Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

Recoding UAG to selenocysteine in Saccharomyces cerevisiae.

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.

Creating Selenocysteine-Specific Reporters Using Inteins.

The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systems to facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote high-throughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins.

Post-translational modifications (PTMs) can occur on almost all amino acids in eukaryotes as a key mechanism for regulating protein function. The ability to study the role of these modifications in various biological processes requires techniques to modify proteins site-specifically. One strategy for this is genetic code expansion (GCE) in bacteria. The low frequency of post-translational modifications in bacteria makes it a preferred host to study whether the presence of a post-translational modification influences a protein's function. Genetic code expansion employs orthogonal translation systems engineered to incorporate a modified amino acid at a designated protein position. Selenoproteins, proteins containing selenocysteine, are also known to be post-translationally modified. Selenoproteins have essential roles in oxidative stress, immune response, cell maintenance, and skeletal muscle regeneration. Their complicated biosynthesis mechanism has been a hurdle in our understanding of selenoprotein functions. As technologies for selenocysteine insertion have recently improved, we wanted to create a genetic system that would allow the study of post-translational modifications in selenoproteins. By combining genetic code expansion techniques and selenocysteine insertion technologies, we were able to recode stop codons for insertion of N ε-acetyl-l-lysine and selenocysteine, respectively, into multiple proteins. The specificity of these amino acids for their assigned position and the simplicity of reverting the modified amino acid via mutagenesis of the codon sequence demonstrates the capacity of this method to study selenoproteins and the role of their post-translational modifications. Moreover, the evidence that Sec insertion technology can be combined with genetic code expansion tools further expands the chemical biology applications.

The selenocysteine toolbox: A guide to studying the 21st amino acid.

Selenocysteine (Sec), the 21st genetically encoded amino acid, is structurally similar to cysteine (Cys) but with a sulfur to selenium replacement. This small change confers Sec with related chemical properties to Cys but often with enhanced reactivity. In organisms, Sec is present in selenoproteins taking on various roles such as cellular maintenance, immune response, hormone regulation, and oxidative stress. The detailed reactions of Sec in these functions remains unclear and has been a difficult question to answer. This is related to the low natural expression of selenoproteins and their complicated biosynthesis pathway. As a result, the focus in selenoprotein research has been on the expansion of tools and techniques to promote research in this area. Over the past two decades there has been immense progress in the development of selenoprotein expression systems, Sec-detection methods, and genomic databases. In this review we have compiled these tools systematically, highlighting their strengths and clarifying the limitations, as a resource for future selenoprotein research.

Unconventional genetic code systems in archaea.

Archaea constitute the third domain of life, distinct from bacteria and eukaryotes given their ability to tolerate extreme environments. To survive these harsh conditions, certain archaeal lineages possess unique genetic code systems to encode either selenocysteine or pyrrolysine, rare amino acids not found in all organisms. Furthermore, archaea utilize alternate tRNA-dependent pathways to biosynthesize and incorporate members of the 20 canonical amino acids. Recent discoveries of new archaeal species have revealed the co-occurrence of these genetic code systems within a single lineage. This review discusses the diverse genetic code systems of archaea, while detailing the associated biochemical elements and molecular mechanisms.

Using selenocysteine-specific reporters to screen for efficient tRNASec variants.

The unique properties of selenocysteine (Sec) have generated an interest in the scientific community to site-specifically incorporate Sec into a protein of choice. Current technologies have rewired the natural Sec-specific translation factor-dependent selenoprotein biosynthesis pathway by harnessing the canonical elongation factor (EF-Tu) to simplify the requirements for Sec incorporation in Escherichia coli. This strategy is versatile and can be applied to Sec incorporation at any position in a protein of interest. However, selenoprotein production is still limited by yield and serine misincorporation. This protocol outlines a method in E. coli to design and optimize tRNA libraries which can be selected and screened for by the use of Sec-specific intein-based reporters. This provides a fast and simple way to engineer tRNAs with enhanced Sec-incorporation ability.

Intein-based Design Expands Diversity of Selenocysteine Reporters.

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.

Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Gld2 activity and RNA specificity is dynamically regulated by phosphorylation and interaction with QKI-7.

In the cell, RNA abundance is dynamically controlled by transcription and decay rates. Posttranscriptional nucleotide addition at the RNA 3' end is a means of regulating mRNA and RNA stability and activity, as well as marking RNAs for degradation. The human nucleotidyltransferase Gld2 polyadenylates mRNAs and monoadenylates microRNAs, leading to an increase in RNA stability. The broad substrate range of Gld2 and its role in controlling RNA stability make the regulation of Gld2 activity itself imperative. Gld2 activity can be regulated by post-translational phosphorylation via the oncogenic kinase Akt1 and other kinases, leading to either increased or almost abolished enzymatic activity, and here we confirm that Akt1 phosphorylates Gld2 in a cellular context. Another means to control Gld2 RNA specificity and activity is the interaction with RNA binding proteins. Known interactors are QKI-7 and CPEB, which recruit Gld2 to specific miRNAs and mRNAs. We investigate the interplay between five phosphorylation sites in the N-terminal domain of Gld2 and three RNA binding proteins. We found that the activity and RNA specificity of Gld2 is dynamically regulated by this network. Binding of QKI-7 or phosphorylation at S62 relieves the autoinhibitory function of the Gld2 N-terminal domain. Binding of QKI-7 to a short peptide sequence within the N-terminal domain can also override the deactivation caused by Akt1 phosphorylation at S116. Our data revealed that Gld2 substrate specificity and activity can be dynamically regulated to match the cellular need of RNA stabilization and turnover.

