RLuc RNA reporters synthesized for in vitro translation were analyzed by TBE-Urea gel electrophoresis and SYBR gold staining. To control for circularity, reporters were digested with RNaseR. (C) Drosophila cell-free in vitro translation assay comparing circularized circMbl reporter, a circMbl deletion reporter (del-circMbl, negative control) and a reversed circMbl reporter (rev-(del-circMbl, negative control) to linear m7Gppp-capped RLuc and linear Appp-capped CrPV reporters (positive control). Assay was carried out in the presence or absence of cap-dependent translation inhibitor (m7Gppp added in trans). m7 Gppp-capped monocistronic FLuc reporter was used as internal translation control (lower panel). Error bars represent standard deviation of three biological replicates. Some of the data have been published previously in Pamudurti et al., 2017 [1].
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