Changing views of translation: from ribosome profiling to high resolution imaging of single molecules in vivo

09:05-09:40 Ribosome recycling, rescue and homeostasis

Dr Rachel Green, John Hopkins University School of Medicine

Abstract

There are several mRNA surveillance pathways in eukaryotes (NGD, NSD and NMD) that moderate the effects of natural errors in the cell and more broadly regulate gene expression. Dr Green has previously defined biochemical parameters of the factors Dom34, Hbs1 and Rli1 in her in vitro reconstituted yeast translation system. She has correlated these biochemical observations with ribosome profiling experiments in yeast to broadly define the in vivo targets of these same mRNA surveillance pathways. In the process of studying these pathways, she has learned that different ribosome footprint (RPFs) sizes represent distinct states of the ribosome in a cell, and that these signatures can inform us on the molecular stresses that the ribosomes encounter. She is currently defining how various stresses encountered by cells trigger specific translational distress, and how the cell responds to these molecular triggers. Other studies in mammalian platelets and reticulocytes revealed the accumulation of ribosomes in the 3’ UTR of mRNAs and we connected this phenomenon with diminished levels of ribosome rescue (DOM34/HBS1L) and recycling (ABCE1) factors in these systems. She is currently drawing together related observations in the yeast and mammalian systems to better define the factors and mechanisms that maintain cellular ribosome homeostasis and to explore how it impacts development and stress-responses.

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09:40-10:15 Probing translational dysregulation in neurological disorders with high-sensitivity ribosome profiling

Assistant Professor Peter A Sims, Columbia University Medical Center, USA

Abstract

Dysregulation of protein synthesis is a common theme in many neurological disorders. In tuberous sclerosis complex, alterations in TSC cause mTOR, a master regulator of translation, to become hyperactivated. Fragile X syndrome involves loss of function of the polysome-associated RNA-binding protein FMRP, which is thought to regulate translation elongation. Professor Sim's developed a new, high-sensitivity ribosome profiling technique and used it for genome-wide analysis of protein synthesis in animal models of these two neurological disorders. His analysis reveals new insights into the molecular mechanisms through which the associated genetically altered proteins impact protein synthesis in the brain.

10:15-10:30 Discussion

11:00-11:35 Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions

Professor Thomas Preiss, Australian National University, Australia

Abstract

Regulation of protein synthesis on mRNA is central to eukaryotic expression of genes. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation consists of dynamic and complex interactions between eukaryotic initiation factors (eIFs) and the small ribosomal subunit (SSU) and concludes with joining of the large ribosomal subunit (LSU) to form a full ribosome and translate the mRNA code. While control of protein synthesis is important for fast-paced cell adaptation, methods to study the mechanistic intricacies of translation initiation in vivo transcriptome-wide were lacking.

A translation complex profile sequencing (TCP-seq) has been developed; a method related to the ribosome profiling approach. TCP-seq uniquely allows to resolve all translation intermediates, including the elusive mRNA ‘scanning’ by SSUs, visualize start codon recognition events, and capture diverse conformations of elongating ribosomes in vivo. Thus, the method can be used to pinpoint mRNAs with high potential for functional control and visualize locations of the regulatory elements in their 5'UTRs.

Combining TCP-seq with eIF-selective purification of complexes, Professor Preiss now aims to discern the fundamental mechanistic problems of initiation, such as what proteins confer directionality to the SSUs during scanning, and how ribosomal recruitment to mRNA is controlled during nutrient stress conditions. He will further adapt TCP-seq to use in mammalian cells and aim to obtain snapshots of translation in normal and cancer cells.

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11:35-12:10 Co-translational interactions of nascent chains studied by ribosome profiling

Professor Bernd Bukau, Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center, Germany

Abstract

Professor Bakau reported the dissection of the flux of newly synthesized proteins through the system of co-translationally engaged factors in E. coli and S. cerevisiae using ribosome profiling technology. In E. coli, the SRP targets inner membrane proteins to the membrane, whereas the chaperone trigger factor (TF) associates primarily with cytosolic, periplasmic and outer membrane proteins, indicating nascent chains are triaged between SRP and TF pathways. The Hsp70 chaperone DnaK interacts in concerted action with TF, typically with longer nascent chains and reflecting domain boundaries of the emerged nascent chains. The assembly of protein complexes initiates co-translationally once the domain interfaces of the interacting polypeptides are exposed. The organisation of the subunit-encoding genes in operons facilitates the assembly process. In S. cerevisiae the Hsp70 chaperone Ssb shows a different pattern of early nascent chain association as compared to its prokaryotic DnaK counterpart. Co-translational protein assembly is also observed, despite operon organisation of genes is less common, pointing to differences in the principles governing protein assembly in pro- and eukaryotes.

