The sequence context of microsatellite repeats seems to have a central role in RAN translation;
14,31,41,50 hence, it was essential to show that CTG·CAG repeats embedded in the third intron of
TCF4 are also translated via non-ATG initiation. CTG·CAG repeats in the context of the
TCF4 gene, cloned under the control of the CMV promoter and in frame with a 3× FLAG C-terminal tag, could be translated in transfected cells without an initiating ATG from the polyC and polyQ frames (
Fig. 3). Some RAN translation products are known to have poor solubility and peculiar mobility in SDS-polyacrylamide gels,
14 but we were unable to detect any reactive polypeptides with the anti-FLAG and anti-AF antibodies in cells transfected with the A99 and A175 constructs, in either the soluble or insoluble fraction of these cell lysates (
Fig. 3A, data not shown). The polyC frame contains an ATG downstream of the repeats, but we showed that an M to K mutation for this residue did not prevent the translation of a polypeptide identified by anti-FLAG antibodies (
Fig. 4C). We tried to force the expression of a polyC-containing polypeptide by introducing an ATG upstream of the repeats, but initiation by RAN translation was preferred, despite a strong Kozak sequence (
Fig. 4C). Interestingly, an anti-FLAG-reactive polypeptide was detectable when 29 repeats were present, but at a much lower level (
Fig. 4B). This observation, together with the larger size (∼15 kDa) of the polypeptide in the C220 extract compared to the C112 extract, points to the size of the translation product being dependent on repeat length and suggests a repeat length threshold in RAN translation, as previously reported.
14,31,50 We also detected an anti-FLAG-reactive polypeptide and some high molecular weight species for the polyS frame (
Fig. 3D), but due to the presence of an ATG in this ORF downstream of the repeats, we could not unequivocally demonstrate that these polypeptides were produced by RAN translation, although their mobility argues for the latter.