Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Structural insights into human co-transcriptional capping.

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Genome-scale molecular principles of mRNA half-life regulation in yeast.

Precise control of protein and messenger RNA (mRNA) degradation is essential for cellular metabolism and homeostasis. Controlled and specific degradation of both molecular species necessitates their engagements with the respective degradation machineries; this engagement involves a disordered/unstructured segment of the substrate traversing the degradation tunnel of the machinery and accessing the catalytic sites. However, while molecular factors influencing protein degradation have been extensively explored on a genome scale, and in multiple organisms, such a comprehensive understanding remains missing for mRNAs. Here, we analyzed multiple genome-scale experimental yeast mRNA half-life data in light of experimentally derived mRNA secondary structures and protein binding data, along with high-resolution X-ray crystallographic structures of the RNase machines. Results unraveled a consistent genome-scale trend that mRNAs comprising longer terminal and/or internal unstructured segments have significantly shorter half-lives; the lengths of the 5'-terminal, 3'-terminal, and internal unstructured segments that affect mRNA half-life are compatible with molecular structures of the 5' exo-, 3' exo-, and endoribonuclease machineries. Sequestration into ribonucleoprotein complexes elongates mRNA half-life, presumably by burying ribonuclease engagement sites under oligomeric interfaces. After gene duplication, differences in terminal unstructured lengths, proportions of internal unstructured segments, and oligomerization modes result in significantly altered half-lives of paralogous mRNAs. Side-by-side comparison of molecular principles underlying controlled protein and mRNA degradation in yeast unravels their remarkable mechanistic similarities and suggests how the intrinsic structural features of the two molecular species, at two different levels of the central dogma, regulate their half-lives on genome scale.

In vivo imaging of tagged mRNA in plant tissues using the bacterial transcriptional antiterminator BglG.

RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants.

RNA structure-wide discovery of functional interactions with multiplexed RNA motif library.

Biochemical assays and computational analyses have discovered RNA structures throughout various transcripts. However, the roles of these structures are mostly unknown. Here we develop folded RNA element profiling with structure library (FOREST), a multiplexed affinity assay system to identify functional interactions from transcriptome-wide RNA structure datasets. We generate an RNA structure library by extracting validated or predicted RNA motifs from gene-annotated RNA regions. The RNA structure library with an affinity enrichment assay allows for the comprehensive identification of target-binding RNA sequences and structures in a high-throughput manner. As a proof-of-concept, FOREST discovers multiple RNA-protein interaction networks with quantitative scores, including translational regulatory elements that function in living cells. Moreover, FOREST reveals different binding landscapes of RNA G-quadruplex (rG4) structures-binding proteins and discovers rG4 structures in the terminal loops of precursor microRNAs. Overall, FOREST serves as a versatile platform to investigate RNA structure-function relationships on a large scale.

ArfB can displace mRNA to rescue stalled ribosomes.

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.

Structure of the bacterial ribosome at 2 Å resolution.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Inside cells, proteins are produced by complex molecular machines called ribosomes. Techniques that allow scientists to visualize ribosomes at the atomic level, such as cryogenic electron microscopy (cryo-EM), help shed light on the structure of these molecular machines, revealing details of how they build proteins. Understanding how ribosomes work has many benefits, including the development of new antibiotics that can kill bacteria without affecting animal cells. Watson et al. used cryo-EM techniques with increased resolution to examine the ribosomes of the bacterium Escherichia coli in a higher level of detail than has been seen before. The results revealed two chemical modifications in proteins that form the ribosome that had not been observed in ribosomes previously. Additionally, a protein segment with a previously undescribed structure was identified close to the site where the ribosome reads the genetic instructions needed to make proteins. Further genetic analyses suggested these structures are in many related species, and may play important roles in how the ribosome works. Watson et al. were also able to see how paromomycin, an antibiotic used to treat parasitic infections, is positioned in the ribosome. The antibiotic interacts with a site near where the genetic code is read out, which might explain why certain changes to the antibiotic can interfere with its potency. Finally, the new ribosome structure reveals thousands of water molecules and metal ions that help keep the ribosome together as it produces proteins. This study shows the value of advances in cryo-EM technology and illustrates the importance of applying these techniques to other cell components. The results also reveal details of the ribosome useful for further research into this essential molecular machine.

Timing of Cytosine Methylation on Newly Synthesized RNA by Electron Microscopy.

Increasing evidence demonstrates that RNA nucleotides undergo epigenetic modifications, such as methylation on cytosine. Although the presence of modified bases on mRNA has been proven, their molecular significance is largely undefined. We describe here a methodology to dissect the timing of modification of cytosine to 5-methylcytosine (5mC or m5C) in relation to RNA elongation and processing. To do this we use chlorouridine and iodouridine, two synthetically modified nucleotide bases which can be recognized by RNA polymerase II and incorporated into nascent RNA. These modified bases are added to a cell culture for defined intervals of time, and then immunocytochemical staining using antibodies against the modified nucleotides is carried out. This procedure allows us to identify the range of time in which 5mC is produced in nascent mRNA. This method provides the ultra-resolution of electron microscopy and allows following nascent RNA molecules during their elongation.

