Our analyses revealed that circMbl mediates cap-independent as well as internal translation initiation in a fly in vitro translation system. Although internal translation initiation of circMbl was rather weak, we hypothesized it might still play a role during conditions in which cap-dependent translation is limited. And indeed, while cap-independent and internal translation initiation of circMbl reporters was only 12 % and 10 % of cap-dependent translation under regular conditions, internal translation initiation was stimulated to increase by a factor of ~3.5 when cap-dependent translation was inhibited. Since the translation of capped linear control mRNAs was repressed by a factor of 2.5 (FLuc reporter) to 6.3 (RLuc reporter) under the same conditions, the results suggest that circMbl-driven translation has the potential to compete with cap-dependent mRNA translation under specific cellular conditions.
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The circMbl sequence that exhibits cap-independent and internal translation initiation activity spans 456 nt and is located within the 5’UTR of Mbl. More specifically, the sequence is contained within the second exon of Mbl where it is located directly upstream of the start codon and the first 189 protein coding nts. The sequence has a low minimum free energy of -77.4 kcal/mol and a low GC content of 34 % [902]. Interestingly, it contains several stretches of multiple adenosine nucleotides: two oligo(A)s with the length of five nucleotides, two oligo(A)s with the length of six nucleotides and one oligo(A) with the length of 17 nucleotides. It was shown previously that PABP can bind the consensus motifs AAAAA and ACDAAYM (D = U, A or G; Y = G, U or C; M = A or C) in human and UAUAUA in yeast [903], [904]. Further, the PABP binding affinity increases with increasing oligo(A) length of up to twelve nucleotides [905].
As circMbl contains several oligo(A) stretches and four times the UAUAUA-binding motif it might interact with one or several PABPs. In general, PABP binding to the poly(A) tail of mRNAs is associated with higher mRNA stability and mRNA translation [906]–[908]. Enhanced translation is mediated through the interaction of PABP with eIF4G which is thought to generate a ‘closed-loop’ conformation between the mRNA 5’ and 3’ end [909]. It was previously reported that PABP can also bind 5’UTRs of a small subset of mice mRNAs regulating transcription, DNA binding, nuclear processes and cell cycle control including Mbnl1 (three 5’UTR CLIP tags) and Mbnl3 (one 5’UTR CLIP tag) [910]. Hence, PABP binding to the 5’UTR of Mbl might be conserved in flies. Interestingly, PABP also binds to A-rich sequences in its own 5’UTR, establishing a feed-back loop that suppresses PABP synthesis [911], [912]. Thus, translational regulation by PABP might be determined by interaction partners who either promote or repress translation, although more promoting interactions are known until now.
To verify that PABP binding to circMbl is involved in internal translation initiation, deletion experiments could be performed to investigate if circMbl translation is reduced upon removal of the 17 residues long oligo(A) stretch. Further, in vitro translations could be carried out in a system previously depleted of PABP. Also, circMbl RNA pull-down assays could be performed using biotinylated DNA oligo probes against the circMbl backsplice junction, followed by PABP Western blotting. Radiolabeled circMbl in vitro translation reactions could be separated on sucrose density gradients to not only identify potential PABP interaction but also analyze the composition of initiation complexes assembled on the circMbl IRES. To test which factors are required for initiation, the system can be manipulated to lack activity of individual eIFs due to depletion or targeted inhibition.
Interestingly, a recent non-peer reviewed publication reported that 10-nt AU-rich motifs are capable of driving translation initiation of circRNAs [913]. A 10 nt long oligo(A) stretch upstream of a circRNA encoding GFP enhanced reporter expression when PABPC1 was
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transfected and tethering of a Puf-PABPC1 fusion protein to circGFP similarly enhanced reporter expression [913]. Unfortunately, the experiments lack appropriate controls ruling out that reporter expression is not driven by linear concatemers, which tend to be generated by circRNA reporter plasmids [1]. It remains uncertain how the short AU-rich motifs mediate circRNA translation and whether the mechanism is active in natural circRNAs. Even though AU-rich hexamers were enAU-riched in circRNAs compared to linear mRNAs and analyses predicted that an AU-rich hexamer will occur by chance every 50 nts, no endogenous circRNAs were investigated for motif-mediated internal translation [913]. Hence, it is unclear whether the identified motifs are active in natural circRNA contexts. Nevertheless, the results indicate a potential positive effect of PABP on circRNA translation.
