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).
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.
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.
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•Increased levels of GDF11 cause increased circulating Activin A and GDF15 in mice•Supraphysiologic levels of GDF11 induce cachexia, anorexia, and muscle loss•Blockade of GDF15 spares anorexia, but not muscle loss•Blockade of the GDF11 receptor ActRII spares muscle loss and decreases anorexia
Jones et al. find that high levels of GDF11 in mice induce symptoms of cachexia: skeletal muscle loss and anorexia. The anorexia is due to GDF11-dependent upregulation of GDF15. Downregulation of GDF11 in settings in which it exists at high levels is predicted to be beneficial.
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-β-β-β-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.
► Immunoprecipitation of lncRNA-containing protein complexes via either a protein component or an lncRNA component. ► Insertion of MS2 coat protein-binding sites into lncRNAs to provide an RNA-based ...affinity-handle. ► Analysis of lncRNAs by RT coupled to either semi-quantitative or quantitative PCR.
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.
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.
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.
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.
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•LncMyoD is directly activated by MyoD during myogenesis•LncMyoD binds to IMPs and regulates mRNA translation•Human and mouse LncMyoD are functionally conserved despite low sequence homology
Long non-coding RNAs are regulators of various biological functions. Gong and Li et al. show that LncMyoD is a LncRNA target of MyoD during myogenesis and is required for myoblast differentiation by affecting IMP2-mediated mRNA translation. LncMyoD is functionally conserved between mouse and human, despite limited sequence homology.
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