We show here that SF2/ASF and miR-7 can form a negative feedback circuit. SF2/ASF directly binds to the primary miR-7 transcript to promote its maturation; mature miR-7 in turn represses the translation of SFRS1
mRNA by targeting its 3′UTR (). Notably, negative feedback does not always lead to a stable steady state, as overcorrection and/or time delay could result in oscillation (Elowitz and Leibler, 2000
). Because gene repression by miRNA is relatively modest, with little time delay, miRNA-mediated negative feedback loops are advantageous for noise dampening and have been shown as a recurrent circuit motif in mammalian gene regulatory networks (Tsang et al., 2007
Negative feedback is also a common mechanism to maintain the steady-state levels of SR proteins (Jumaa and Nielsen, 1997
; Sureau et al., 2001
). In the case of SF2/ASF, autofeedback has been proposed to occur at multiple levels, including unproductive alternative splicing (Lareau et al., 2007
; Ni et al., 2007
) and inhibition of translation initiation (Sun et al., 2010
). Thus, the miR-7-mediated negative feedback loop is expected to synergize with other feedback mechanisms to precisely control the protein level of SF2/ASF in cells. The relative contribution of each mechanism may vary under different conditions, such as in different cell types or physiological states. This may explain the different phenotypes we observed when knocking down endogenous miR-7 in HEK293T and HeLa cells. Because the regulation observed in the SFRS1/miR-7 circuit is relatively modest (2-fold effects in both directions), miR-7-mediated negative feedback does not appear to be a major contributor to the robust feedback regulation observed for SF2/ASF. Interestingly, Dicer null embryonic stem cells (ESCs) showed compromised but detectable feedback of SF2/ASF on its own expression (Sun et al., 2010
), consistent with the notion that both miRNA-dependent and -independent mechanisms are involved.
Given the reciprocal nature of the SFRS1/miR-7 circuit, an alternative possibility is that SF2/ASF may be critical for the homeostasis of miR-7, an important regulatory molecule orchestrating diverse cellular functions (Li et al., 2009
). In this scenario, SF2/ASF serves as a “rheostat” to sense the cellular level of mature miR-7. Fluctuations in miR-7 level are expected to drive the expression of endogenous SF2/ASF in an opposite direction. Because SF2/ASF is required for efficient pri-miR-7 maturation, the negative feedback loop in effect buffers the noise in miR-7 expression to better maintain its steady state.
Besides the SFRS1/miR-7 feedback loop, a circuit with the same architecture has been reported between the transcriptional factor E2F and the miR-17-92 cluster (Woods et al., 2007
). Mathematical modeling showed that miR-17-92 is essential for the E2F/Myc cancer network to balance between cell proliferation and apoptosis (Aguda et al., 2008
). Therefore, it will be of interest to further investigate the SFRS1/miR-7 feedback loop from a network perspective in order to fully understand its functional significance.
One key finding of this study is a splicing-independent function of SF2/ASF in pri-miRNA processing. We provided multiple lines of evidence that SF2/ASF promotes miR-7 maturation at the Drosha cleavage step. HnRNPA1, another well-known alternative splicing factor, serves as an auxiliary factor in pri-miR-18a processing (Guil and Caceres, 2007
). In fact, binding of hnRNPA1 to pri-miR-18a introduces a conformational change in its terminal loop to allow more efficient Drosha cleavage (Michlewski et al., 2008
). It is plausible that SF2/ASF may function in a similar manner in promoting pri-miR-7 maturation. In addition, we observed a dominant-negative effect of SF2ΔRS on miR-7 production. Since the RS domain of SR proteins often mediates protein-protein interactions, this result suggests that an additional factor(s) might also be involved to enhance pri-miRNA maturation.
Both alternative splicing and miRNA processing coincide in the last intron of the hnRNPK gene, which provides a unique opportunity to examine the potential cooperation and/or competition between the spliceosome and microprocessor (Drosha/DGCR8 complex). Our results showed that SF2/ASF promotes proximal 3′ splice-site usage of the miR-7-containing intron. However, the ability of SF2/ASF to promote miR-7 expression is slightly reduced when the proximal 3′ss is used (), suggesting a context dependence. It has been shown that processing of intronic miRNAs takes place before intron removal (Kim and Kim, 2007
). Therefore, our data imply that spliceosome assembly at the nearby splice sites may affect the processing of intronic miRNAs. One attractive model is that the spliceosome might compete with the miRNA processing machinery for common auxiliary factors (e.g., SF2/ASF). Alternatively, intronic miRNAs might adopt different local conformations depending on alternative splice-site usage. We therefore propose that the functions of SF2/ASF in mRNA and miRNA processing might not be mutually exclusive; instead they might modulate each other in a context-dependent manner.
The functional involvement of SF2/ASF in pri-miRNA processing is not limited to miR-7. We acquired initial evidence that SF2/ASF is also involved in the maturation of miR-221, miR-222, and miR-29b-1 (). While enhanced Drosha cleavage is likely to be involved in the case of miR-221 and miR-222, SF2/ASF might promote the expression of miR-29b-1 (but not miR-29b-2) at a postcropping step (e.g., pre-miRNA export and/or Dicer cleavage). Conversely, our profiling results showed that the expression of a subset of miRNAs can also be repressed by SF2/ASF. It will be interesting to find out whether SF2/ASF is directly involved and can play a negative role in pri-miRNA processing in a substrate-specific manner. Supporting these notions, SF2/ASF is a shuttling protein that plays diverse roles in both the nucleus and cytoplasm (Caceres et al., 1998
; Sanford et al., 2004
). Furthermore, there is a precedent that KSRP, a well-known factor involved in alternative splicing and mRNA degradation, regulates the biogenesis of a subset of miRNAs at multiple distinct steps (Trabucchi et al., 2009
). Lastly, RNA-binding proteins other than SF2/ASF (e.g., hnRNP A1 or KSRP) also participate in miRNA processing. It will be important to determine the substrate specificities of, as well as the potential cooperation/competition between, different RNA-binding proteins in controlling tissue- and cell type-specific miRNA expression.
Both splicing factors and miRNAs regulate the expression of a large number of protein-coding genes. Therefore, they may share common downstream targets and/or signaling pathways. Such a “wiring” structure has been reported between transcriptional factors and their regulated miRNAs and is a recurring motif in transcriptional gene networks (Lee et al., 2007
; Shalgi et al., 2007
). One well-known example is the miR-34 family of miRNAs, which are direct transcriptional targets of p53 and act in concert with other p53 downstream effectors to inhibit inappropriate cell proliferation (Chang et al., 2007
; He et al., 2007
). Notably, SF2/ASF can also act as an oncoprotein by activating the mTOR pathway (Karni et al., 2007
). In addition, several SF2/ASF-upregulated miRNAs (e.g., miR-221 and miR-222) have been implicated in tumorigenesis (Sun et al., 2009
; Terasawa et al., 2009
). It is possible that these miRNAs may contribute to SF2/ASF-driven tumorigenesis. One attractive scenario is that splicing regulation and miRNA-mediated gene repression may be broadly coordinated in posttranscriptional gene regulatory networks, a possibility that deserves systematic characterization.