12:10-12:30 Discussion

13:30-14:05 Real-time quantification of single RNA translation dynamics in living cells

Assistant Professor Timothy Stasevich, Colorado State University, USA

Abstract

Professor Stasevich has developed technology to image single RNA translation dynamics in living cells. Using high-affinity antibody-based probes, multimerized epitope tags, and single molecule microscopy, we are able to visualize and quantify the emergence of nascent protein chains from single pre-marked RNA1. In this talk, he will describe this technology as well as a new multi-frame tag that extends the technology to enable real-time quantification of single RNA translation kinetics in any two of the three possible open reading frames. As a first application of the multi-frame tag, he uses it to dissect the kinetics of the HIV-1 frameshift sequence. Whereas previous bulk assays have shown this sequence leads to ~10% frameshifted product, it was not clear if all RNA frameshift with ~10% efficiency or if instead ~10% of RNA frameshift with ~100% efficiency. Interestingly, our live-cell data suggest the latter scenario, where a small subset of genetically identical RNA frameshift with high efficiency. The origin of this heterogeneity is not yet clear, but experiments are beginning to implicate a ribosomal pause that leads to a higher than normal density of ribosomes near the frameshift sequence on frameshifting RNA.

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14:05-14:40 Imaging the life and death of mRNAs in single cells

Dr Jeffrey Chao, Friedrich Miescher Institute for Biomedical Research, Switzerland

Abstract

After transcription, an mRNA's fate is determined by an orchestrated series of events (processing, export, localisation, translation and degradation) that is regulated both temporally and spatially within the cell. In order to more completely understand these processes and how they are coupled, it is necessary to be able to observe these events as they occur on single molecules of mRNA in real-time in living cells. To expand the scope of questions that can be addressed by RNA imaging, we are developing multi-color RNA biosensors that allow that status of a single mRNA molecules (e.g. translation or degradation) to be directly visualised and quantified.  

In order to image the first round of translation, Dr Chao has developed TRICK (translating RNA imaging by coat protein knock-off) which relies on the detection of two fluorescent signals that are placed within the coding sequence and the 3′UTR.  In this approach, an untranslated mRNA is dual labeled and the fluorescent label in the coding sequence is displaced by the ribosome during the first round of translation resulting in translated mRNAs being singly labeled. A conceptually similar approach was used for single-molecule imaging of mRNA decay, where dual-colored mRNAs identify intact transcripts, while a single-colored stabilized decay intermediate marked degraded transcripts (TREAT, 3′ RNA end accumulation during turnover). Dr Chao is using these tools to characterise localised translation and degradation during normal cell growth and stress. 

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14:40-15:00 Discussion

15:30-16:05 Paradoxical role for ribosomes in Ago2-mediated mRNA cleavage

Marvin Tanenbaum, Hubrecht Institute, The Netherlands

Abstract

Guided by a small RNA, Argonaute 2 (Ago2) interacts with its target mRNA, resulting in mRNA cleavage and decay. In vitro measurements have provided important insights in the binding and cleavage kinetics of Ago2, but much less is known about the behavior of Ago2 in living cells. Here, Marvin describes a method based on his previously developed SunTag translation imaging system, which allows him to observe cleavage of single mRNAs by Ago2 in living cells. He found that Ago2 frequently cleaves its target mRNA within minutes after nuclear export, at a time that precisely coincides with the arrival of the first translating ribosome at the Ago2 binding site. Using translation drugs and different mRNA reporters, he showed that ribosomes can stimulate mRNA decay by Ago2, by promoting the release of cleaved mRNA fragments from Ago2. However, the role of ribosomes in modulating Ago2 activity is paradoxical, as ribosomes also inhibit mRNA cleavage by Ago2, by displacing Ago2 molecules from the mRNA before cleavage can occur. Whether ribosomes promote or inhibit cleavage depends on mRNA release kinetics of the small RNA/Ago2 complex, and Ago2 showed distinct release kinetics when associated with different small RNAs. In summary, through live-cell single molecule imaging, he found that ribosomes profoundly alter Ago2’s mRNA cleavage activity by modulating distinct rate constants of the Ago2 cleavage cycle.

