Temporal dynamics of normalization reweighting.

For decades, neural suppression in early visual cortex has been thought to be fixed. But recent work has challenged this assumption by showing that suppression can be reweighted based on recent history; when pairs of stimuli are repeatedly presented together, suppression between them strengthens. Here we investigate the temporal dynamics of this process using a steady-state visual evoked potential (SSVEP) paradigm that provides a time-resolved, direct index of suppression between pairs of stimuli flickering at different frequencies (5 and 7 Hz). Our initial analysis of an existing electroencephalography (EEG) dataset (N = 100) indicated that suppression increases substantially during the first 2-5 seconds of stimulus presentation (with some variation across stimulation frequency). We then collected new EEG data (N = 100) replicating this finding for both monocular and dichoptic mask arrangements in a preregistered study designed to measure reweighting. A third experiment (N = 20) used source-localized magnetoencephalography and found that these effects are apparent in primary visual cortex (V1), consistent with results from neurophysiological work. Because long-standing theories propose inhibition/excitation differences in autism, we also compared reweighting between individuals with high versus low autistic traits, and with and without an autism diagnosis, across our three datasets (total N = 220). We find no compelling differences in reweighting that are associated with autism. Our results support the normalization reweighting model and indicate that for prolonged stimulation, increases in suppression occur on the order of 2-5 seconds after stimulus onset.

Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales.

In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The dimers must be resolved before cell separation to ensure genomic stability and cell viability. Dimer resolution is achieved by the broadly conserved dif/Xer system, which catalyzes one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here, we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (here named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Finally, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. IMPORTANCE The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif within the same molecule, leading to rapid site-specific recombination and conversion back into chromosome monomers. The apparent absence of chromosome dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.

Legionella pneumophila CRISPR-Cas Suggests Recurrent Encounters with One or More Phages in the Family Microviridae.

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires' disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella mobile element 1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila's adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants-all targeted by previously and newly identified CRISPR-Cas spacers-but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae. These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires' disease is an often-fatal pneumonia caused by Legionella pneumophila, which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the "missing" L. pneumophila phages belong to the microvirus subfamily Gokushovirinae. Gokushoviruses are known to infect another intracellular pathogen, Chlamydia. How do gokushoviruses access L. pneumophila (and Chlamydia) inside their "cozy niches"? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

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.

DRUID: a pipeline for transcriptome-wide measurements of mRNA stability.

Control of messenger RNA (mRNA) stability is an important aspect of gene regulation. The gold standard for measuring mRNA stability transcriptome-wide uses metabolic labeling, biochemical isolation of labeled RNA populations, and high-throughput sequencing. However, difficult normalization procedures have inhibited widespread adoption of this approach. Here, we present DRUID (for determination of rates using intron dynamics), a new computational pipeline that is robust, easy to use, and freely available. Our pipeline uses endogenous introns to normalize time course data and yields reproducible half-lives, even with data sets that were otherwise unusable. DRUID can handle data sets from a variety of organisms, spanning yeast to humans, and we even applied it retroactively on published data sets. We anticipate that DRUID will allow broad application of metabolic labeling for studies of transcript stability.

Determining mRNA half-lives on a transcriptome-wide scale.

Every step in the life cycle of an RNA transcript provides opportunity for regulation. One important aspect of post-transcriptional control is the regulation of RNA stability. Of the many strategies for determining mRNA stability, transcription inhibition and metabolic labeling have proved the most amenable to high-throughput analysis and have opened the door to dissecting mRNA decay transcriptome-wide. Here, we describe experimental and computational methods to determine transcriptome-wide RNA stabilities using both pharmacological inhibition of transcription and metabolic labeling. To aid in the analysis of these experiments, we discuss key characteristics of high-quality experiments and address other experimental and computational considerations for the analysis of mRNA stability. Broader application of these approaches will further our understanding of mRNA decay and illuminate its contribution to different biological processes.

The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

BACKGROUND: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells. RESULTS: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression. CONCLUSIONS: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.

Exploring the architecture of viral RNA genomes.

