Control of gene expression by small RNAs within mammalian cells is usually associated with gene silencing in the cytoplasm. miRNAs (31
) and AGO protein (16
), however, both exist in the nucleus, suggesting that recognition of nuclear RNA targets might also help regulate cellular function. We (15
) and others (33
) have observed that small RNAs can either silence or activate gene transcription through recognition of non-coding transcripts that overlap genes. These data and our demonstration that duplex RNAs can affect splicing through recognition of pre-mRNA establish the potential for potent RNA-mediated regulation in mammalian cell nuclei.
Our findings are the first example of RNA-mediated exclusion of introns or exons from mRNA. There has been one previous report describing RNAs that affect exon inclusion (23
), but the mechanism appears different from the one suggested by our data. Given the rich variety of RNA targets in the nucleus, it should not be surprising that RNA can employ multiple mechanisms to regulate splicing. The precedent that gene silencing by small RNAs can be achieved by blocking transcription through binding to non-coding transcripts or post-transcriptionally through recognition of mRNA also supports the hypothesis that multiple routes may also exist for RNA-mediated control of splicing.
AGO2 is involved in RNA-mediated exon exclusion. This may be seen as surprising because the ability of AGO2 to induce cleavage of mRNA targets in the cytoplasm is well known. In nuclear extracts, however, Tuschl reported that cleavage was much weaker relative to cytoplasmic activity and cleavage site selectivity was greatly reduced (35
). Fluorescence correlation spectroscopy suggests that nuclear AGO complexes are much smaller than AGO complexes in the cytoplasm (36
), providing one explanation why activities might differ. Other reports have described post-translational modifications that affect AGO2 activity (37–39
), and such modifications may play a role in targeting and activity in the nucleus. Our results emphasize the need for experimenters to have an open mind regarding how AGO might function in the nucleus and design experiments accordingly.
We have not observed evidence of target transcript cleavage during our studies of AGO2-mediated transcriptional silencing and activation (15
). Cleavage was not detected by 5′ RACE and expression of the targeted non-coding transcript was unchanged during gene activation. When RIP is performed with anti-AGO2 antibody, the target transcript is detected even though detection involves primers that flank the RNA binding site where cleavage would be predicted to occur.
Our observation that AGO2 mediates RNA-mediated exon exclusion without causing destruction of transcripts in the nucleus, regardless of whether the target sequence is within an intron or an exon, further establishes that nuclear AGO2 function can differ significantly from the function of AGO2 in the cytoplasm. Understanding the molecular basis for the different properties of AGO2 in the nucleus and cytoplasm will be an important goal for future studies. Nuclear AGO2 may have different post-translational modifications or may bind to different proteins or other cofactors. The duplex RNAs used in our studies target sequences near splice sites and it is likely that binding of the AGO2/RNA complex blocks the sites and alters splicing preferences.
Synthetic RNA duplexes are widely used to modulate gene expression and our results demonstrate that they can also control splicing. Typically, for gene silencing, duplexes are designed to be fully complementary and mismatch-containing duplexes are used as controls. We have previously shown that the introduction of mismatched bases relative to their mRNA targets can permit RNA duplexes to achieve high levels of allele selectivity (39
). We now show that mismatched RNA duplexes can alter splicing and help tailor the ratio of spliced products. The use of mismatched RNA duplexes to achieve desired selectivities emphasizes the potential to achieve improved important properties of RNAs through mechanism-guided duplex design.
Our data demonstrating RNA-mediated alteration of splicing have several implications (): (i) Small RNAs can function together with nuclear AGO2 to recognize pre-mRNA transcripts and alter splicing. This finding expands the range of RNA-mediated control of gene expression to alternative splicing; (ii) regulation of splicing is robust and reproducible, consistent with the robustness found in endogenous regulatory pathways. (iii) Small miRNA-like mismatch-containing duplexes also alter splicing. miRNAs exist in the nucleus (31
) and our data suggest that miRNAs have the potential to modulate splicing; (iv) redirecting splicing using duplex RNAs provides an alternative to using antisense oligonucleotides that may prove advantageous for development of nucleic acid therapeutics and (v) mismatched duplexes can be used if it is necessary to preserve spliceforms that retain targeted exons, while fully complementary duplexes can be used if maximal biasing toward exon-excluded spliceforms are required. The potential of small RNAs to modulate alternative splicing by small RNAs offers another layer to the subtle pattern of RNA-mediated regulation that exists inside cells and a new option for therapeutic development.
Scheme showing redirection of splicing by fully complementary and mismatch-containing duplex RNAs.