16:05-16:40 3'UTR-mediated protein-protein interactions regulate protein functions

Dr Christine Mayr, Memorial Sloan Kettering Cancer Center, USA

Abstract

At least half of human genes use alternative cleavage and polyadenylation to generate mRNA transcripts that differ in the length of their 3' untranslated regions (3'UTRs) while producing the same protein.  We found that 3'UTRs can mediate protein-protein interactions, and thus, can determine protein localization and protein functions. Dr Mayr showed that the long 3'UTR of CD47 is required for the formation of a protein complex between CD47 and SET. This interaction enables CD47 protein to efficiently localise to the cell surface, but it also changes protein function as only CD47 protein that was generated from the long 3'UTR isoform (CD47-LU) is able to activate the Ras GTPase RAC1.  

Dr Mayr investigated the mechanism of 3'UTR-dependent interaction between SET and CD47 and found that the RNA-binding protein TIS11B is necessary for this process. TIS11B is known to bind to AU-rich element (ARE) containing mRNAs and destabilizes specific mRNAs. Using super-resolution live cell imaging, we observed that TIS11B assembles into a large tubule-like meshwork that is intertwined with the endoplasmic reticulum (ER). The TIS11B granules enrich membrane protein-encoding mRNAs that contain multiple AREs in their 3'UTRs, including CD47-LU, and they exclude mRNAs lacking these features. The TIS11B granules also enrich specific proteins including chaperones, but they exclude SET.  The spatial arrangement of the TIS11B granules and the ER enables the ER to act as diffusion boundary for SET. This traps SET at the ER surface and facilitates 3'UTR-mediated protein-protein interactions between SET and newly made membrane proteins, including CD47-LU and PD-L1.

The function of TIS11B in the destabilization of mRNAs does not require TIS11B granule formation. Therefore, when RNA-binding proteins are soluble or present as single entities, they can regulate mRNA-based processes, including mRNA stability. However, assembly of RNA-binding proteins into larger aggregates, including RNA granules, allows them to acquire new properties that are necessary for the regulation of protein functions through 3'UTR-dependent protein-protein interactions.

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16:40-17:00 Discussion

09:00-09:35 Translational control of adaptive responses in mycobacteria

Dr Teresa Cortes, London School of Hygiene and Tropical Medicine, UK

Abstract

Mycobacterium tuberculosis causes human tuberculosis but can also persist for decades as an asymptomatic latent infection. An ability to adapt to diverse growth conditions encountered during infection is central to its success as a human pathogen. The mechanisms underlying persistence are poorly understood, and the emergence of drug-resistant tuberculosis makes the development of effective new treatments an urgent challenge. Understanding the ability of M tuberculosis to switch between replicating and non-replicating states during infection and disease is central to the search for improved treatments. 

The number of copies of a protein produced by a cell is generally viewed as being determined by the number of mRNA transcripts, but recent findings suggest that ‘specialised ribosomes’ can modify proteome profiles by preferential translation of particular mRNA subsets, particularly in response to stress. mRNA molecules contain specific signals that optimise their interaction with ribosomes; known as leader sequences, these include the Shine-Dalgarno (SD) sequence required for canonical translation initiation in bacteria. Dr Cortes has previously demonstrated that M tuberculosis expresses an unexpectedly high number of leaderless mRNA transcripts that lack the SD sequence. In Escherichia coli, only a few leaderless transcripts have been described and they are selectively translated. Proteins with secondary adaptive functions are generally leaderless in M tuberculosis. Furthermore, in a starvation model of growth arrest, transcriptome and translatome analysis reveal a preferential translation of leaderless encoding transcripts. Dr Cortes postulates that translational regulation contributes to the phenotypic adaptation of this pathogen, presenting the latest advances in our work. 

