Electrophoretic mobility shift assays (EMSAs) for in vitro detection of protein-nucleic acid interactions.

Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al.1.

The LARP1 homolog Slr1p controls the stability and expression of proto-5'TOP mRNAs in fission yeast.

Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the translational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.

Crosstalk between the tRNA methyltransferase Trm1 and RNA chaperone La influences eukaryotic tRNA maturation.

tRNAs undergo an extensive maturation process involving posttranscriptional modifications often associated with tRNA structural stability and promoting the native fold. Impaired posttranscriptional modification has been linked to human disease, likely through defects in translation, mitochondrial function, and increased susceptibility to degradation by various tRNA decay pathways. More recently, evidence has emerged that bacterial tRNA modification enzymes can act as tRNA chaperones to guide tRNA folding in a manner independent from catalytic activity. Here, we provide evidence that the fission yeast tRNA methyltransferase Trm1, which dimethylates nuclear- and mitochondrial-encoded tRNAs at G26, can also promote tRNA functionality in the absence of catalysis. We show that WT and catalytic-dead Trm1 are active in an in vivo tRNA-mediated suppression assay and possess RNA strand annealing and dissociation activity in vitro, similar to previously characterized RNA chaperones. Trm1 and the RNA chaperone La have previously been proposed to function synergistically in promoting tRNA maturation, yet we surprisingly demonstrate that La binding to nascent pre-tRNAs decreases Trm1 tRNA dimethylation in vivo and in vitro. Collectively, these results support the hypothesis for tRNA modification enzymes that combine catalytic and noncatalytic activities to promote tRNA maturation, as well as expand our understanding of how La function can influence tRNA modification.

Heterogeneity in leukemia cells that escape drug-induced senescence-like state.

Erythropoietin (EPO) suppresses drug-induced apoptosis in EPO-receptor-positive leukemia cells and allows cells to persist after drug treatment by promoting cellular senescence. Importantly a small proportion of senescent cells can re-enter the cell cycle and resume proliferation after drug treatment, resulting in disease recurrence/persistence. Using a single-cell assay to track individual cells that exit a drug-induced senescence-like state, we show that cells exhibit asynchronous exit from a senescent-like state, and display different rates of proliferation. Escaped cells retain sensitivity to drug treatment, but display inter-clonal variability. We also find heterogeneity in gene expression with some of the escaped clones retaining senescence-associated gene expression. Senescent leukemia cells exhibit changes in gene expression that affect metabolism and senescence-associated secretory phenotype (SASP)-related genes. Herein, we generate a senescence gene signature and show that this signature is a prognostic marker of worse overall survival in AML and multiple other cancers. A portion of senescent leukemia cells depend on lysosome activity; chloroquine, an inhibitor of lysosome activity, promotes senolysis of some senescent leukemia cells. Our study indicates that the serious risks associated with the use of erythropoietin-stimulating agents (ESAs) in anemic cancer patients may be attributed to their ability to promote drug-tolerant cancer cells through the senescence program.

The fission yeast methyl phosphate capping enzyme Bmc1 guides 2'-O-methylation of the U6 snRNA.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5' γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2'-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2'-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5' γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2'-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2'-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.

Altered tRNA processing is linked to a distinct and unusual La protein in Tetrahymena thermophila.

Nascent pre-tRNAs are transcribed by RNA polymerase III and immediately bound by La proteins on the UUU-3'OH sequence, using a tandem arrangement of the La motif and an adjacent RNA recognition motif-1 (RRM1), resulting in protection from 3'-exonucleases and promotion of pre-tRNA folding. The Tetrahymena thermophila protein Mlp1 has been previously classified as a genuine La protein, despite the predicted absence of the RRM1. We find that Mlp1 functions as a La protein through binding of pre-tRNAs, and affects pre-tRNA processing in Tetrahymena thermophila and when expressed in fission yeast. However, unlike in other examined eukaryotes, depletion of Mlp1 results in 3'-trailer stabilization. The 3'-trailers in Tetrahymena thermophila are uniquely short relative to other examined eukaryotes, and 5'-leaders have evolved to disfavour pre-tRNA leader/trailer pairing. Our data indicate that this variant Mlp1 architecture is linked to an altered, novel mechanism of tRNA processing in Tetrahymena thermophila.

The methyl phosphate capping enzyme Bmc1/Bin3 is a stable component of the fission yeast telomerase holoenzyme.

The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bmc1/Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bmc1 associates with the telomerase holoenzyme and U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bmc1 with telomerase is independent of its methyltransferase activity, but rather that Bmc1 functions in telomerase holoenzyme assembly by promoting TER1 accumulation and Pof8 recruitment to TER1. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation.