The genomes of RNA viruses contain local structural elements and long-range interactions that control various steps in virus replication. While many individual RNA elements have been characterized, it remains less clear how the structure and activity of such elements are integrated and regulated within the complex context of complete viral genomes. Recent technical advances, particularly the development of high-throughput solution structure mapping methods, have made secondary structural analysis of entire viral RNA genomes feasible. As a consequence, whole-genome structural models have been deduced for a number of plus-strand RNA viruses and retroviruses and these structures have provided intriguing functional and evolutionary insights into global genome architecture.

Functional long-range RNA-RNA interactions in positive-strand RNA viruses.

Positive-strand RNA viruses are important human, animal and plant pathogens that are defined by their single-stranded positive-sense RNA genomes. In recent years, it has become increasingly evident that interactions that occur between distantly positioned RNA sequences within these genomes can mediate important viral activities. These long-range intragenomic RNA-RNA interactions involve direct nucleotide base pairing and can span distances of thousands of nucleotides. In this Review, we discuss recent insights into the structure and function of these intriguing genomic features and highlight their diverse roles in the gene expression and genome replication of positive-strand RNA viruses.

Translational readthrough in Tobacco necrosis virus-D.

The plus-strand RNA genome of Tobacco necrosis virus-D (TNV-D) expresses its polymerase via translational readthrough. The RNA signals involved in this readthrough process were characterized in vitro using a wheat germ extract translation system and in vivo via protoplast infections. The results indicate that (i) TNV-D requires a long-range RNA-RNA interaction between an extended stem-loop (SL) structure proximal to the readthrough site and a sequence in the 3'-untranslated region of its genome; (ii) stability of the extended SL structure is important for its function; (iii) TNV-D readthrough elements are compatible with UAG and UGA, but not UAA; (iv) a readthrough defect can be rescued by a heterologous readthrough element in vitro, but not in vivo; and (v) readthrough elements can also mediate translational frameshifting. These results provide new information on determinants of readthrough in TNV-D and further support the concept of a common general mechanism for readthrough in Tombusviridae.

Tombusvirus Y-shaped translational enhancer forms a complex with eIF4F and can be functionally replaced by heterologous translational enhancers.

Certain plus-strand RNA plant viruses that are uncapped and nonpolyadenylated rely on RNA elements in their 3' untranslated region, termed 3'-cap-independent translational enhancers (3'CITEs), for efficient translation of their proteins. Here, we have investigated the properties of the Y-shaped class of 3'CITE present in the tombusvirus Carnation Italian ringspot virus (CIRV). While some types of 3'CITE have been found to function through recruitment of translation initiation factors to the viral genome, no trans-acting translation-related factors have yet been identified for the Y-shaped 3'CITE. Our results indicate that the CIRV 3'CITE complexes with eIF4F and eIFiso4F, with the former mediating translation more efficiently than the latter. In nature, some classes of 3'CITE are present in several different viral genera, suggesting that these elements hold a high degree of modularity. Here, we test this concept by engineering chimeric viruses containing heterologous 3'CITEs and show that the Y-shaped class of 3'CITE in CIRV can be replaced by two alternative types of 3'CITE, i.e., a Panicum mosaic virus-like 3'CITE or an I-shaped 3'CITE, without any major loss in in vitro translation or replication efficiency in protoplasts. The heterologous 3'CITEs also mediated whole-plant infections of Nicotiana benthamiana, where distinct symptoms were observed for each of the alternative 3'CITEs and 3'CITE evolution occurred during serial passaging. Our results supply new information on Y-shaped 3'CITE function and provide insights into 3'CITE virus-host compatibilities.

Internal RNA Replication Elements are Prevalent in Tombusviridae.

Internal replication elements (IREs) are RNA structures that are present at internal positions in the genomes of different types of plus-strand RNA viruses. Members of the genus Tombusvirus (family Tombusviridae) contain an IRE within the polymerase coding region of their genomes and this RNA element participates in both genome targeting to sites of replication and replicase complex assembly. Here we propose that other members of the virus family Tombusviridae also possess comparable IREs. Through sequence and structural analyses, candidate IREs in several genera of this family were identified, including aureusviruses, necroviruses, carmoviruses, and pelarspoviruses. The results from subsequent mutational analysis of selected proposed IREs were consistent with a critical role for these structures in viral genome accumulation during infections. Our study supports the existence of IREs in several genera in Tombusviridae and points to previously unappreciated similarities in genome replication strategies between members of this virus family.