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09:35-10:10 Ribosome profiling studies of bacterial translational initiation

Dr Allen Buskirk, John Hopkins University School of Medicine, USA

Abstract

Although ribosome profiling shows tremendous potential to yield mechanistic insight into protein synthesis in vivo, its effectiveness in bacteria has been hindered by poor resolution and technical artifacts. Dr Birskirk has developed improvements in the method that reveal ribosome density at single nucleotide resolution in bacteria. He found that the methods used to harvest cells and prevent translation in lysates (with antibiotics) induce sequence-specific pauses. Using updated methods that eliminate these artifacts, we have begun a series of studies on initiation of protein synthesis. The classical model is that the Shine-Dalgarno (SD) sequence recruits 30S subunits to mRNAs through base-pairing interactions. Paradoxically, ribosome profiling studies show no correlation between the strength of the SD-aSD interaction and translation efficiency (a measure of ribosome density per mRNA). To address this paradox, we developed a ribosome profiling method to study the translational landscapes of ribosomes with mutant anti-Shine-Dalgarno sequences (so-called orthogonal ribosomes). Overall, he observe that translational efficiencies are well correlated for the wild-type and mutant ribosomes, suggesting that the SD sequence may not be the most important factor in determining initiation rates. When he sorted mRNAs based on their SD strength, however, he saw that mutant ribosomes are more dense on messages with weak SDs and less dense than wild-type on messages with strong SDs. His studies show for the first time a contribution of SD sequences to translational efficiency genome wide.

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10:10-10:30 Discussion

11:00-11:35 Translation from the 5'-untranslated region is an extracellular confession of health and disease

Dr Shelley Starck, NGM Biopharmaceuticals, USA

Abstract

Cellular adaptation to stress by phosphorylation of eIF2-alpha and downregulation of eIF2-dependent protein synthesis by a diverse set of stimuli is collectively described as the integrated stress response (ISR) and is a salient feature associated with neurodegeneration, cancer, and autoimmunity. Yet, despite translational repression during the ISR, select mRNAs sustain translation and are associated with translation upstream of the open reading frame (uORF).

Dr Starck re-purposed the sensitivity and specificity of T cells to monitor translation from unanticipated regions of the mRNA, such as uORFs. Tracing Translation by T cells (3T) is a method whereby insertion of a tracer peptide coding sequence into select candidate DNA sequences supports translation of the nested tracer peptide. The tracer peptide translation products are processed and loaded onto MHC I molecules in the ER and are presented on the cell surface where they can be detected by specific T cell hybridomas using a colorimetric reagent.  

Using 3T, Dr Starck showed that the essential endoplasmic reticulum (ER)-resident chaperone BiP harbors uORFs that are exclusively initiated by the non-AUG start codons UUG and CUG. BiP uORF expression bypasses a requirement for eIF2 and is dependent on the alternative initiation factor eIF2A. Both translation of the UUG-initiated uORF and eIF2A are necessary for BiP expression during the ISR. Unexpectedly, the products of uORF translation are predicted to generate MHC I peptides active in adaptive immunity. Dr Starck proposes that this phenomenon represents an extracellular signature during the ISR and could elicit autoimmunity and be a source of tumor-associated antigens (TAAs).

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11:35-12:10 Exploring the bacterial universe of functional RNA with Grad-seq

Professor Jörg Vogel, University of Würzburg, Germany

Abstract

RNA-seq can rapidly profile the expression of theoretically all RNA molecules in any given organism but the primary sequence of these transcripts is a poor predictor of cellular function. This has been particularly evident for the regulatory small RNAs of bacteria which dramatically vary in length and sequence within and between organisms. Professor Vogel has established a new method (gradient profiling by sequencing; Grad-seq) to partition the full ensemble of cellular RNAs based on their biochemical behavior. His approach enabled him to draw an RNA landscape of the model pathogen Salmonella Typhimurium, identifying clusters functionally related noncoding RNAs irrespective of their primary sequence. The map revealed a previously unnoticed class of transcripts that commonly interact with the osmoregulatory protein ProQ in Salmonella enterica. He has shown that ProQ is a conserved abundant global RNA-binding protein with a wide range of targets, including a new class of ProQ-associated small RNAs that are highly structured, and mRNAs from many cellular pathways. His functional characterization of these small RNAs that suggests that they constitute a previously unrecognized third domain of RNA-mediated control in bacteria which rivals the scope of the well-established regulons of the small RNA-binding proteins, Hfq and CsrA. By its ability to describe a functional RNA landscape based on expressed cellular transcripts irrespective of their primary sequence, our generic gradient profiling approach promises to aid the discovery of major functional RNA classes and RNA-binding proteins in many organisms.

