A distinctive stage in evolution was the formation of the cell nucleus, which stores the genetic information encoded by DNA. This compartmentalization resulted in the segregation of key steps in the synthetic pathways from DNA to protein. A similarity exists with the biogenesis of miRNA molecules. The appropriate subcellular localization of the pre-miRNA is essential to their complete processing, their function and regulation. Compartmentalization can control access to binding partners, concentrate cofactors or temporarily segregate components of the pathway away from the rest of the cellular environment. The exquisite spatiotemporal control of miRNA abundance is made possible, in part, by regulation of the miRNA biogenesis pathway. While pri-miRNA processing is a nuclear event, pre-miRNA processing occurs in the cytoplasm. Thus, pre-miRNAs need to transit from the nucleus into the cytoplasm, a process that requires the nuclear export receptor XPO5.34,35
Frameshift mutations occur in protein-coding sequences, which may render affected proteins nonfunctional and thus drive carcinogenesis through the inactivation of tumor suppressor genes. We have reported frameshift mutations in the XPO5 gene in two MSI+
cell lines and primary tumors.31
The mutations found in exon 32 alter and truncate the protein sequence and prevent XPO5 from associating with its pre-miRNA cargo and exiting the nucleus (). In XPO5 heterozygous mutant cells, less pre-miRNA was hence accessible to processing by the cytoplasmic machinery, resulting in decreased mature miRNA levels and enhanced tumorigenicity. Restoration of XPO5 wild-type protein levels in the defective cells rescued pre-miRNA export and processing defects and had tumor suppressor features. These cancer cells exhibited impaired miRNA processing but failed to lose the wild-type XPO5 allele. Recent work has suggested that other components of the miRNA biogenesis pathway, DICER1 and TARBP2, are haploinsufficient tumor suppressors.28–30
Moreover, biallelic deletion was found to impair cell viability, hence preventing the phenomenon of loss-of-heterozygosity (LOH).28–30
This is also the case for XPO5, where mimicking LOH by RNA interference against the XPO5 wild-type transcript rendered cells unviable.31
In addition, the miRISC components AGO2, TNRC6A and TNRC6C can also be mutated in MSI+
although the functional consequences remain to be evaluated. The presence of mutations in the miRNA pathway genes, including TARBP2 and XPO5 genes, in MSI+
cancer samples has also been reported in this separate study of Korean patients.36
Inefficient nuclear export of a precursor microRNA by the presence of an inactivating mutation in the exportin 5 gene that prevents the formation of a functional XPO5/RAN/GTP/pre-miRNA complex.
The mutated status of this central component turned out to be crucial in malignant transformation since it clogged pre-miRNA flow between the nucleus and cytoplasm. The disruption of this process resulted in a dramatic deregulation of cellular functions that triggered tumorigenesis. Additionally, we also characterized a minimal region in XPO5 required for pre-miRNA binding and/or recognition and therefore for nuclear envelope transversion. Others have reported that XPO5 is expressed at low levels in many tumor types.37
Therefore, the XPO5 heterozygous mutational event found in human cancer clogs the miRNA-nuclear export complex in the nucleus and prevents the export of pre-miRNAs to the cytoplasm and further processing. Nonetheless, analysis of miRNA array data shows that there are pre-miRNAs that the processing does not seem to alter by this impairment of the export machinery. Recent work in C. elegans
has suggested additional nuclear export pathways for pre-miRNAs that could explain why some miRNA levels remained unaffected by this mutation.38
Many pre-miRNAs are also targeted by ADARs at various stages of their processing, and the modification can also prevent export of pre-miRNAs.
Similar to what we demonstrated, the expression of over 200 precursor and mature miRNAs demonstrated a wide discrepancy between in a high percentage of human primary tumors.10
Our results are also in line with the finding that, after profiling 225 precursor and mature miRNAs in 22 human primary tumors and 16 pancreatic and liver tissues/tumors, many of the miRNAs analyzed are processed to the precursor but these precursors are retained in the nucleus.7
However, this study did not provide any possible explanation that could shed light on the mechanism underlying this pre-miRNA processing blockage and nuclear retention. In light of our results we can now speculate about possible impairment at the level of the nucleocytoplasmic pre-miRNA export machinery in some of the analyzed samples. Two other groups have also reported the accumulation of let7 miRNA precursors at various stages during fruit fly and sea urchin development.39,40
Although this could represent a defect in RNA processing, it is also possible that the nuclear export of the let7 precursor, and hence access to cytoplasmic DICER1 and TRBP, are developmentally regulated.