Introducing Selenocysteine into Recombinant Proteins in Escherichia coli.

Selenoproteins contain the 21st amino acid, selenocysteine. Selenocysteine is the only amino acid that is synthesized on its cognate tRNA, and it is inserted at specific recoded UGA stop codons via a complex translation system. Although highly similar to cysteine, selenocysteine has unique properties, including a stronger nucleophilic ability and lower reduction potential. Efforts to site-specifically incorporate selenocysteine to create recombinant selenoproteins involve a recoded UAG stop codon and expression of the necessary selenocysteine translation machinery. This article presents a protocol for expressing and purifying selenoproteins in Escherichia coli. © 2021 Wiley Periodicals LLC. Basic Protocol: Recombinant selenoprotein production in E. coli using a rewired translation system.

Using Genetic Code Expansion for Protein Biochemical Studies.

Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue- or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein-protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein-protein interactions and offer considerations for future work in expanding genetic code expansion methods.

Gld2 activity is regulated by phosphorylation in the N-terminal domain.

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3'-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

The Role of 3' to 5' Reverse RNA Polymerization in tRNA Fidelity and Repair.

The tRNAHis guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3' to 5' synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3' to 5' chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed.

RNA surveillance by uridylation-dependent RNA decay in Schizosaccharomyces pombe.

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3'-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5'-NxAUUAAAA-3' RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.

MiRAR-miRNA Activity Reporter for Living Cells.

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase.

tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

Tipping the balance of RNA stability by 3' editing of the transcriptome.

BACKGROUND: The regulation of active microRNAs (miRNAs) and maturation of messenger RNAs (mRNAs) that are competent for translation is a crucial point in the control of all cellular processes, with established roles in development and differentiation. Terminal nucleotidyltransferases (TNTases) are potent regulators of RNA metabolism. TNTases promote the addition of single or multiple nucleotides to an RNA transcript that can rapidly alter transcript stability. The well-known polyadenylation promotes transcript stability while the newly discovered but ubiquitious 3'-end polyuridylation marks RNA for degradation. Monoadenylation and uridylation are essential control mechanisms balancing mRNA and miRNA homeostasis. SCOPE OF REVIEW: This review discusses the multiple functions of non-canonical TNTases, focusing on their substrate range, biological functions, and evolution. TNTases directly control mRNA and miRNA levels, with diverse roles in transcriptome stabilization, maturation, silencing, or degradation. We will summarize the current state of knowledge on non-canonical nucleotidyltransferases and their function in regulating miRNA and mRNA metabolism. We will review the discovery of uridylation as an RNA degradation pathway and discuss the evolution of nucleotidyltransferases along with their use in RNA labeling and future applications as therapeutic targets. MAJOR CONCLUSIONS: The biochemically and evolutionarily highly related adenylyl- and uridylyltransferases play antagonizing roles in the cell. In general, RNA adenylation promotes stability, while uridylation marks RNA for degradation. Uridylyltransferases evolved from adenylyltransferases in multiple independent evolutionary events by the insertion of a histidine residue into the active site, altering nucleotide, but not RNA specificity. GENERAL SIGNIFICANCE: Understanding the mechanisms regulating RNA stability in the cell and controlling the transcriptome is essential for efforts aiming to influence cellular fate. Selectively enhancing or reducing RNA stability allows for alterations in the transcriptome, proteome, and downstream cellular processes. Genetic, biochemical, and clinical data suggest TNTases are potent targets for chemotherapeutics and have been exploited for RNA labeling applications. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2).

The nontemplated addition of single or multiple nucleotides to RNA transcripts is an efficient means to control RNA stability and processing. Cytoplasmic RNA adenylation and the less well-known uridylation are post-transcriptional mechanisms regulating RNA maturation, activity, and degradation. Gld2 is a member of the noncanonical poly(A) polymerases, which include enzymes with varying nucleotide specificity, ranging from strictly ATP to ambiguous to exclusive UTP adding enzymes. Human Gld2 has been associated with transcript stabilizing miRNA monoadenylation and cytoplasmic mRNA polyadenylation. Most recent data have uncovered an unexpected miRNA uridylation activity, which promotes miRNA maturation. These conflicting data raise the question of Gld2 nucleotide specificity. Here, we biochemically characterized human Gld2 and demonstrated that it is a bona fide adenylyltransferase with only weak activity toward other nucleotides. Despite its sequence similarity with uridylyltransferases (TUT4, TUT7), Gld2 displays an 83-fold preference of ATP over UTP. Gld2 is a promiscuous enzyme, with activity toward miRNA, pre-miRNA, and polyadenylated RNA substrates. Apo-Gld2 activity is restricted to adding single nucleotides and processivity likely relies on additional RNA-binding proteins. A phylogeny of the PAP/TUTase superfamily suggests that uridylyltransferases, which are derived from distinct adenylyltransferase ancestors, arose multiple times during evolution via insertion of an active site histidine. A corresponding histidine insertion into the Gld2 active site alters substrate specificity from ATP to UTP.