N-RAP, a muscle-specific protein concentrated at myotendinous junctions in skeletal muscle and intercalated disks in cardiac muscle, has been implicated in myofibril assembly. N-RAP is concentrated in myofibril precursors during sarcomere assembly in cultured cardiomyocytes and early embryos. Distinct regions of N-RAP bind specific cytoskeletal proteins, and overexpression of these regions in cultured chick cardiomyocytes inhibits or arrests myofibril assembly. Thus, we hypothesize that N-RAP is a scaffolding protein that coordinates organization of alpha-actinin and actin in the initial premyofibril complexes that form at the cell periphery, and that deletion of N-RAP will yield cardiomyocytes incapable of assembling mature myofibrils. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by ~70% within two days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching ~50% of control levels within six days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations, while cell spreading was not affected. The effect of N-RAP knockdown on alpha-actinin organization was partially rescued by expression of N-RAP-LIM-IB, a deletion mutant containing the N-RAP LIM domain and simple repeats, but omitting the super repeats. This construct allows normal assembly of alpha-actinin into mature striations, but disrupts sarcomeric actin organization. N-RAP-LIM-IB expression prevented N-RAP knockdown by siRNA at both the mRNA and protein levels. Therefore, the mechanism by which N-RAP-LIM-IB rescues myofibril assembly is likely to be by its effect on endogenous N-RAP expression. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days six and seven, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that myofibril assembly is closely linked to N-RAP protein levels, and that N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-transcriptional mechanisms. Primary cultured mouse skeletal muscle cells contain mononuclear myoblasts that begin to fuse on the second day of culture to form multi-nucleated myotubes. As a prelude to RNAi knockdown experiments, we characterized myofibril assembly and N-RAP gene expression and localization during differentiation of primary cultured myotubes. In undifferentiated myoblasts, N-RAP colocalized with alpha-actinin at the cell periphery. Premyofibrils characterized by punctate alpha-actinin and N-RAP staining were concentrated at the ends or edges of newly formed myotubes. Within 3-4 days, mature myofibrils, characterized by broad striations of alpha-actinin, were abundant. Quantitative real-time PCR was used to quantitate expression of selected genes during myotube differentiation. These include putative catalysts of myofibril assembly such as N-RAP and Krp1, transient structural elements thought to be essential for assembly such as non-muscle myosin heavy chain IIb (nmMHCIIb), mature structural components such as sarcomeric alpha-actinin, and proteins that promote myogenic differentiation, such as muscle LIM protein (MLP). Expression of each of these genes increased 50-900 fold during the first 3-5 days in culture, with the exception of nmMHCIIb, which remained relatively constant. The initial time course of gene expression was similar for N-RAP, Krp1, and MLP, with each transcript increasing linearly over the first three days. However, MLP expression peaked at day 3 and then decreased to 20% of maximum, while N-RAP and Krp1 transcripts remained at their maximum level through day 6. Sarcomeric alpha-actinin expression lagged that of the other transcripts by one day, but reached a constant maximum level by day 3. The results show that the primary mouse myotube cultures should be an excellent system in which to apply RNAi technology to dissect the molecular mechanism of myofibril assembly. Future investigations will focus on preventing the initial accumulation of putative myofibril assembly catalysts using RNA interference.