Summary
In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.
by
Patricia J. Campbell;
Constantinos S. Kyriakis;
Nicolle Marshall;
Suganthi Suppiah;
Jill Seladi-Schulman;
Shamika Danzy;
Anice Carmen Lowen;
John Steel
Tetrahydrobiopterin (BH4) responsiveness is currently defined as a decrease in plasma phenylalanine concentrations in patients with phenylketonuria (PKU). This definition does not offer insight beyond the initial assessment of patients, which may lead to treatment ambiguity in patients who only experience an initial decrease in plasma phenylalanine concentrations. We present our experience with a novel classification approach using sequentially-applied criteria. Plasma phenylalanine concentrations were measured at baseline and after one month of BH4 therapy (20 mg/kg/day) in 58 PKU patients (34M, 24F; age 17.3 ± 11.0 years). Thirty-two patients (55.2%) were classified as –preliminary responders“ at one month, experiencing at least a 15% decrease in plasma phenylalanine concentrations. Preliminary responders’ ability to liberalize their dietary restrictions was then systematically assessed. –Definitive responders“ were defined as preliminary responders who could increase their dietary phenylalanine tolerance by at least 300 mg/day and lower prescribed medical food needs by at least 25% while maintaining metabolic control (plasma phenylalanine <360 μmol/L) and consuming adequate dietary protein. Preliminary responders who could not liberalize their diets according to these criteria were classified as –provisional responders.“ Nineteen patients (32.8% of patients initiating BH4 therapy) met the definitive responder criteria, increasing dietary phenylalanine tolerance from 704 ± 518 mg/day to 1922 ± 612 mg/day and reducing medical food to 16.7 ±19.5% of their baseline prescription. Nine patients (15.5% of patients initiating BH4 therapy) were classified as provisional responders, all remaining on 100% of their baseline medical food prescription. From this classification approach, a subgroup of provisionally responsive patients emerged who experienced an initial decrease in plasma phenylalanine concentrations but who could not substantially increase their dietary phenylalanine tolerance or decrease medical food needs. Diet liberalization is an essential component of BH4-responsiveness classification.
Background: NR5A nuclear receptors are important pharmaceutical targets with poorly understood ligand regulation. Sequence divergence has potentially altered their ligand response in model organisms.
Results: Sequence divergence has differentially impacted ligand binding and protein dynamics in NR5A orthologs.
Conclusion: Mouse LRH-1 is a phospholipid-responsive receptor, whereas Drosophila NR5A2 is not.
Significance: Mice are viable therapeutic models for LRH-1-dependent diseases.
Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize 'individual' poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and α-actinin, and the elongation factor, EF-1α (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a 'microdomain' where mRNA is attached and functionally expressed.
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem-loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2α subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.
Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). The therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs). To understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. We isolated CPCs from cardiac biopsies of patients with congenital heart disease (n = 29) and the lead-in patients with HLHS in the CHILD trial (n = 5). We sequenced CPC-EVs, and measured EV inflammatory, fibrotic, angiogeneic, and migratory responses. Overall, CPC-EV RNAs involved in pro-reparative outcomes had a significant fit to cardiac development and signaling pathways. Using a model trained on previously collected CPC-EVs, we predicted in vitro outcomes for the CHILD clinical samples. Finally, CPC-EV angiogenic performance correlated to clinical improvements in right ventricle performance.
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William H. Hudson;
Mark R. Pickard;
Ian Mitchelle S. de Vera;
Emily G. Kuiper;
Mirna Mourtada-Maarabouni;
Graeme Conn;
Douglas J. Kojetin;
Gwyn T Williams;
Eric Ortlund
The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.
The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate premRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.
In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.