Translation at first sight: the influence of leading codons.

First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.

A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate.

Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.

Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression.

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

The energy landscape of -1 ribosomal frameshifting.

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3' messenger RNA (mRNA) structures, but how these mRNA structures enhance -1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal -1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

Live-cell imaging of single mRNA dynamics using split superfolder green fluorescent proteins with minimal background.

The MS2 system, with an MS2 binding site (MBS) and an MS2 coat protein fused to a fluorescent protein (MCP-FP), has been widely used to fluorescently label mRNA in live cells. However, one of its limitations is the constant background fluorescence signal generated from free MCP-FPs. To overcome this obstacle, we used a superfolder GFP (sfGFP) split into two or three nonfluorescent fragments that reassemble and emit fluorescence only when bound to the target mRNA. Using the high-affinity interactions of bacteriophage coat proteins with their corresponding RNA binding motifs, we showed that the nonfluorescent sfGFP fragments were successfully brought close to each other to reconstitute a complete sfGFP. Furthermore, real-time mRNA dynamics inside the nucleus as well as the cytoplasm were observed by using the split sfGFPs with the MS2-PP7 hybrid system. Our results demonstrate that the split sfGFP systems are useful tools for background-free imaging of mRNA with high spatiotemporal resolution.

The landscape of eukaryotic mRNPs.

The proper regulation of mRNA processing, localization, translation, and degradation occurs on mRNPs. However, the global principles of mRNP organization are poorly understood. We utilize the limited, but existing, information available to present a speculative synthesis of mRNP organization with the following key points. First, mRNPs form a compacted structure due to the inherent folding of RNA. Second, the ribosome is the principal mechanism by which mRNA regions are partially decompacted. Third, mRNPs are 50%-80% protein by weight, consistent with proteins modulating mRNP organization, but also suggesting the majority of mRNA sequences are not directly interacting with RNA-binding proteins. Finally, the ratio of mRNA-binding proteins to mRNAs is higher in the nucleus to allow effective RNA processing and limit the potential for nuclear RNA based aggregation. This synthesis of mRNP understanding provides a model for mRNP biogenesis, structure, and regulation with multiple implications.

RDI Calculator: An Analysis Tool to Assess RNA Distributions in Cells.

Localization of RNAs to various subcellular destinations has emerged as a widely used mechanism that regulates a large proportion of transcripts in polarized cells. A number of methodologies have been developed that allow detection and imaging of RNAs at single-molecule resolution. However, methodologies to quantitatively describe RNA distributions are limited. Such approaches usually rely on the identification of cytoplasmic and nuclear boundaries which are used as reference points. Here, we describe an automated, interactive image analysis program that facilitates the accurate generation of cellular outlines from single cells and the subsequent calculation of metrics that quantify how a population of RNA molecules is distributed in the cell cytoplasm. We apply this analysis to mRNAs in mouse and human cells to demonstrate how these metrics can highlight differences in the distribution patterns of distinct RNA species. We further discuss considerations for the practical use of this tool. This program provides a way to facilitate and expedite the analysis of subcellular RNA localization for mechanistic and functional studies.

In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release.

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.

Conformational Dynamics of the RNA G-Quadruplex and its Effect on Translation Efficiency.

During translation, intracellular mRNA folds co-transcriptionally and must refold following the passage of ribosome. The mRNAs can be entrapped in metastable structures during these folding events. In the present study, we evaluated the conformational dynamics of the kinetically favored, metastable, and hairpin-like structure, which disturbs the thermodynamically favored G-quadruplex structure, and its effect on co-transcriptional translation in prokaryotic cells. We found that nascent mRNA forms a metastable hairpin-like structure during co-transcriptional folding instead of the G-quadruplex structure. When the translation progressed co-transcriptionally before the metastable hairpin-like structure transition to the G-quadruplex, function of the G-quadruplex as a roadblock of the ribosome was sequestered. This suggested that kinetically formed RNA structures had a dominant effect on gene expression in prokaryotes. The results of this study indicate that it is critical to consider the conformational dynamics of RNA-folding to understand the contributions of the mRNA structures in controlling gene expression.

Imaging cell-type-specific dynamics of mRNAs in living mouse brain.

We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose ß-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.

Role of a Stem-Loop Structure in Helicobacter pylori cagA Transcript Stability.

Helicobacter pylori CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among H. pylori strains in the steady-state levels of CagA and that a strain-specific motif downstream of the cagA transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The cagA 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on cagA transcript levels and cagA mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the cagA transcript and the CagA protein. Additionally, these mutations resulted in a decreased cagA mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state cagA transcript and CagA protein levels but did not affect cagA transcript stability. cagA transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment cagA transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the cagA 5' untranslated region influence the levels of cagA expression.

Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway.

mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single-molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while pharmacologically dissociating ribosomes or sequestering them into stress granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localized 5' and 3' ends, indicating either that mRNAs are not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs.