Calcium regulator hidden in a long “noncoding” RNA

Sci. Signal.  03 Mar 2015:
Vol. 8, Issue 366, pp. ec48
DOI: 10.1126/scisignal.aab0080

Muscle contraction is controlled by Ca2+ released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). The amount of Ca2+ stored in the SR is controlled by Ca2+-ATPases of the SERCA family in the SR membrane. In cardiac muscle and in slow twitch skeletal muscle, small structurally and functionally related proteins called phospholamban (PLN) and sarcolipin (SLN), respectively, inhibit SERCA to limit the amount of Ca2+ stored and thus regulate contractility. Anderson et al. identified the protein that performs this function in fast twitch skeletal muscle and named it myoregulin (MLN). Unexpectedly, this 46-amino-acid protein was encoded within an RNA that was annotated as a long noncoding RNA. In vitro transcription and translation of the MLN RNA produced a 5-kD peptide of the expected size for MLN, and introduction of a tagged MLN by CRISPR/Cas9-mediated recombination in the myoblast cell line C2C12 also produced a protein of the expected size. RNA analysis showed that mice expressed PLN specifically in heart muscle, SLN early in development in slow twitch skeletal muscle, and MLN in fetal and adult skeletal muscle after the switch to fast twitch. Structural modeling predicted that MLN adopted a helical motif and could dock onto SERCA in the same manner as PLN or SLN. Green fluorescent protein (GFP)–tagged MLN introduced into mouse skeletal muscle colocalized with SERCA, and hemagglutinin-tagged MLN coimmunoprecipitated with SERCA in a manner dependent on the residues conserved with SLN and PLN. Ca2+ imaging of C2C12 cells or a nonmuscle cell line indicated that overexpression of MLN reduced Ca2+ uptake into internal stores and the peak of Ca2+ released by RyRs, respectively. The MLN promoter region contains consensus binding sites for the transcription factors MyoD and MEF2. Reporter analysis showed that these were functional in stimulating expression, and chromatin immunoprecipitation analysis confirmed the binding of MyoD in C2C12 cells. A LacZ transgene controlled by the MLN promoter showed muscle-specific expression in mice that depended on the MEF2- and MyoD-binding sites. Mice in which MLN was knocked down by TALEN-mediated recombination exhibited increased performance in a forced running test, and myoblasts isolated from these mice exhibited increased SR Ca2+ and an increased peak of Ca2+ released from the SR. This study reveals the challenges of classifying RNAs and the need to continue the search for short functionally important proteins or “micropeptides” that may be encoded in sequences considered to be noncoding.

D. M. Anderson, K. M. Anderson, C.-L. Chang, C. A. Makarewich, B. R. Nelson, J. R. McAnally, P. Kasaragod, J. M. Shelton, J. Liou, R. Bassel-Duby, E. N. Olson, A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell 160, 595–606 (2015). [PubMed]