The major goal of this study was to establish a comprehensive profile of miRNAs localized to the nucleus of a human cell line. The quality of the subcellular fractions was critical and entirely validated by multiple immunoblot assays with representative protein markers. MicroRNAs showing potential nuclear localization at levels higher than average were selected by a summary of microarray analyses; and the existence of nuclear miRNA was subsequently verified by RT-PCR and northern blot analyses.
The current model or consensus is that the major cellular function of miRNA involves post-transcriptional regulation of target messenger RNA.9
Depending on the mode of interaction with target mRNA, miRNA could lead to cleavage of the mRNA or inhibition of translation from the target mRNA.3,4,21
Interestingly, several recent studies provide strong evidence for the nuclear localization of a certain class of miRNAs in a short mature form.10–14
Most of those studies suggested sporadic cases of nuclear detection of miRNA(s). In order to establish the biological functions of such nuclear miRNAs, it would be beneficial to generate a comprehensive list of miRNAs that localize to the nucleus of mammalian cells. The potential role of nuclear miRNAs in the regulation of gene transcription could uncover an entirely new and exciting application for this family of small non-coding RNAs.
We speculate that the subcellular localization of miRNA to nuclei is regulated in a defined manner by quite specific mechanisms. It is interesting that after completion of the maturation process in the cytoplasm by Dicer and incorporation into RISC, the mature forms of certain miRNAs are trafficked back into the nucleus.10,14,22
Given the complicated and various mechanisms involved in the nucleocytoplasmic transport of RNA molecules, it would not be surprising that specific pathways exist to traffic mature miRNAs from cytoplasm back to nuclei. In fact, they may be in complexes with protein factors related to the function of miRNAs or their transport between nucleus and cytoplasm.23
Despite the small size of miRNAs, it is highly unlikely that the nuclear pore complex (NPC) allows significant random diffusion of regulator molecules into the nucleus. Although a small 9-nm channel remains open in the NPC, even small tRNAs cannot pass through without gated transport.24
As recently suggested, karyopherins such as CRM1 (Exportin-1) could be involved in the shuttling system of miRNAs back and forth between nucleus and cytoplasm.14
Additionally, Ago2, the major component of RISC was detected as a simple complex with a short RNA molecule in the nucleus besides the large 3-MDa complex of RISC in the cytoplasm.13
Identification of such a nucleocytoplasmic transport system for miRNA could provide clues into the potential function of nuclear miRNAs.
The target sequences of probes on a microarray are by nature included in the precursor forms of the miRNAs.5,6
Thus, it was critical to rule out signals from nuclear precursors, such as pri-miRNA or pre-miRNA in the detection of mature species. Control probes complimentary to the antisense strand of precursor miRNA (strand complementary to the mature miRNA sequence) could be used to evaluate the detection of such precursors in the microarray analysis since the antisense strand would only exist in the precursor forms. By subtracting the estimated signals from probes specific for precursor sequences, the fluorescent signals from the authentic miRNA probes could be corrected for nuclear fractions, which would include precursor elements. However, in the hybridization conditions of microarray analysis, miRNA sense strand sequences in the precursor would hybridize primarily with their antisense counterparts in the same molecule of precursors due to the close proximity between those complementary sequences in the single molecule. In addition, the pri- or pre-miRNA precursors in the nucleus appear to be in a rapid cycle of turnover during miRNA biogenesis since we were able to detect only barely discernible signals of the precursor forms on the northern blot where mature short forms of miRNA were easily detected in both fractions (). In support of this, precursor forms of miRNA could be only detected by RT-PCR following the knockdown of Drosha or DGCR8 that are components of pri-miRNA processing complex.25
Unexpectedly, we were not able to recapitulate the nuclear enrichment of human miR-29b as previously reported.10
This might have been been due to cell-specific regulation in the subcellular localization of a particular miRNA since we examined HCT116 cells and not HeLa cells, as originally reported for detection of nuclear miR-29b.10
Even though our microarray also included probes for other miRNAs that were very similar to miR-29b in sequence, including cytoplasmic miR-29a, the probe design in our microRNA microarray platform was able to discriminate 2-nt differences between similar sequences of miRNAs from the same family.
It is also interesting that nucleolar-enriched miRNAs identified in rat myoblasts12
do not overlap significantly with our list of potential nuclear miRNAs derived from human HCT116 cells. Again, this could be attributed to differences in the origin of the cell lines since our data were acquired from a human colorectal cancer cell line, in contrast to rat myoblasts that are replete with nucleoli. As exemplified by miR-206, a miRNA could be enriched both in the nucleoli and cytoplasm simultaneously,11
but the nuclear signal would be masked by strong cytoplasmic signal in our analysis.
It should be noted that Xist RNA was detected in a complex with RISC and miR-210,26
(). Xist is a long non-coding RNA which is well known for its crucial function in the mammalian X-chromosome inactivation and has been proven to be localized in the nucleus,27
painting the inactive X-chromosomes.28
This observation suggests that certain non-coding RNAs in the nucleus could be potential targets of nuclear miRNAs. Currently, most computational methods of miRNA target prediction use stringent seed pairing at the potential target 3′-UTR, as well as evolutionary conservation of the target pairing sites.29
With such predictive algorithms, it is impossible to identify potential noncoding nuclear RNA targets in the absence of associated UTRs. MicroRNA binding to Xist RNA was established by immunoprecipitation and biochemical analysis.26
Thus, the identification of nuclear factors, including proteins, RNA and DNA, which can interact with microRNAs, could provide valuable clues in understanding the potential function(s) of nuclear-associated miRNAs.
In summary, we have provided strong evidence for the nuclear localization of numerous miRNAs in a human cell line. These data will undoubtedly provide a basis for future studies to investigate the possible functions of nuclear miRNAs in mammalian cells. Further investigations such as in situ hybridization in a comprehensive scale and identification of nuclear (or nuclear envelope components) protein factors associated with mature miRNAs could provide valuable clues to elucidating their mode of nuclear trafficking and potential biological functions.