-
RNA (New York, N.Y.) Jun 2024Cap-independent, or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog,...
Cap-independent, or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. Internal-ribosome-entry-site (IRES)-like cap-independent translation initiation does not require an unpaired 5' end for eIF binding, as these eIFs recruit the 40S ribosome at or near the start codon. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem loops, and the RNA helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. FGF-9 is a member of a subset of mRNAs that are cap-independently upregulated in breast and colorectal cancer cells, likely using an IRES-like mechanism. However, the DAP5 binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require proximity to an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186-nt FGF-9 5' UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV-crosslinking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5' UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5 binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.
PubMed: 38866431
DOI: 10.1261/rna.080013.124 -
Biochemistry Jul 2024Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that...
Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing riboswitch in by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.
Topics: Riboswitch; Thiamine Pyrophosphate; Escherichia coli; RNA Folding; Nucleic Acid Conformation; Transcription, Genetic; RNA, Bacterial; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Bacterial Proteins
PubMed: 38864595
DOI: 10.1021/acs.biochem.3c00665 -
Biomacromolecules Jun 2024In biology, nanomachines like the ribosome use nucleic acid templates to synthesize polymers in a sequence-specific, programmable fashion. Researchers have long been... (Review)
Review
In biology, nanomachines like the ribosome use nucleic acid templates to synthesize polymers in a sequence-specific, programmable fashion. Researchers have long been interested in using the programmable properties of nucleic acids to enhance chemical reactions via colocalization of reagents using complementary nucleic acid handles. In this review, we describe progress in using nucleic acid templates, handles, or splints to enhance the covalent coupling of peptides to other peptides or oligonucleotides. We discuss work in several areas: creating ribosome-mimetic systems, synthesizing bioactive peptides on DNA or RNA templates, linking peptides into longer molecules and bioactive antibody mimics, and scaffolding peptides to build protein-mimetic architectures. We close by highlighting the challenges that must be overcome in nucleic acid-templated peptide chemistry in two areas: making full-length, functional proteins from synthetic peptides and creating novel protein-mimetic architectures not possible through macromolecular folding alone.
PubMed: 38860980
DOI: 10.1021/acs.biomac.4c00372 -
Analytical Chemistry Jun 2024Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing...
Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials and reaching the market has led to a growing interest to better characterize the ON diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, and for the first time, we identify higher-order structures as the major cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions and melting profiles of several representative full-length ONs to first analyze ON folding and then run mass spectrometry and HILIC to underpin the link between their folding and diastereomer separation. On top, we show how one can either enhance or suppress diastereomer separation depending on chromatographic settings, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier. This work will significantly facilitate future HILIC-based characterization of PS-containing ONs; e.g., enabling monitoring of batch-to-batch diastereomer distributions in full-length siRNAs, a complex task that is now for the first time shown as possible on this delicate class of therapeutic double-stranded ONs.
Topics: Hydrophobic and Hydrophilic Interactions; Stereoisomerism; Oligonucleotides; RNA, Small Interfering; Nucleic Acid Conformation; Chromatography, Liquid
PubMed: 38855895
DOI: 10.1021/acs.analchem.4c01384 -
BioRxiv : the Preprint Server For... May 2024Recent studies have demonstrated that the mechanisms through which biopolymers like RNA interconvert between multiple folded structures are critical for their cellular...
Recent studies have demonstrated that the mechanisms through which biopolymers like RNA interconvert between multiple folded structures are critical for their cellular functions. A major obstacle to elucidating these mechanisms is the lack of experimental approaches that can resolve these interconversions between functionally relevant biomolecular structures. Here, using a nano-electronic device with microsecond time resolution, we dissect the complete set of structural rearrangements executed by an ultra-stable RNA, the UUCG stem-loop, at the single-molecule level. We show that the stem-loop samples at least four conformations along two folding pathways leading to two distinct folded structures, only one of which has been previously observed. By modulating its flexibility, the stem-loop can adaptively select between these pathways, enabling it to both fold rapidly and resist unfolding. This paradigm of stabilization through compensatory changes in flexibility broadens our understanding of stable RNA structures and is expected to serve as a general strategy employed by all biopolymers.
PubMed: 38853856
DOI: 10.1101/2024.05.27.595525 -
BioRxiv : the Preprint Server For... May 2024Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration,...
Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration, creating an elevated potential for intermolecular base pairing. However, the type and abundance of intermolecular base pairing in mRNA clusters is poorly characterized. Using single-molecule super-resolution microscopy, chemical probing for base accessibility, phase separation assays, and simulations, we demonstrated that mRNAs remain well-folded upon localization to germ granules. While most base pairing is intramolecular, mRNAs still display the ability for intermolecular base pairing, facilitating clustering without high sequence complementarity or significant melting of secondary structure. This base pairing among mRNAs is driven by scattered and discontinuous stretches of bases appearing on the surface of folded RNAs, providing multivalency to clustering but exhibits low probability for sustained interactions. Notably, engineered germ granule mRNAs with exposed GC-rich complementary sequences (CSs) presented within stable stem loops induce sustained base pairing in vitro and enhanced intermolecular interactions in vivo. However, the presence of these stem loops alone disrupts fly development, and the addition of GC-rich CSs exacerbates this phenotype. Although germ granule mRNAs contain numerous GC-rich CSs capable of stable intermolecular base pairing, they are primarily embedded by RNA folding. This study emphasizes the role of RNA folding in controlling the type and abundance of intermolecular base pairing, thereby preserving the functional integrity of mRNAs within the germ granules.
PubMed: 38853845
DOI: 10.1101/2024.05.31.596852 -
ELife Jun 2024CRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard...
CRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard guide RNA () that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.
Topics: Zebrafish; Animals; Gene Editing; RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; CRISPR-Associated Protein 9; Embryo, Nonmammalian; RNA Folding
PubMed: 38847802
DOI: 10.7554/eLife.90948 -
Journal of Molecular Biology Jun 2024HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and...
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
PubMed: 38838849
DOI: 10.1016/j.jmb.2024.168639 -
Brain, Behavior, and Immunity Jun 2024Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are...
Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are currently no FDA-approved cessation therapeutics. Repeated methamphetamine (METH) use induces long-term gene expression changes in brain regions associated with reward processing and drug-seeking behavior, and recent evidence suggests that methamphetamine-induced neuroinflammation may also shape behavioral and molecular responses to the drug. Microglia, the resident immune cells in the brain, are principal drivers of neuroinflammatory responses and contribute to the pathophysiology of substance use disorders. Here, we investigated transcriptional and morphological changes in dorsal striatal microglia in response to methamphetamine-taking and during methamphetamine abstinence, as well as their functional contribution to drug-taking behavior. We show that methamphetamine self-administration induces transcriptional changes associated with protein folding, mRNA processing, immune signaling, and neurotransmission in dorsal striatal microglia. Importantly, many of these transcriptional changes persist through abstinence, a finding supported by morphological analyses. Functionally, we report that microglial ablation increases methamphetamine-taking, possibly involving neuroimmune and neurotransmitter regulation. In contrast, microglial depletion during abstinence does not alter methamphetamine-seeking. Taken together, these results suggest that methamphetamine induces both short and long-term changes in dorsal striatal microglia that contribute to altered drug-taking behavior and may provide valuable insights into the pathophysiology of MUD.
PubMed: 38838836
DOI: 10.1016/j.bbi.2024.05.038 -
Science Advances Jun 2024Folding of the cerebral cortex is a key aspect of mammalian brain development and evolution, and defects are linked to severe neurological disorders. Primary folding...
Folding of the cerebral cortex is a key aspect of mammalian brain development and evolution, and defects are linked to severe neurological disorders. Primary folding occurs in highly stereotyped patterns that are predefined in the cortical germinal zones by a transcriptomic protomap. The gene regulatory landscape governing the emergence of this folding protomap remains unknown. We characterized the spatiotemporal dynamics of gene expression and active epigenetic landscape (H3K27ac) across prospective folds and fissures in ferret. Our results show that the transcriptomic protomap begins to emerge at early embryonic stages, and it involves cell-fate signaling pathways. The H3K27ac landscape reveals developmental cell-fate restriction and engages known developmental regulators, including the transcription factor . Manipulating expression in cortical progenitors changed their proliferation and the folding pattern in ferret, caused by selective transcriptional changes as revealed by single-cell RNA sequencing analyses. Our findings highlight the key relevance of epigenetic mechanisms in defining the patterns of cerebral cortex folding.
Topics: Animals; Cerebral Cortex; Ferrets; Gene Expression Regulation, Developmental; Epigenesis, Genetic; Transcription Factors; Transcriptome; Homeodomain Proteins; Histones; Gene Regulatory Networks
PubMed: 38838158
DOI: 10.1126/sciadv.adn1640