5'UTR G-quadruplex structure enhances translation in size dependent manner.

Translation initiation in bacteria is frequently regulated by various structures in the 5' untranslated region (5'UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5'UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5'UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5'UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression.

Single-molecule observation of G-quadruplex and R-loop formation induced by transcription.

Potential G-quadruplex forming sequences (PQS) are enriched in cancer-related genes and immunoglobulin class-switch recombination. They are prevalent in the 5'UTR of transcriptionally active genes, thereby contributing to the regulation of gene expression. We and others previously demonstrated that the PQS located in the non-template strand leads to an R-loop formation followed by a G-quadruplex (G4) formation during transcription. These structural changes increase mRNA production. Here, we present how single-molecule technique was used to observe cotranscriptional G4 and R-loop formation and to examine the impact on transcription, particularly for the initiation and elongation stages.

The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation.

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

BG4 antibody can recognize telomeric G-quadruplexes harboring destabilizing base modifications and lesions.

BG4 is a single-chain variable fragment antibody shown to bind various G-quadruplex (GQ) topologies with high affinity and specificity, and to detect GQ in cells, including GQ structures formed within telomeric TTAGGG repeats. Here, we used ELISA and single-molecule pull-down (SiMPull) detection to test how various lengths and GQ destabilizing base modifications in telomeric DNA constructs alter BG4 binding. We observed high-affinity BG4 binding to telomeric GQ independent of telomere length, although three telomeric repeat constructs that cannot form stable intramolecular GQ showed reduced affinity. A single guanine substitution with 8-aza-7-deaza-G, T, A, or C reduced affinity to varying degrees depending on the location and base type, whereas two G substitutions in the telomeric construct dramatically reduced or abolished binding. Substitution with damaged bases 8-oxoguanine and O6-methylguanine failed to prevent BG4 binding although affinity was reduced depending on lesion location. SiMPull combined with FRET revealed that BG4 binding promotes folding of telomeric GQ harboring a G to T substitution or 8-oxoguanine. Atomic force microscopy revealed that BG4 binds telomeric GQ with a 1:1 stoichiometry. Collectively, our data suggest that BG4 can recognize partially folded telomeric GQ structures and promote telomeric GQ stability.

Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates.

Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions. For complete details on the use and execution of this protocol, please refer to Djaja et al.1.

5'UTR G-quadruplex structure enhances translation in size dependent manner.

Translation initiation in bacteria is frequently regulated by various structures in the 5' untranslated region (5'UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aimed to explore how G4 formation in mRNA (RG4) at 5'UTR impacts translation using a T7-based in vitro translation system and in E. coli. We showed that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We found that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We proposed a physical barrier model in which bulky structures in 5'UTR prevent ribosome dislodging and thereby increase the translation output. This study provides biophysical insights into the regulatory role of 5'UTR structures in bacterial translation, highlighting their potential applications in tuning gene expression.

Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level.

Protein-protein and protein-RNA interactions are essential for cell function and survival. These interactions facilitate the formation of ribonucleoprotein complexes and biomolecular condensates via phase separation. Such assembly is involved in transcription, splicing, translation and stress response. When dysregulated, proteins and RNA can undergo irreversible aggregation which can be cytotoxic and pathogenic. Despite technical advances in investigating biomolecular condensates, achieving the necessary spatiotemporal resolution to deduce the parameters that govern their assembly and behavior has been challenging. Many laboratories have applied advanced microscopy methods for imaging condensates. For example, single molecule imaging methods have enabled the detection of RNA-protein interaction, protein-protein interaction, protein conformational dynamics, and diffusional motion of molecules that report on the intrinsic molecular interactions underlying liquid-liquid phase separation. This review will outline advances in both microscopy and spectroscopy techniques which allow single molecule detection and imaging, and how these techniques can be used to probe unique aspects of biomolecular condensates.

Nucleation and dissolution mechanism underlying amyotrophic lateral sclerosis/frontotemporal lobar dementia-linked fused in sarcoma condensates.

Fused in sarcoma (FUS) is a nuclear RNA-binding protein. Mutations in FUS lead to the mislocalization of FUS from the nucleus to the cytosol and formation of pathogenic aggregates in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), yet with unknown molecular mechanisms. Using mutant and stress conditions, we visualized FUS localization and aggregate formation in cells. We used single-molecule pull-down (SiMPull) to quantify the native oligomerization states of wildtype (WT) and mutant FUS in cells. We demonstrate that the NLS mutants exhibited the highest oligomerization (>3) followed by other FUS mutants (>2) and WT FUS which is primarily monomeric. Strikingly, the mutant FUS oligomers are extremely stable and resistant to treatment by high salt, hexanediol, RNase, and Karyopherin-ß2 and only soluble in GdnHCl and SDS. We propose that the increased oligomerization units of mutant FUS and their high stability may contribute to ALS/FTLD pathogenesis.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral parts of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area, and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

Switch-like compaction of poly(ADP-ribose) upon cation binding.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na+, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.

