In this study, we identify small regulatory RNAs that are processed by a non-canonical biogenesis pathway that involves Drosha, but does not require DGCR8 or the other components of the canonical biogenesis pathway. These miRNAs were originally predicted to be mirtrons, due to their predicted hairpin structure, location within short introns, and identification from deep-sequencing analysis (40
). Although we confirm that the predicted mammalian mirtrons, miR-877 and miR-1226 are, indeed, splicing-dependent in the context of their natural host gene, two other predicted mirtrons, miR-1225 and miR-1228 do not require splicing for their biogenesis. We classify these miRNAs as ‘simtrons’ (splicing-independent mirtron-like miRNAs), for their resemblance to mirtrons in their structure and genomic context spanning introns. To our knowledge, simtrons are the only class of miRNAs characterized to date that are processed in a manner that does not require splicing, the Microprocessor, Dicer or Ago2.
A number of non-canonical miRNAs have been described. However, they all require Dicer to produce the mature miRNA. Flynt et al
) discovered a miRNA in Drosophila
, whose 5′ pre-miRNA end is generated as a result of splicing and whose 3′ pre-miRNA end is generated by exosome-mediated trimming. These 3′ tailed mirtrons are distinct from simtrons because they require splicing. Endogenous shRNAs are non-canonical miRNAs that do not require Drosha or splicing but are cleaved by Dicer to generate a mature miRNA (25
). Another class of non-canonical miRNAs is encoded by a murine herpesvirus and processed by tRNAseZ rather than by Drosha (26
). Likewise, endogenous siRNAs are generated from the sequential processing of long hairpins by Dicer but are not considered miRNAs because they produce many different small RNAs (1
). Finally, miRNAs have been identified that are derived from snoRNAs and tRNAs by non-canonical pathways that do not require the Microprocessor but require Dicer (25
). These examples highlight the diversity of small RNAs and their processing pathways in the cells.
We demonstrated that simtrons are distinct from other classes of small RNAs in that they are not processed by Dicer or Ago2 (), and yet are functional in gene silencing ( and Supplementary Figure S4C
). Although we identified simtrons during our examination of mammalian mirtrons, it is possible, and perhaps likely, that the processing pathway responsible for the biogenesis of this non-canonical type of miRNA has a wider range of substrates. Indeed, several studies have observed miRNAs whose abundance, as determined by large-scale sequencing analysis, is not altered in Dicer
knockout or DGCR8
knockout cells (25
), suggesting that additional processing pathways exist.
Our data indicate that Drosha is involved in the processing of simtrons, though this may not be a strict requirement. We also show that DGCR8 is not required for simtron processing, though we cannot exclude the possibility that DGCR8 may be involved in simtron biogenesis at some level (). These findings distinguish simtrons from other miRNAs. A Drosha complex that does not contain DGCR8 has been identified but was reported to be less efficient at miRNA processing than the complex containing both DGCR8 and Drosha (8
). It is possible that this alternative Drosha complex, that lacks DGCR8, processes simtrons. Although Drosha contains a double-stranded RNA binding domain (DSB), it appears to require an adaptor, such as DGCR8, for proper positioning and precise cleavage (62
). DGCR8 functions as an anchor that recognizes both dsRNA and ssRNA and directs Drosha to cleave the stemloop ~11
bp from the dsRNA/ssRNA junction (5
). Drosha is capable of cleaving hairpin-structured mRNA without producing miRNAs (61
), however, this is the first evidence that Drosha can process a subclass of miRNAs in the absence of DGCR8. It is possible that another RNA binding protein plays a role similar to DGCR8 in the processing of some miRNAs such as simtrons. A number of splicing factors, for example have been shown to promote Drosha cleavage and silencing (63–66
). Such a factor, which binds in the intron, could potentially act as a cofactor for Drosha cleavage of simtrons. Indeed, though DGCR8 is not required for simtron processing (A), it appears to enhance processing in vitro
(B) possibly by co-immunoprecipitating an important co-factor or by stabilizing Drosha in the in vitro
Interestingly, simtrons generated from both the wt and splicing-deficient minigene transcripts immunoprecipitated with Drosha (A), suggesting that Drosha processing by the simtron pathway may occur even when splicing is active. In this case, splicing and Drosha processing may be in competition. Competition between splicing and Drosha processing is further supported by results from experiments with the dominant negative form of Drosha. Only miR-1225 derived from the splicing deficient transcripts (Δss) was reduced as a result of TN-Drosha expression. Reduction of Drosha activity is not expected to affect miR-1225 levels from the wt transcript because splicing can generate the pre-miR-1225 via the mirtron pathway. Processing of the miR-1225 by the mirtron pathway is also suggested by the interaction of mature miR-1225 with Dicer. The simtron pathway, on the other hand, does not require Dicer (A), and miR-1225 generated by this pathway does not interact with Dicer (Δss, B). Taken together, these results suggest that miR-1225 can be excised from the host gene RNA transcript by either the mirtron pathway (Drosha/DGCR8-independent, Dicer-dependent) or the simtron pathway (involving Drosha but DGCR8 and Dicer-independent) ().
