It is becoming largely accepted that the noncoding portion of the genome rather than its coding counterpart is likely to account for the greater complexity of higher eukaryotes. Many new functions have been assigned to noncoding RNAs both in the nucleus and in the cytoplasm (Mattick, 2011; Nagano and Fraser, 2011
). Likewise, similar to what happened for the well-known small noncoding RNAs, long noncoding RNAs are now attracting much interest. Recent data suggest that coding and noncoding RNAs can regulate one another through their ability to compete for miRNA binding; these molecules have been termed competing endogenous RNA (ceRNA, Salmena et al., 2011
). ceRNAs can sequester miRNAs, thereby protecting their target RNAs from repression (Karreth et al., 2011
[this issue of Cell
]; Sumazin et al., 2011
[this issue of Cell
]; Tay et al., 2011
[this issue of Cell
In this paper, we identify a muscle-specific long noncoding RNA (linc-MD1) that displays decoy activity for two specific miRNAs and, in doing so, regulates their targets in a molecular circuitry affecting the differentiation program.
We show that linc-MD1 is encoded by a genomic locus containing the miR-206 and miR-133b coding regions and demonstrate that there is a complex architecture in terms of transcriptional control in this locus: while miR-206 is expressed autonomously from its own proximal promoter, miR-133b is cotranscribed with linc-MD1 RNA which derives from a 13 Kb distal promoter.
Here, we provide evidence of the existence of two distinct promoters: (1) miR-206 is already expressed in growing myoblasts, whereas miR-133b and linc-MD1 are activated only upon differentiation; (2) 5′ RACE and promoter fusion experiments indicate the existence of two transcriptional regulatory elements (DIST and PROX); (3) ChIP experiments for RNA Polymerase II and different markers of chromatin activity indicate a well-defined chromatin organization of the two transcriptional units. In particular, in growth conditions, only the PROX promoter displayed markers of transcriptional activation, and no RNAPII loading was detected on the miR-133b region. These data suggest that miR-133b mainly originates from the DIST promoter by processing of linc-MD1.
linc-MD1 accumulates as a cytoplasmic poly-A+ RNA, supporting the conclusion that this species is the remaining portion of the transcript that escaped Drosha cleavage inside the nucleus. We prove that indeed miR-133b is produced with ectopic expression of linc-MD1. In order to avoid a possible confusion between the effect of linc-MD1 and that of miR-133b release, a mutant linc-MD1 derivative lacking the ability to release miR-133b was utilized in most of the overexpression experiments. Future work will address the mechanism regulating the relative ratio between miR-133b processing and the export of the unprocessed precursor.
Notably, we show that transcriptional activation of the linc-MD1 promoter correlates with the formation of a DNA loop in which the distal and proximal promoters (and the polyadenylation region) are connected in a functional/structural interaction. So far, gene loops have been shown to be transcription-dependent, because they are absent in nontranscribing conditions and have been suggested to represent specific structural domains of active chromatin (Tan-Wong et al., 2008; West and Fraser, 2005
). Therefore, a drastic structural change occurs in the miR-206/miR-133b locus; in growth conditions, only the proximal promoter is active and no long-distance interactions occur, while upon differentiation, a DNA loop is observed between distantly located regions, and this correlates with activation of the distal promoter and consolidation of the overall transcription of the locus.
As far as the function of linc-MD1 is concerned, we show that its modulation impinged on myogenesis. linc-MD1 RNAi-dependent downregulation in mouse myoblasts produced a decrease in the accumulation of myogenic markers, while its overexpression led to increased synthesis. linc-MD1 was found to be conserved in human cells: high levels were observed upon induction of differentiation in wild-type cells, whereas strongly reduced levels were found in Duchenne myoblasts. This observation is in line with the well-known delay observed in the differentiation program of DMD myoblasts (Cacchiarelli et al., 2011
). Notably, when linc-MD1 expression was restored to wild-type levels in DMD myoblasts, the timing and expression level of the myogenic factors were partially rescued toward wild-type levels.
According to the ceRNA hypothesis, lncRNAs may elicit their biological activity through their ability to act as endogenous decoys for miRNAs; such activity would in turn affect the distribution of miRNAs on their targets (Salmena et al., 2011
). We searched for miRNA recognition motifs in the linc-MD1 sequence and found that the presence of recognition sites for miR-133 and miR-135 could be reliably predicted. linc-MD1 was validated as target for both these miRNAs since they induced translational repression of a reporter gene.
Among the many different putative targets for these miRNAs, we discovered two mRNAs encoding for proteins with a relevant function in myogenesis: the Myocyte-specific enhancer factor 2C (MEF2C), targeted by miR-135 and Mastermind-like-1 (MAML1) controlled by miR-133.
Consistent with linc-MD1 being a decoy for miR-133 and miR-135, we proved that its depletion reduced the levels of both MAML1 and MEF2C while its overexpression produced an increase in protein accumulation. These data are consistent with the idea that decoy lincRNAs are transmodulators of gene expression through miRNA binding.
The identification of the targets indirectly controlled by linc-MD1 can be instrumental to explain the myogenic alterations observed upon its deregulation. MEF2C belongs to a family of transcription factors that bind the control regions of numerous muscle-specific genes activating their expression (Lin et al., 1997
). Moreover, it was shown to play a key role in differentiation of muscle cells (Lilly et al., 1995
) and in the maintenance of sarcomere integrity (Potthoff et al., 2007
On the other side, the Mastermind-like genes encode critical transcriptional coactivators for Notch signaling. Additionally, the MAML proteins were described as transcriptional coactivators in other signal transduction pathways including muscle differentiation: mice with a targeted disruption of the MAML1 gene had severe muscular dystrophy and MAML1-null
embryonic fibroblasts failed to undergo MyoD-induced myogenic differentiation (Shen et al., 2006
). Moreover, ectopic MAML1 expression in mouse myoblasts dramatically enhanced myotube formation and increased the expression of muscle-specific genes, while MAML1 knockdown inhibited differentiation.
Even more interesting is the finding that MAML1 and MEF2C specifically interact and act synergistically to activate several genes required for muscle development and function, including muscle creatine kinase (MCK). The MAML1 promyogenic effects were completely blocked upon activation of Notch signaling, which was associated with recruitment of MAML1 away from MEF2C to the Notch transcriptional complex (Wilson-Rawls et al., 1999
). Therefore, a crosstalk between MAML1 and Notch was postulated to influence myogenic differentiation.
In light of these notions, we proved that depletion of linc-MD1 led to repression of both MAML1 and MEF2C, while its overexpression restored their synthesis at high levels. Notably, in conditions of linc-MD1 excess, titrated repression of both MAML1 and MEF2C could be obtained by increasing miR-133 and miR-135 levels. This indicated a direct competition for miRNA binding between linc-MD1 and mRNAs, allowing us to conclude that the three components crosstalk with one another at the post-trascriptional level. Notably, MCK, a known target of MEF2C, coherently behaved as part of the circuitry: it increased upon linc-MD1 overexpression and decreased upon linc-MD1 RNAi.
In Duchenne muscle cells the rescue of linc-MD1 through lentiviral-mediated expression produced the recovery of both MAML1 and MEF2C synthesis and partial rescue of the correct timing of the differentiation program. These data allowed us to conclude, that also long noncoding RNAs play a relevant role in the complex network of regulatory interactions governing muscle terminal differentiation. Moreover, the discovery of the decoy role of lncRNA opens the road to the prediction and identification of new regulatory networks acting through miRNA competition.