Computationally reconstructing cotranscriptional RNA folding from experimental data reveals rearrangement of non-native folding intermediates.

The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.

Use of the 'double diamond' design framework to nurture creativity in life sciences research.

Designers' work processes are shaped by a four-phase 'discover, define, develop, and deliver' model that alternates between divergent and convergent thinking. We suggest consideration of this conceptual scaffold in 'design sprint' workshops for graduate students in the life sciences and in design to promote creativity, interdisciplinary collaboration, and knowledge cocreation.

An outreach activity to enhance biochemistry pedagogy.

Students are self-motivated to learn when provided opportunities that connect theory and real-world applications. Here, we describe for biochemistry majors a newborn screening-focused outreach activity that seeks to develop students' mastery of disciplinary content and soft skills (e.g., critical thinking, teamwork, effective communication, community engagement) and to enhance student engagement.

The many faces of RNA-based RNase P, an RNA-world relic.

RNase P is an essential enzyme that catalyzes removal of the 5' leader from precursor transfer RNAs. The ribonucleoprotein (RNP) form of RNase P is present in all domains of life and comprises a single catalytic RNA (ribozyme) and a variable number of protein cofactors. Recent cryo-electron microscopy structures of representative archaeal and eukaryotic (nuclear) RNase P holoenzymes bound to tRNA substrate/product provide high-resolution detail on subunit organization, topology, and substrate recognition in these large, multisubunit catalytic RNPs. These structures point to the challenges in understanding how proteins modulate the RNA functional repertoire and how the structure of an ancient RNA-based catalyst was reshaped during evolution by new macromolecular associations that were likely necessitated by functional/regulatory coupling.

A tribute to Sidney Altman, one of the architects of modern RNA biology.

This special issue of JBC pays tribute to Sidney Altman, whose discovery of a catalytic role for RNA, a breakthrough made independently by Thomas Cech, overturned the long-held dogma that only proteins can serve as catalysts in biological systems. The discovery of RNA catalysis galvanized biologists to think expansively in new directions and has given rise to a remarkable RNAissance in science and medicine. The collection of articles begins with the story of the discovery of RNase P and builds up to the emerging picture of an unexpectedly vast repertoire of RNase P variants in the three domains of life, including insights derived from recent high-resolution structures on how RNAs, ribonucleoproteins, or protein scaffolds can be used variably to generate an active site for catalyzing the same RNA processing reaction. The series of articles ends with a discussion of more recently discovered endonucleases (Argonautes, Cas), whose parallels with RNase P underscore recurring themes in diverse biological contexts.

Structural basis for impaired 5' processing of a mutant tRNA associated with defects in neuronal homeostasis.

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.

Alternative Protein Topology-Mediated Evolution of a Catalytic Ribonucleoprotein.

The high-resolution structures of yeast RNase for mitochondrial RNA processing (MRP), a catalytic ribonucleoprotein (RNP), recently reported by Lan et al. and Perederina et al. illustrate how RNA-mediated selection of alternative protein conformations, sampled during stochastic excursions by polymorphic/metamorphic proteins, enabled RNAs and proteins to mutually influence their functional repertoires and shape RNP evolution.

Transfer RNAs: A treasure trove that keeps on giving.

Transfer RNAs are the adaptors in protein synthesis that provide the key link between the nucleic acid-based genetic blueprint and proteins. While the central role of tRNAs in protein synthesis has been known for over 60 years, recent discoveries of their many non-canonical functions and therapeutic potential have heightened interest in tRNAs. In this thematic series, we highlight some of the developments presented at the recent biennial "International tRNA Workshop". The topics chosen reflect advances that were enabled by the latest technological breakthroughs in structure determination and small RNA sequencing and emphasize the prospects and challenges of tRNA-based medicines to treat human diseases.

Mapping the MOB proteins' proximity network reveals a unique interaction between human MOB3C and the RNase P complex.

Distinct functions mediated by members of the monopolar spindle-one-binder (MOB) family of proteins remain elusive beyond the evolutionarily conserved and well-established roles of MOB1 (MOB1A/B) in regulating tissue homeostasis within the Hippo pathway. Since MOB proteins are adaptors, understanding how they engage in protein-protein interactions and help assemble complexes is essential to define the full scope of their biological functions. To address this, we undertook a proximity-dependent biotin identification approach to define the interactomes of all seven human MOB proteins in HeLa and human embryonic kidney 293 cell lines. We uncovered >200 interactions, of which at least 70% are unreported on BioGrid. The generated dataset reliably recalled the bona fide interactors of the well-studied MOBs. We further defined the common and differential interactome between different MOBs on a subfamily and an individual level. We discovered a unique association between MOB3C and 7 of 10 protein subunits of the RNase P complex, an endonuclease that catalyzes tRNA 5' maturation. As a proof of principle for the robustness of the generated dataset, we validated the specific interaction of MOB3C with catalytically active RNase P by using affinity purification-mass spectrometry and pre-tRNA cleavage assays of MOB3C pulldowns. In summary, our data provide novel insights into the biology of MOB proteins and reveal the first interactors of MOB3C, components of the RNase P complex, and hence an exciting nexus with RNA biology.

Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein.

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

Demonstrating the utility of sugar-phosphate phosphatases in coupled enzyme assays: galactose-1-phosphate uridylyltransferase as proof-of-concept.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P.

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Analysis of Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry.

Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein-specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to test multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography-mass spectrometry-based method for direct analysis of target proteins in cell lysates. This approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS). While we illustrate a proof of concept here using relatively straightforward examples, the use of IMAC-OBE-nMS to optimize conditions for large-scale protein production may become invaluable for expediting structural biology and biotherapeutic initiatives.

Piece by piece: Building a ribozyme.

The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient biological assemblies that were already present in the last universal common ancestor of all life. The large subunit rRNA in the ribosome and the RNA subunit of RNase P are the ribozyme components required for catalysis. Here, we explore the idea that these two large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA "fragments" smaller than the contemporary full-length versions and that they transitioned through distinct stages along a pathway that may also be relevant for the evolution of other non-coding RNAs.

Structure Tuning, Strong Second Harmonic Generation Response, and High Optical Stability of the Polar Semiconductors Na1-xKxAsQ2.

The mixed cation compounds Na1-xKxAsSe2 (x = 0.8, 0.65, 0.5) and Na0.1K0.9AsS2 crystallize in the polar noncentrosymmetric space group Cc. The AAsQ2 (A = alkali metals, Q = S, Se) family features one-dimensional (1D) 1/∞[AQ2-] chains comprising corner-sharing pyramidal AQ3 units in which the packing of these chains is dependent on the alkali metals. The parallel 1/∞[AQ2-] chains interact via short As···Se contacts, which increase in length when the fraction of K atoms is increased. The increase in the As···Se interchain distance increases the band gap from 1.75 eV in γ-NaAsSe2 to 2.01 eV in Na0.35K0.65AsSe2, 2.07 eV in Na0.2K0.8AsSe2, and 2.18 eV in Na0.1K0.9AsS2. The Na1-xKxAsSe2 (x = 0.8, 0.65) compounds melt congruently at approximately 316 °C. Wavelength-dependent second harmonic generation (SHG) measurements on powder samples of Na1-xKxAsSe2 (x = 0.8, 0.65, 0.5) and Na0.1K0.9AsS2 suggest that Na0.2K0.8AsSe2 and Na0.1K0.9AsS2 have the highest SHG response and exhibit significantly higher laser-induced damage thresholds (LIDTs). Theoretical SHG calculations on Na0.5K0.5AsSe2 confirm its SHG response with the highest value of d33 = 22.5 pm/V (χ333(2) = 45.0 pm/V). The effective nonlinearity for a randomly oriented powder is calculated to be deff = 18.9 pm/V (χeff(2) = 37.8 pm/V), which is consistent with the experimentally obtained value of deff = 16.5 pm/V (χeff(2) = 33.0 pm/V). Three-photon absorption is the dominant mechanism for the optical breakdown of the compounds under intense excitation at 1580 nm, with Na0.2K0.8AsSe2 exhibiting the highest stability.

The rice RNase P protein subunit Rpp30 confers broad-spectrum resistance to fungal and bacterial pathogens.

RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars.

Both kinds of RNase P in all domains of life: surprises galore.

RNase P, an essential housekeeping endonuclease needed for 5'-processing of tRNAs, exists in two distinct forms: one with an RNA- and the other with a protein-based active site. The notion that the protein form of RNase P exists only in eukaryotes has been upended by the recent discovery of a protein-only variant in Bacteria and Archaea. The use of these two divergent scaffolds, shaped by convergent evolution, in all three domains of life inspires questions relating to the ancestral form of RNase P, as well as their origins and function(s) in vivo. Results from our analysis of publicly available bacterial and archaeal genomes suggest that the widespread RNA-based ribonucleoprotein variant is likely the ancient form. We also discuss the possible genetic origins and function of RNase P, including how the simultaneous presence of its variants may contribute to the fitness of their host organisms.

Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits.

RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5' leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.

Chance and necessity in the evolution of RNase P.

RNase P catalyzes 5'-maturation of tRNAs in all three domains of life. This primary function is accomplished by either a ribozyme-centered ribonucleoprotein (RNP) or a protein-only variant (with one to three polypeptides). The large, multicomponent archaeal and eukaryotic RNase P RNPs appear disproportionate to the simplicity of their role in tRNA 5'-maturation, prompting the question of why the seemingly gratuitously complex RNP forms of RNase P were not replaced with simpler protein counterparts. Here, motivated by growing evidence, we consider the hypothesis that the large RNase P RNP was retained as a direct consequence of multiple roles played by its components in processes that are not related to the canonical RNase P function.