Figure 8. Proposed model of simtron biogenesis compared to other miRNA processing pathways. The pathways shown begin with the primary transcript and end with the mature product. Left: simtron pathway, Middle: mirtron pathway, Right: canonical miRNA pathway. Exons (more ...)
Our results indicate that Drosha is involved in the processing of simtrons, however, it seems unlikely that Drosha alone completes all steps of simtron processing. Known canonical and non-canonical miRNA processing pathways require two endonucleolytic cleavage events, carried out by distinct enzymes, to generate a mature miRNA. There are a number of human proteins that are predicted to have RNase activity (67
). For example, human RNase P and tRNAse Z generate the 5′ and 3′-ends of tRNAs, respectively (68
), and could feasibly recognize the highly structured simtronic introns and cleave the intron to generate the pre-miRNA. Additionally, the human tRNA splicing endonuclease complex cleaves both the 5′ and 3′ splice sites of a pre-tRNA intron to release the intron from the transcript (70
). It is therefore possible, that one of these enzymes also plays a role in simtron biogenesis.
The simtron biogenesis pathway appears to follow traditional miRNA assembly into the RISC complex with Ago proteins ( and Supplementary Figure S4D
) and targeting ( and Supplementary Figure S4C
). Even though miR-1225 and miR-1228 abundance was not affected by the absence of Argonaute-2 in the knockout cell lines (C), they do associate with Ago2 when both are present ( and Supplementary Figure S4D
) suggesting that simtronic miRNAs in the Ago2−/−
cells sort into Agos-1,3 and 4, which can compensate for the lack of Ago2.
One implication of our results is that, in certain situations, splicing and miRNA biogenesis may be in competition with one another. Competition between the splicing of pre-mRNA and the production of the miRNA would impact not only miRNA target gene expression, but also miRNA host gene expression. The generation of simtrons, by virtue of their intron-spanning location and processing pathway, could have an effect on the expression of the mRNA from which it is removed. Although excision of miRNAs from introns has been shown to be compatible with splicing of the intron, yielding both a pre-miRNA and spliced mRNA, with little if any impact on expression of either RNA (72
), these studies examined miRNAs which are housed in larger introns and spatially removed from the essential 5′ splice site, 3′ splice site, branchpoint sequence and polypyrimidine tracts. Simtrons, on the other hand, encompass and excise all of these splice site sequences during processing. Thus, simtron biogenesis and splicing would appear to be mutually exclusive if splicing does not occur first ().
Considering that many small RNAs are processed from the introns and non-coding sequences of pre-mRNA transcripts, a mechanism by which excision and expression of these RNAs is linked to mRNA expression is important to consider when investigating such phenomenon as genetic modifiers to disease. For example, PKD1
, the host gene for miR-1225, is the most frequently mutated gene in autosomal dominant polycystic kidney disease (85% of cases) (74
). Polycystic kidney disease, for reasons that are not clear, has variable disease severity (74
). One intriguing possibility is that miR-1225 is a disease modifier that, depending on the type of mutation in the gene, could influence the penetrance of the disease (74
). Furthermore, mutations found only within the simtron containing intron are associated with disease (76–78
). Likewise, miR-1228 is housed within the LRP1
gene, which has diverse functions in the cell and has been implicated to play a role in atherosclerosis and Alzheimer's disease (79
). The idea that two separate biogenesis pathways (mirtronic and simtronic) can generate the same miRNA may indicate the importance of these miRNAs in the maintenance of homeostasis.
The discovery of the simtron biogenesis pathway demonstrates the complexity of RNA processing and uncovers another mechanism by which small non-coding RNAs can be produced from an RNA transcript. Our results also caution against the assumption of a biogenesis mechanism based on sequence, location and predicted structure alone. It is possible that other predicted mirtrons are not splicing-dependent, but are instead processed by the simtron pathway.