Regulation of Biomolecular Condensates by Poly(ADP-ribose).

Biomolecular condensates are reversible compartments that form through a process called phase separation. Post-translational modifications like ADP-ribosylation can nucleate the formation of these condensates by accelerating the self-association of proteins. Poly(ADP-ribose) (PAR) chains are remarkably transient modifications with turnover rates on the order of minutes, yet they can be required for the formation of granules in response to oxidative stress, DNA damage, and other stimuli. Moreover, accumulation of PAR is linked with adverse phase transitions in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In this review, we provide a primer on how PAR is synthesized and regulated, the diverse structures and chemistries of ADP-ribosylation modifications, and protein-PAR interactions. We review substantial progress in recent efforts to determine the molecular mechanism of PAR-mediated phase separation, and we further delineate how inhibitors of PAR polymerases may be effective treatments for neurodegenerative pathologies. Finally, we highlight the need for rigorous biochemical interrogation of ADP-ribosylation in vivo and in vitro to clarify the exact pathway from PARylation to condensate formation.

Switch-like Compaction of Poly(ADP-ribose) Upon Cation Binding.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a post-translational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na + , Mg 2+ , Ca 2+ , and spermine). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR. Significance: Poly(ADP-ribose) (PAR) is an RNA-like homopolymer that regulates DNA repair, RNA metabolism, and biomolecular condensate formation. Dysregulation of PAR results in cancer and neurodegeneration. Although discovered in 1963, fundamental properties of this therapeutically important polymer remain largely unknown. Biophysical and structural analyses of PAR have been exceptionally challenging due to the dynamic and repetitive nature. Here, we present the first single-molecule biophysical characterization of PAR. We show that PAR is stiffer than DNA and RNA per unit length. Unlike DNA and RNA which undergoes gradual compaction, PAR exhibits an abrupt switch-like bending as a function of salt concentration and by protein binding. Our findings points to unique physical properties of PAR that may drive recognition specificity for its function.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral components of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While all-atom molecular dynamics method can, in principle, elucidate the conformational changes responsible for the aging of the condensate, the applications of this method to protein condensate systems is conditioned by the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force field that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

A minimal construct of nuclear-import receptor Karyopherin-ß2 defines the regions critical for chaperone and disaggregation activity.

Karyopherin-ß2 (Kapß2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals of diverse cytoplasmic cargo for transport to the nucleus. Kapß2 cargo includes several disease-linked RNA-binding proteins with prion-like domains, such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RNA-binding proteins with prion-like domains are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Remarkably, Kapß2 prevents and reverses aberrant phase transitions of these cargoes, which is cytoprotective. However, the molecular determinants of Kapß2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapß2 is a super-helical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapß2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8 to 20 of Kapß2 recapitulate all salient features of Kapß2 activity. By contrast, Kapß2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapß2 construct for delivery in adeno-associated viruses as a potential therapeutic for amyotrophic lateral sclerosis/frontotemporal dementia, multisystem proteinopathy, and related disorders.

Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates.

Many biomolecular condensates, including nucleoli and stress granules, form via dynamic multivalent protein-protein and protein-RNA interactions. These molecular interactions nucleate liquid-liquid phase separation (LLPS) and determine condensate properties, such as size and fluidity. Here, we outline the experimental procedures for single-molecule fluorescence experiments to probe protein-RNA interactions underlying LLPS. The experiments include single-molecule Förster (Fluorescence) resonance energy transfer (smFRET) to monitor protein-induced conformational changes in the RNA, protein-induced fluorescence enhancement (PIFE) to measure protein-RNA encounters, and single-molecule nucleation experiments to quantify the association and buildup of proteins on a nucleating RNA. Together, these experiments provide complementary approaches to elucidate a molecular view of the protein-RNA interactions that drive ribonucleoprotein condensate formation.

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics.

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Helicase mediated vectorial folding of telomere G-quadruplex.

The G-rich single-stranded telomere overhang can self-fold into G-quadruplex (G4) structure both in vivo and in vitro. In somatic cells, telomeres shorten progressively due to the end-replication. In stem cells, however, telomeres are replenished by a special enzyme, telomerase which synthesizes single-stranded telomere overhang. The active extension by the telomerase releases G-rich overhang segmentally in 5' to 3' direction as the overhang folds into G4 structure after successive elongation. To replicate such vectorial G4 folding process, we employed a superhelicase, Rep-X to release the G-rich sequence gradually. Using single-molecule assay we demonstrated that the folded conformation achieved by the vectorial folding is inherently different from the post-folding where the entire overhang is allowed to fold at once. In addition, the vectorially folded overhangs are less stable and more accessible to a complementary C-rich strand and the telomere binding protein, POT1 compared to the post-folded state. The higher accessibility may have implications for the facile loading of shelterin proteins after DNA replication.

Vectorial folding of telomere overhang promotes higher accessibility.

Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5' to 3') and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5' to 3' direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.