Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery
for their maturation. However, the molecular details of this Drosha-independent processing
are still not fully understood; as an example, the Microprocessor complex cannot process
the mirtronic pre-miRNA from the transcript even if splice site mutations are present.
To investigate the influence of alternative splicing sites on mirtron formation, we
generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial
introns to compare the processing of canonical miRNAs and mirtrons. Although mutations
of both splice sites generated a complex pattern of alternative transcripts, mirtron
formation was always severely affected as opposed to the normal processing of the
canonical hsa-mir-33b miRNA. However, we also detected that while its formation was
also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain
mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that
this phenomenon is not dependent on Microprocessor activity but rather points toward
the potential stability difference between the miRNAs from the different arms. Our
results indicate that when the major splice sites are mutated, mirtron formation cannot
be rescued by nearby alternative splice sites, and stability differences between 5p
and 3p species should also be considered for functional studies of mirtrons.
