Control of myogenesis by rodent SINE-containing lncRNAs.

Staufen1-mediated mRNA decay (SMD) degrades mRNAs that harbor a Staufen1-binding site (SBS) in their 3' untranslated regions (UTRs). Human SBSs can form by intermolecular base-pairing between a 3' UTR Alu element and an Alu element within a long noncoding RNA (lncRNA) called a ½-sbsRNA. Since Alu elements are confined to primates, it was unclear how SMD occurs in rodents. Here we identify mouse mRNA 3' UTRs and lncRNAs that contain a B1, B2, B4, or identifier (ID) element. We show that SMD occurs in mouse cells via mRNA-lncRNA base-pairing of partially complementary elements and that mouse ½-sbsRNA (m½-sbsRNA)-triggered SMD regulates C2C12 cell myogenesis. Our findings define new roles for lncRNAs as well as B and ID short interspersed elements (SINEs) in mice that undoubtedly influence many developmental and homeostatic pathways.

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements.

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).

SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs.

UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which we show here are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with our finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. We discuss the remarkable balance between SMD and the two types of NMD in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis.

Biochemical analysis of long non-coding RNA-containing ribonucleoprotein complexes.

Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA.

Gene expression networks: competing mRNA decay pathways in mammalian cells.

Nonsense-mediated mRNA decay and Staufen1-mediated mRNA decay are mechanistically related pathways that serve distinct purposes. In the present article, we give an overview of each pathway. We describe how a factor that is common to both pathways results in their competition. We also explain how competition between the two pathways contributes to the differentiation of C2C12 myoblasts to multinucleated myotubes.

Supraphysiologic Administration of GDF11 Induces Cachexia in Part by Upregulating GDF15.

The age-related effects of GDF11 have been a subject of controversy. Here, we find that elevated GDF11 causes signs of cachexia in mice: reduced food intake, body weight, and muscle mass. GDF11 also elicited a significant elevation in plasma Activin A, previously shown to contribute to the loss of skeletal muscle. The effects of GDF11 on skeletal muscle could be reversed by administration of antibodies to the Activin type II receptors. In addition to the effects on muscle, GDF11 increased plasma GDF15, an anorectic agent. The anorexia, but not the muscle loss, could be reversed with a GDF15-neutralizing antibody. GDF15 upregulation is due to GDF11-induced recruitment of SMAD2/3 to the GDF15 promoter. Inhibition of GDF15 can restore appetite but cannot restore the GDF11-induced loss of muscle mass, which requires blockade of ActRII signaling. These findings are relevant for treatment of cachexia.

Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein.

Enhanced green fluorescent protein (EGFP) from Aequorea victoria was fused to the C terminal region of protein ZZ, an artificial synthetic IgG Fc fragment binding protein derived from tandem repeats of the B domain of protein A. The ZZ-EGFP fusion protein was expressed in Escherichia coli with a His(6) tag and purified in high yield by one-step Ni(2+) chelating affinity chromatography. It was then used in the immunoblot analysis of GST and TNFalpha as well as in immunofluorescent assays of 293T cells transfected with IRF3, an interferon regulatory factor which localized in cytoplasm without virus infection. The fusion protein also performed effectively in FACS analysis of surface integrin beta3 subunit on 293 T cells. The chimeric protein bound various antibodies from different animal sources, directed against a variety of proteins. Thus, ZZ-EGFP showed broad promise in potential immunological applications.

Affinity purification of long noncoding RNA-protein complexes from formaldehyde cross-linked mammalian cells.

Long noncoding RNAs (lncRNAs) are a class of recently identified untranslated RNA molecules that have been shown to function in diverse cellular processes. The purification and analysis of lncRNA-protein (lncRNP) complexes is critical toward understanding the normal physiological function of these molecules. Here, we describe the purification of lncRNP complexes from human cells using a FLAG-tagged MS2-phage coat protein (MS2 CP) that binds in sequence-specific fashion to MS2-phage coat protein-binding sites (MS2bs) with high affinity. In these experiments, a FLAG-tagged MS2 CP is transiently co-expressed with a version of the lncRNA into which 12 copies of the MS2bs have been inserted near its 3'-end. The lncRNA-FLAG-tagged MS2 CP complex is then isolated using an anti-FLAG antibody, allowing for characterization of associated cellular proteins and RNAs.

A long non-coding RNA, LncMyoD, regulates skeletal muscle differentiation by blocking IMP2-mediated mRNA translation.

Increasing evidence suggests that long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation, largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well conserved between human and mouse, its locus, gene structure, and function are preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation.

mRNA-mRNA duplexes that autoelicit Staufen1-mediated mRNA decay.

We report a new mechanism by which human mRNAs cross-talk: an Alu element in the 3' untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA, thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3'-UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3'-UTR SBS created by mRNA-mRNA base-pairing, we show that SMD targets both mRNAs in the duplex, provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the functional importance of mRNA-mRNA-triggered SMD in cell migration and invasion.

Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay.

Staufen1 (STAU1)-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded RNA (dsRNA)-binding protein STAU1 in their 3' untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human STAU1 (hSTAU1) homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, which we named the Staufen-swapping motif (SSM), and the dsRNA-binding domain 5 ('RBD'5) mediate protein dimerization: the two SSM α-helices of one molecule interact primarily through a hydrophobic patch with the two 'RBD'5 α-helices of a second molecule. 'RBD'5 adopts the canonical α-ß-ß-ß-α fold of a functional RBD, but it lacks residues and features required to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound healing and many other cellular processes.

Distance and helical phase dependence of synergistic transcription activation in cis-regulatory module.

Deciphering of the spatial and stereospecific constraints on synergistic transcription activation mediated between activators bound to cis-regulatory elements is important for understanding gene regulation and remains largely unknown. It has been commonly believed that two activators will activate transcription most effectively when they are bound on the same face of DNA double helix and within a boundary distance from the transcription initiation complex attached to the TATA box. In this work, we studied the spatial and stereospecific constraints on activation by multiple copies of bound model activators using a series of engineered relative distances and stereospecific orientations. We observed that multiple copies of the activators GAL4-VP16 and ZEBRA bound to engineered promoters activated transcription more effectively when bound on opposite faces of the DNA double helix. This phenomenon was not affected by the spatial relationship between the proximal activator and initiation complex. To explain these results, we proposed the novel concentration field model, which posits the effective concentration of bound activators, and therefore the transcription activation potential, is affected by their stereospecific positioning. These results could be used to understand synergistic transcription activation anew and to aid the development of predictive models for the identification of cis-regulatory elements.

Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays.

In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C-terminus of protein ZZ, a synthetic artificial IgG-Fc-fragment-binding protein derived from the B-domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N-terminus, was expressed in Escherichia coli and easily purified by one-step Ni2+-chelating affinity chromatography. Its fluorescence and IgG-binding activities were validated using fluorescence-spectrum analysis, ELISA and dot-blot analysis. Furthermore, in subsequent dot-blotting immunoanalysis of glutathione S-transferase and tumour necrosis factor-alpha, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence-conjugated antibodies, ZZ-DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)-conjugated antibodies, using ZZ-DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ-DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP-conjugated anti-IgGs.