Multifaceted regulation of translational readthrough by RNA replication elements in a tombusvirus.

Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family.

3' Cap-independent translation enhancers of positive-strand RNA plant viruses.

Positive-strand RNA plant viruses that are neither 5'-capped nor 3'-polyadenylated use nontraditional mechanisms to recruit ribosomes to the 5'-end of their viral genomes. One strategy employed by some of these viruses involves a type of RNA element, termed the 3' cap-independent translation enhancer (3'CITE), located in or near the 3'-untranslated region of viral RNA genomes. 3'CITEs function to mediate efficient translation of 5'-proximally encoded viral proteins and function by recruiting either translation initiation factors or the 60S ribosomal subunit to the viral RNA. Recent mechanistic and structural studies have revealed important new insights and details of how 3'CITEs are able to facilitate viral translation and allow these viruses to compete efficiently against cellular mRNAs for the host translational machinery.

Tombusvirus recruitment of host translational machinery via the 3' UTR.

RNA viruses recruit the host translational machinery by different mechanisms that depend partly on the structure of their genomes. In this regard, the plus-strand RNA genomes of several different pathogenic plant viruses do not contain traditional translation-stimulating elements, i.e., a 5'-cap structure and a 3'-poly(A) tail, and instead rely on a 3'-cap-independent translational enhancer (3'CITE) located in their 3' untranslated regions (UTRs) for efficient synthesis of viral proteins. We investigated the structure and function of the I-shaped class of 3'CITE in tombusviruses--also present in aureusviruses and carmoviruses--using biochemical and molecular approaches and we determined that it adopts a complex higher-order RNA structure that facilitates translation by binding simultaneously to both eukaryotic initiation factor (eIF) 4F and the 5' UTR of the viral genome. The specificity of 3'CITE binding to eIF4F is mediated, at least in part, through a direct interaction with its eIF4E subunit, whereas its association with the viral 5' UTR relies on complementary RNA-RNA base-pairing. We show for the first time that this tripartite 5' UTR/3'CITE/eIF4F complex forms in vitro in a translationally relevant environment and is required for recruitment of ribosomes to the 5' end of the viral RNA genome by a mechanism that shares some fundamental features with cap-dependent translation. Notably, our results demonstrate that the 3'CITE facilitates the initiation step of translation and validate a molecular model that has been proposed to explain how several different classes of 3'CITE function. Moreover, the virus-host interplay defined in this study provides insights into natural host resistance mechanisms that have been linked to 3'CITE activity.

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

Context-influenced cap-independent translation of Tombusvirus mRNAs in vitro.

Tomato bushy stunt virus (TBSV) possesses a positive-strand RNA genome that is not 5'-capped or 3'-polyadenylated. Previous analysis revealed that the TBSV genome contains a 3'-cap-independent translational enhancer (3'CITE) in its 3'-untranslated region (3'UTR) that facilitates translation of viral mRNAs in vivo. A long-range 5'-3' RNA-RNA interaction between the 3'CITE and the 5'UTR of viral mRNAs is necessary for function, and this RNA bridge has been proposed to mediate delivery of translation-related factors bound to the 3'CITE to the 5'-end of the message. Although fully functional when assayed in plant protoplasts, the TBSV 3'CITE was previously found to be unable to activate translation in vitro in wheat germ extract (wge). In the current report we have determined that (i) another Tombusvirus, Carnation Italian ringspot virus (CIRV), contains a TBSV-like 3'CITE that is active in wge; (ii) the CIRV 3'CITE functions in vitro in a manner analogous to the TBSV 3'CITE in vivo; (iii) the TBSV 3'CITE is able to competitively inhibit CIRV 3'CITE-dependent translation in wge and (iv) the TBSV 3'CITE can enhance translation in wge when present in short viral messages. These results reveal the contrasting activities of different TBSV-like 3'CITEs in vitro and shed light on the nature of the defect in TBSV.