MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Serum response factor (SRF), a cardiac enriched transcription factor, is required for the appearance of beating sarcomeres in the heart. SRF may also direct the expression of microRNAs (miRs) that inhibit the expression of cardiac regulatory factors. The recent knockout of miR-1-2, an SRF gene target, showed defective heart development, caused in part by the induction of GATA6, Irx4/5, and Hand2, that may alter cardiac morphogenesis, channel activity and cell cycling. SRF and co-factors play an obligatory role in cardiogenesis, as major determinants of myocyte differentiation not only by regulating the biogenesis of muscle contractile proteins but also by driving the expression of silencer miRNA.

Myocardin, a serum response factor (SRF)-dependent cofactor, is a potent activator of smooth muscle gene activity but a poor activator of cardiogenic genes in pluripotent 10T1/2 fibroblasts. Posttranslational modification of GATA4, another myocardin cofactor, by sumoylation strongly activated cardiogenic gene activity. Here, we found that myocardin's activity was strongly enhanced by SUMO-1 via modification of a lysine residue primarily located at position 445 and that the conversion of this residue to arginine (K445R) impaired myocardin transactivation. PIAS1 was involved in governing myocardin activity via its E3 ligase activity that stimulated myocardin sumoylation on an atypical sumoylation site(s) and by its physical association with myocardin. Myocardin initiated the expression of cardiac muscle-specified genes, such as those encoding cardiac ␣-actin and ␣-myosin heavy chain, in an SRF-dependent manner in 10T1/2 fibroblasts, but only in the presence of coexpressed SUMO-1/PIAS1. Thus, SUMO modification acted as a molecular switch to promote myocardin's role in cardiogenic gene expression.SUMOs (small ubiquitin-like modifiers) are implicated in numerous physiological and pathological processes through altering the functions of its target proteins. SUMO covalent linkage is usually through the lysine residue(s) in the consensus sequence KXE ( stands for a bulky hydrophobic amino acid, and X represents any residue) (37, 47). Some targets are involved with gene regulation, such as the coactivator P300 (14), the corepressor CtBP (32), and other transcription factors (38). SUMOs govern transcription target activity by altering DNA binding activity, subcellular localization, and/or protein stability. In contrast to what is seen for invertebrates, which have only one SUMO gene, at least four members of the SUMO gene family have been identified in vertebrates (7,13,16,25). SUMO-1 has about 50% homology to SUMO-2, -3, and -4. These SUMO members exert preferential target activities. For instance, SUMO modulation of RanGAP1 was favored by SUMO-1 but poorly modified by SUMO-2/-3 (49), while SUMO-2/-3 became highly active upon oxidative stress but SUMO-1 did not (49); thus, distinct roles for SUMO members under physiological and pathological states are exhibited.SUMO conjugation is part of an enzymatic cascade basically involving two enzymes, heterodimer E1-activating enzyme (SAE1/2) and E2-conjugating enzyme (Ubc9). Unlike ubiquitination, some sumoylation assays revealed that in the presence of E1 and E2, the E3 ligase was dispensable to accomplish SUMO conjugation. However, SUMO E3 ligases, such as PIAS1, contributed to the efficiency and specificity of SUMO conjugation (29, 57) and were attributed to the RING domain, which is similar to the corresponding structure in E3 ligases involved in the ubiquitination. The following three distinct classes of E3 ligase have been revealed: (i) the PIAS family (19), whose members, in addition to having their E3 ligase activity, may influence targets independently of sumoylation, as...