But not only circMbl mediated internal translation, also the inverted circMbl sequence that was used as a negative control was able to promote internal translation with ~60 % of the efficiency of circMbl in forward orientation. Unexpectedly, reverse circMbl was also stimulated by cap-dependent translation inhibition in the same way as circMbl in forward orientation. As nucleotide sequences in the reverse orientation will fold into different secondary structures due to asymmetrical free energies of stacking base pairs (e.g. 5’-GC/GC-3’ ≃ -3.4 kcal/mol and 5’CG/CG-3’ ≃ -2.4 kcal/mol) it is rather unlikely that circMbl is forming an IRES that drives translation through structural elements in both forward and reverse orientation [914]. As oligo(A) stretches are palindromic they could also function in reverse orientation to recruit PABP. The long 17 nt oligo(A) stretch of circMbl is still located upstream of the authentic Mbl start codon in the reserve sequence, however, several upstream AUGs and upstream ORFs could reduce translation initiation from the authentic start codon or could cause synthesis of an N-terminally extended reporter. Interaction of PABP with reverse circMbl could be investigated as mentioned above and translation of N-terminally extended reporters could be examined by Western blots for RLuc.
In Drosophila, the Mbl gene locus gives rise to 30 circRNAs in total (circBase as of May 2019 [736]). Of these, eight contain the second exon and thus have capacity to encode potential C-terminally truncated protein isoforms [1]. Putative circMbl encoded peptides were found in fly synaptosomes and circRNAs have also been found to be enriched in synaptosomes and synaptic genes in several other organisms [737], [742], [747], [749], [915]. Hence, circMbl translation might play a role in synaptic functions under conditions in which cap-dependent translation is diminished or linear mRNAs might be degraded. As circMbl sequence is also present in the 5’UTR of linear Mbl transcripts, effects of circRNA translation might be more pronounced during localized mRNA degradation or destabilization. Growing evidence suggests that local mRNA and protein metabolism are involved in neuronal functions and synaptic plasticity [916],
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[917]. This regulation is substantially shaped by functional sequence elements within 3’UTRs [918]–[920]. As circMbl lacks 3’UTR sequences it has the potential to be differentially regulated from linear mRNA counterparts. Furthermore, circMbl encoded peptides could act as dominant negatives of the full-length Mbl protein. As previous studies indicated a role of MBNL proteins in mRNA localization, local translation and stability in addition to its well-established function in splicing, circMbl encoded peptides might interfere with such regulatory mechanisms at synapses potentially shaping underlying processes like e.g. memory formation [921].
Few other studies have recently found indications for endogenously translated circRNAs (see section 1.5.1) [743], [807], [808]. Taken together, the process of circRNA translation seems to be a regulated one instead of happening by chance, as circRNAs of varying abundances can be found associated with ribosomes and translational efficiency of different types of circRNAs varies at least when expressed from minigenes [1], [743], [807]. But only a small proportion of circRNAs has translational potential and the vast majority of circRNAs cannot be detected in association with ribosomes or polysomes [1], [727], [731], [737], [801], [807]. circRNAs are biased to originate from the 5’ terminal part of their host gene and 14 % of human circRNAs as well as 17 % of mouse circRNAs deposited at circBase are annotated to contain 5’UTR sequences [736].
Further, about half of the circRNAs that contain overlapping ORFs with their host gene have the potential to encode known protein domains involved in protein interaction or protein-RNA interaction [913]. In addition, there are indications that polysome-associated circprotein-RNAs have longer putative ORFs compared to total circRNAs and that ribosome-associated circRNAs often share the start codon and parts of the 5’UTR with their host gene [1], [807]. Together, this suggest that only few circRNAs function in translation and that these circRNAs are likely to originate from the 5’-terminal end of the host transcript likely sharing the same regulatory 5’UTR, start codon and N-terminal protein domains.
Moreover, the studies suggest that circRNA translation is inefficient under regular conditions but can be activated when cap-dependent translation is compromised [1], [743].
Evidence exists that upregulation of circRNA translation involves circRNA splicing and/or methylation [743], [807]. The exon-junction complex, eIF4G2 and eIF3A might play a role in regulating circRNA translation but so far there are at most indirect hints [743], [807]. Hence, a couple of open questions remain regarding the mode of action of circRNA translation and involved interaction partners: Which factors are required to drive internal translation from circRNAs? Which cellular conditions must be met to allow for functionally relevant circRNA translation? Do circRNA encoded peptides perform a function or is circRNA translation indirectly affecting the regulation of surrounding transcripts competing for involved factors?
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Future investigations will provide mechanistical insights that will help to elucidate the function of circRNAs in translation helping to further define regulatory roles of this new class of RNA. A better understanding of circRNA-mediated protein synthesis might eventually enable the development of therapeutic applications of circRNAs that might have potential in the treatment of neurological disorders as circRNA might fulfill regulatory functions in neuronal processes also in healthy organisms [737], [742], [784], [922], [923].