Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α.

Endoplasmic reticulum (ER) to nucleus homeostatic signaling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1α (inositol-requiring enzyme 1 alpha). While IRE1α can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1α complexes as well as the targeting mechanism that recruits XBP1 to IRE1α oligomers remains unknown. Here, we have developed a single-molecule imaging approach to monitor the recruitment of individual XBP1 transcripts to the ER surface. Using this methodology, we confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2 (hydrophobic region 2)-dependent targeting and relies on active translation. In addition, we show that IRE1α-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1α clusters, which are only observed upon overexpression of fluorescently tagged IRE1α during ER stress. Our findings support a model where ribosome-engaged, immobilized XBP1 mRNA is processed by small IRE1α assemblies that could be dynamically recruited for processing of mRNA transcripts on the ER.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Single-Molecule Imaging Reveals Translation of mRNAs Localized to Stress Granules.

Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.

Quantification of mRNA Turnover in Living Cells: A Pipeline for TREAT Data Analysis.

mRNA turnover plays an important role in the regulation of post-transcriptional gene expression. While many protein factors involved in mRNA degradation have been identified, we still lack a basic understanding of the principles that regulate the spatiotemporal dynamics of mRNA turnover within single cells. To overcome this limitation, we have developed the TREAT biosensor, which allows for discrimination of intact reporter transcripts and stabilized decay intermediates using single RNA imaging. Here, we present an image analysis pipeline that performs semiautomated detection and tracking of individual mRNA particles. It colocalizes tracks and applies the colocalization information to quantify the number of intact transcripts and degradation intermediates. Based on the analysis of control data, the workflow further determines detection efficiencies and uses them to correct RNA particle numbers.

Detection and quantification of RNA decay intermediates using XRN1-resistant reporter transcripts.

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.

Single-Molecule Imaging of mRNA Localization and Regulation during the Integrated Stress Response.

Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules (SGs) and processing bodies (PBs) and its effect on translation and degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics, very few mRNAs directly move between SGs and PBs, and that specific RNA-binding proteins can anchor mRNAs within these compartments. During recovery from stress, we show that mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts. Our work provides a framework for using single-molecule measurements to directly investigate the molecular mechanisms of phase-separated compartments within their cellular environment.

Detection of the First Round of Translation: The TRICK Assay.

Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.

Single-Molecule Quantification of Translation-Dependent Association of mRNAs with the Endoplasmic Reticulum.

It is well established that mRNAs encoding secretory or membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). The extent to which mRNAs that encode cytosolic proteins associate with the ER, however, remains controversial. To address this question, we quantified the number of cytosolic protein-encoding mRNAs that co-localize with the ER using single-molecule RNA imaging in fixed and living cells. We found that a small but significant number of mRNAs that encode cytosolic proteins associate with the ER and show that this interaction is translation dependent. Furthermore, we demonstrate that cytosolic protein-encoding transcripts can remain on the ER with dwell times consistent with multiple rounds of translation and have higher ribosome occupancies than transcripts translated in the cytosol. These results advance our understanding of the diversity and dynamics of localized translation on the ER.

The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells.

RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells.

Intermolecular base stacking mediates RNA-RNA interaction in a crystal structure of the RNA chaperone Hfq.

The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq6:A18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a "speed-dating" fashion, thereby supporting their regulatory function.

Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering.

Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.

Acquisition of an Archaea-like ribonuclease H domain by plant L1 retrotransposons supports modular evolution.

Although a variety of non-LTR retrotransposons of the L1 superfamily have been found in plant genomes over recent decades, their diversity, distribution, and evolution have yet to be analyzed in depth. Here, we perform comprehensive comparative and evolutionary analyses of L1 retrotransposons from 29 genomes of land plants covering a wide range of taxa. We identify numerous L1 elements in these genomes and detect a striking diversity of their domain composition. We show that all known land plant L1 retrotransposons can be grouped into five major families based on their phylogenetic relationships and domain composition. Moreover, we trace the putative evolution timeline that created the current variants and reveal that evolutionary events included losses and acquisitions of diverse putative RNA-binding domains and the acquisition of an Archaea-like ribonuclease H (RNH) domain. We also show that the latter RNH domain is autonomously active in vitro and speculate that retrotransposons may play a role in the horizontal transfer of RNH between plants, Archaea, and bacteria. The acquisition of an Archaea-like RNH domain by plant L1 retrotransposons negates the hypothesis that RNH domains in non-LTR retrotransposons have a single origin and provides evidence that acquisition happened at least twice. Together, our data indicate that the evolution of the investigated retrotransposons can be mainly characterized by repeated events of domain rearrangements and identify modular evolution as a major trend in the evolution of plant L1 retrotransposons.

Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc.

Piwi-interacting RNAs (piRNAs) are a gonad-specific class of small RNAs that associate with the Piwi clade of Argonaute proteins and play a key role in transposon silencing in animals. Since biogenesis of piRNAs is independent of the double-stranded RNA-processing enzyme Dicer, an alternative nuclease that can process single-stranded RNA transcripts has been long sought. A Phospholipase D-like protein, Zucchini, that is essential for piRNA processing has been proposed to be a nuclease acting in piRNA biogenesis. Here we describe the crystal structure of Zucchini from Drosophila melanogaster and show that it is very similar to the bacterial endonuclease, Nuc. The structure also reveals that homodimerization induces major conformational changes assembling the active site. The active site is situated on the dimer interface at the bottom of a narrow groove that can likely accommodate single-stranded nucleic acid substrates. Furthermore, biophysical analysis identifies protein segments essential for dimerization and provides insights into regulation of Zucchini's activity.