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12:10-12:30 Discussion

13:30-14:05 Bacterial ribosome heterogeneity: novel aspects of selective translation

Dr Isabella Moll, University of Vienna, Austria

Abstract

In their natural environment, Bacteria encounter a variety of stressors. Thus, they are equipped with diverse mechanisms to adapt to these dynamic environmental cues. Hitherto, the alteration of gene expression in response to stress was mainly attributed to alternative transcription or the regulation of protein synthesis by means of protein or RNA factors that affect translation initiation. In contrast, we focus on the modulation of the translatome via the reversible formation of ‘specialized ribosomes’ that selectively translate mRNA subsets. Here, we will present different modes of ribosome heterogeneity as well as their potential implication in the regulation of protein synthesis, emphasizing the key role of the translational machinery in the economic integration of environmental signals in the stress response network.

14:05-14:40 Regulation of mRNA translation via control of elongation

Professor Anne Willis, MRC Toxicology Unit, UK

Abstract

Cells respond rapidly to external stress conditions by post-transcriptional control of gene expression; in particular, by the regulation of protein synthesis. Control of translation is achieved through modification of the translational machinery resulting in reprogramming of the translatome and synthesis of specific proteins required for stress protection/apoptosis. Recently, it has been shown that under certain pathophysiological conditions, modulation of translation rates via the elongation (rather than initiation) stage makes the major contribution to protein synthesis control. We have shown that this regulatory step is important in disease mechanisms that range from initiation of tumorigenesis (Faller et al 2015 Nature) to failure of synaptic maintenance in neurodegeneration (Perretti et al 2015 Nature, Bastide et al 2017 Current Biology). Importantly, Professor Willis has shown that it is possible to reduce neurodegeneration in mice with prion disease by modifying the rates of elongation through the induction of mild hypothermia. Mechanistically we have shown, by translational profiling, that cooling results in reprogramming of the translatome, up-regulation of RTN3, which evades cooling-induced translational elongation repression through its interaction of RBM3. In mice, knockdown of RTN3 expression eliminates cooling-induced neuroprotection, whereas RTN3 overexpression prevents synaptic loss and cognitive deficits downstream and independently of RBM3. The use of translational/ribosome profiling to examine elongation control in healthy and diseased states will be discussed.

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14:40-15:00 Discussion

15:30-16:05 Quality control in translation

Professor Ramanujan Hegde FRS, MRC Laboratory of Molecular Biology, UK

Abstract

A major goal of my group’s research efforts is to understand how cells identify failures during protein maturation and dispose of aberrant products to maintain cellular homeostasis. Failures can occur at any step on the pathway to a functional protein, including translation, folding, localization, modifications, and assembly. This talk will focus on how interruptions during the normal translation cycle are detected by the cell to selectively target the partially synthesized nascent protein for degradation. We have been investigating the problem of molecular recognition at two critical steps. Working backwards from nascent protein ubiquitination, we have determined how the ubiquitin ligase Listerin achieves exquisite specificity for nascent chains on stalled ribosomes, while scrupulously avoiding actively translating ribosomes. More recently, we have studied how pathologically slow ribosomes are first identified by the cell. We found that abnormally slow ribosomes are selectively and site-specifically mono-ubiquitinated by the E3 ligase ZNF598 to mark them for downstream quality control. Biochemical studies showed that ZNF598 preferentially engages nuclease-resistant di-ribosomes, a molecular species that arises when a trailing ribosome encounters a slower leading ribosome. Indirect sensing of aberrantly slow translation via di-ribosomes is shown to integrate the elongation speeds of leading and trailing ribosomes with translation initiation frequency to reach a final quality control decision. This multi-parameter model reconciles earlier contradictory findings across different experimental systems, and shows how higher-order polysome structure can be exploited by cellular machinery to monitor translation status.

16:35-17:00 Overview and future directions

16:35-17:00 Short talks