Revisiting tRNA chaperones: New players in an ancient game.

tRNAs undergo an extensive maturation process including post-transcriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding of misfolded substrates into functional conformations. The best characterized tRNA chaperone is the La protein, which interacts with nascent RNA polymerase III transcripts to promote folding and offers protection from exonucleases. More recently, certain tRNA modification enzymes have also been demonstrated to possess tRNA folding activity distinct from their catalytic activity, suggesting that they may act as tRNA chaperones. In this review, we will discuss pioneering studies relating post-transcriptional modification to tRNA stability and decay pathways, present recent advances into the mechanism by which the RNA chaperone La assists pre-tRNA maturation, and summarize emerging research directions aimed at characterizing modification enzymes as tRNA chaperones. Together, these findings shed light on the importance of tRNA folding and how tRNA chaperones, in particular, increase the fraction of nascent pre-tRNAs that adopt a folded, functional conformation.

La proteins couple use of sequence-specific and non-specific binding modes to engage RNA substrates.

La shuttles between the nucleus and cytoplasm where it binds nascent RNA polymerase III (pol III) transcripts and mRNAs, respectively. La protects the 3' end of pol III transcribed RNA precursors, such as pre-tRNAs, through the use of a well-characterized UUU-3'OH binding mode. La proteins are also RNA chaperones, and La-dependent RNA chaperone activity is hypothesized to promote pre-tRNA maturation and translation at cellular and viral internal ribosome entry sites via binding sites distinct from those used for UUU-3'OH recognition. Since the publication of La-UUU-3'OH co-crystal structures, biochemical and genetic experiments have expanded our understanding of how La proteins use UUU-3'OH-independent binding modes to make sequence-independent contacts that can increase affinity for ligands and promote RNA remodeling. Other recent work has also expanded our understanding of how La binds mRNAs through contacts to the poly(A) tail. In this review, we focus on advances in the study of La protein-RNA complex surfaces beyond the description of the La-UUU-3'OH binding mode. We highlight recent advances in the functions of expected canonical nucleic acid interaction surfaces, a heightened appreciation of disordered C-terminal regions, and the nature of sequence-independent RNA determinants in La-RNA target binding. We further discuss how these RNA binding modes may have relevance to the function of the La-related proteins.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function.

La proteins have well-established roles in the maturation of RNA polymerase III transcripts, including pre-tRNAs. In addition to protecting the 3' end of pre-tRNAs from exonuclease digestion, La proteins also promote the native fold of the pre-tRNA using RNA chaperone activity. tRNA-mediated suppression in the fission yeast S. pombe has been an invaluable tool in determining the mechanistic basis by which La proteins promote the maturation of defective pre-tRNAs that benefit from RNA chaperone activity. More recently, tRNA-mediated suppression has been adapted to test for RNA chaperone function in the La-related proteins and in the promoting of tRNA function by tRNA modification enzymes. Thus tRNA-mediated suppression can be a useful assay for the investigation of various proteins hypothesized to promote tRNA folding through RNA chaperone related activities.

C-lection by the DM15 Motif Gets LARP1 to the TOP.

LARP1 proteins integrate the translation and stability of 5'TOP mRNAs with signaling from the mTOR pathway, but the mechanism is not well understood. In this issue of Structure, Cassidy et al. (2019) propose that the LARP1-DM15 motif modulates access to the 5'TOP mRNA's m7G-ppp-Cytosine cap.

An interdomain bridge influences RNA binding of the human La protein.

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3'OH-tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3'OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen-deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein-protein interdomain interaction in La proteins.

FMRP recruitment of ß-catenin to the translation pre-initiation complex represses translation.

Canonical Wnt/ß-catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/ß-catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context-dependent mechanisms regulating this pathway. Since protein-protein interactions underpin ß-catenin function and localization, we sought to identify novel ß-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where ß-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC ß-catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for ß-catenin, particularly in mRNA translation, and we confirm a direct interaction between ß-catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of ß-catenin to the messenger ribonucleoprotein and translational pre-initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering ß-catenin away from the pre-initiation complex. In conclusion, we present evidence that ß-catenin fulfills a previously unrecognized function in translational repression.

Human La binds mRNAs through contacts to the poly(A) tail.

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

The RNA chaperone La promotes pre-tRNA maturation via indiscriminate binding of both native and misfolded targets.

Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3'OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates.

Distinct Dynamic Modes Enable the Engagement of Dissimilar Ligands in a Promiscuous Atypical RNA Recognition Motif.

Conformational dynamics play a critical role in ligand binding, often conferring divergent activities and specificities even in species with highly similar ground-state structures. Here, we employ time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX) to characterize the changes in dynamics that accompany oligonucleotide binding in the atypical RNA recognition motif (RRM2) in the C-terminal domain (CTD) of human La protein. Using this approach, which is uniquely capable of probing changes in the structure and dynamics of weakly ordered regions of proteins, we reveal that binding of RRM2 to a model 23-mer single-stranded RNA and binding of RRM2 to structured IRES domain IV of the hepatitis C viral (HCV) RNA are driven by fundamentally different dynamic processes. In particular, binding of the single-stranded RNA induces helical "unwinding" in a region of the CTD previously hypothesized to play an important role in La and La-related protein-associated RNA remodeling, while the same region becomes less dynamic upon engagement with the double-stranded HCV RNA. Binding of double-stranded RNA also involves less penetration into the RRM2 binding pocket and more engagement with the unstructured C-terminus of the La CTD. The complementarity between TRESI-HDX and Δδ nuclear magnetic resonance measurements for ligand binding analysis is also explored.

Imprint Desorption Electrospray Ionization Mass Spectrometry Imaging for Monitoring Secondary Metabolites Production during Antagonistic Interaction of Fungi.

Direct analysis of microbial cocultures grown on agar media by desorption electrospray ionization mass spectrometry (DESI-MS) is quite challenging. Due to the high gas pressure upon impact with the surface, the desorption mechanism does not allow direct imaging of soft or irregular surfaces. The divots in the agar, created by the high-pressure gas and spray, dramatically change the geometry of the system decreasing the intensity of the signal. In order to overcome this limitation, an imprinting step, in which the chemicals are initially transferred to flat hard surfaces, was coupled to DESI-MS and applied for the first time to fungal cocultures. Note that fungal cocultures are often disadvantageous in direct imaging mass spectrometry. Agar plates of fungi present a complex topography due to the simultaneous presence of dynamic mycelia and spores. One of the most devastating diseases of cocoa trees is caused by fungal phytopathogen Moniliophthora roreri. Strategies for pest management include the application of endophytic fungi, such as Trichoderma harzianum, that act as biocontrol agents by antagonizing M. roreri. However, the complex chemical communication underlying the basis for this phytopathogen-dependent biocontrol is still unknown. In this study, we investigated the metabolic exchange that takes place during the antagonistic interaction between M. roreri and T. harzianum. Using imprint-DESI-MS imaging we annotated the secondary metabolites released when T. harzianum and M. roreri were cultured in isolation and compared these to those produced after 3 weeks of coculture. We identified and localized four phytopathogen-dependent secondary metabolites, including T39 butenolide, harzianolide, and sorbicillinol. In order to verify the reliability of the imprint-DESI-MS imaging data and evaluate the capability of tape imprints to extract fungal metabolites while maintaining their localization, six representative plugs along the entire M. roreri/T. harzianum coculture plate were removed, weighed, extracted, and analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Our results not only provide a better understanding of M. roreri-dependent metabolic induction in T. harzianum, but may seed novel directions for the advancement of phytopathogen-dependent biocontrol, including the generation of optimized Trichoderma strains against M. roreri, new biopesticides, and biofertilizers.

Analysis of metabolic changes in plant pathosystems by imprint imaging DESI-MS.

The response of plants to microbial pathogens is based on the production of secondary metabolites. The complexity of plant-pathogen interactions makes their understanding a challenging task for metabolomic studies requiring powerful analytical approaches. In this paper, the ability of ambient mass spectrometry to provide a snapshot of plant metabolic response to pathogen invasion was tested. The fluctuations of glycoalkaloids present in sprouted potatoes infected by the phytopathogen Pythium ultimum were monitored by imprint imaging desorption electrospray ionization mass spectrometry (DESI-MS). After 8 d from the inoculation, a decrease of the relative abundance of potato glycoalkaloids α-solanine (m/z 706) and α-chaconine (m/z 722) was observed, whereas the relative intensity of solanidine (m/z 398), solasodenone (m/z 412), solanaviol (m/z 430), solasodiene (m/z 396), solaspiralidine (m/z 428), γ-solanine/γ-chaconine (m/z 560) , ß-solanine (m/z 706), and ß-chaconine (m/z 722) increased. The progression of the disease, expressed by the development of brown necrotic lesions on the potato, led to the further decrease of all the glycoalkaloid metabolites. Therefore, the applicability of imprint imaging DESI-MS in studying the plant metabolic changes in a simple pathosystem was demonstrated with minimal sample preparation.