Ribosome decision graphs for the representation of eukaryotic RNA translation complexity.

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, within both annotated protein-coding and noncoding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term ribosome decision graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the latter "translons." Nondeterministic events, such as initiation, reinitiation, selenocysteine insertion, or ribosomal frameshifting, are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions and for analyzing genetic variation and quantitative genome-wide data on translation for characterization of regulatory modulators of translation.

Ribosome Decision Graphs for the Representation of Eukaryotic RNA Translation Complexity.

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, both within annotated protein-coding and non-coding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term Ribosome Decision Graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the later 'translons'. Non-deterministic events, such as initiation, re-initiation, selenocysteine insertion or ribosomal frameshifting are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions, analysis of genetic variation and quantitative genome-wide data on translation for characterisation of regulatory modulators of translation.

The nucleolar protein NOL12 is required for processing of large ribosomal subunit rRNA precursors in Arabidopsis.

BACKGROUND: NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors. RESULTS: In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics. CONCLUSION: Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.

Preliminary Incidence and Trends of Infections Caused by Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2022.

Each year, infections from major foodborne pathogens are responsible for an estimated 9.4 million illnesses, 56,000 hospitalizations, and 1,350 deaths in the United States (1). To evaluate progress toward prevention of enteric infections in the United States, the Foodborne Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-diagnosed infections caused by eight pathogens transmitted commonly through food at 10 U.S. sites. During 2020-2021, FoodNet detected decreases in many infections that were due to behavioral modifications, public health interventions, and changes in health care-seeking and testing practices during the COVID-19 pandemic. This report presents preliminary estimates of pathogen-specific annual incidences during 2022, compared with average annual incidences during 2016-2018, the reference period for the U.S. Department of Health and Human Services' Healthy People 2030 targets (2). Many pandemic interventions ended by 2022, resulting in a resumption of outbreaks, international travel, and other factors leading to enteric infections. During 2022, annual incidences of illnesses caused by the pathogens Campylobacter, Salmonella, Shigella, and Listeria were similar to average annual incidences during 2016-2018; however, incidences of Shiga toxin-producing Escherichia coli (STEC), Yersinia, Vibrio, and Cyclospora illnesses were higher. Increasing culture-independent diagnostic test (CIDT) usage likely contributed to increased detection by identifying infections that would have remained undetected before widespread CIDT usage. Reducing pathogen contamination during poultry slaughter and processing of leafy greens requires collaboration among food growers and processors, retail stores, restaurants, and regulators.

Arabidopsis thaliana NudiXes have RNA-decapping activity.

Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np n Ns (Ap2-5A, Gp3-4G, Ap3-5G, m7Gp3G, m7Gp3A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap4A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap4A, while AtNUDT7 preferentially cleaved Ap4A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap4A and NADH should be searched for in plant RNA.

Preliminary Incidence and Trends of Infections Caused by Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2016-2021.

To evaluate progress toward prevention of enteric infections in the United States, the Foodborne Diseases Active Surveillance Network (FoodNet) conducts active population-based surveillance for laboratory-diagnosed infections caused by Campylobacter, Cyclospora, Listeria, Salmonella, Shiga toxin-producing Escherichia coli (STEC), Shigella, Vibrio, and Yersinia at 10 U.S. sites. This report summarizes preliminary 2021 data and describes changes in annual incidence compared with the average annual incidence for 2016-2018, the reference period for the U.S. Department of Health and Human Services' (HHS) Healthy People 2030 goals for some pathogens (1). During 2021, the incidence of infections caused by Salmonella decreased, incidence of infections caused by Cyclospora, Yersinia, and Vibrio increased, and incidence of infections caused by other pathogens did not change. As in 2020, behavioral modifications and public health interventions implemented to control the COVID-19 pandemic might have decreased transmission of enteric infections (2). Other factors (e.g., increased use of telemedicine and continued increase in use of culture-independent diagnostic tests [CIDTs]) might have altered their detection or reporting (2). Much work remains to achieve HHS Healthy People 2030 goals, particularly for Salmonella infections, which are frequently attributed to poultry products and produce, and Campylobacter infections, which are frequently attributed to chicken products (3).

Alternative splicing as a key player in the fine-tuning of the immunity response in Arabidopsis.

Plants, like animals, are constantly exposed to abiotic and biotic stresses, which often inhibit plant growth and development, and cause tissue damage, disease, and even plant death. Efficient and timely response to stress requires appropriate co- and posttranscriptional reprogramming of gene expression. Alternative pre-mRNA splicing provides an important layer of this regulation by controlling the level of factors involved in stress response and generating additional protein isoforms with specific features. Recent high-throughput studies have revealed that several defence genes undergo alternative splicing that is often affected by pathogen infection. Despite extensive work, the exact mechanisms underlying these relationships are still unclear, but the contribution of alternative protein isoforms to the defence response and the role of regulatory factors, including components of the splicing machinery, have been established. Modulation of gene expression in response to stress includes alternative splicing, chromatin remodelling, histone modifications, and nucleosome occupancy. How these processes affect plant immunity is mostly unknown, but these facets open new regulatory possibilities. Here we provide an overview of the current state of knowledge and recent findings regarding the growing importance of alternative splicing in plant response to biotic stress.

Analysis of mRNA-derived siRNAs in mutants of mRNA maturation and surveillance pathways in Arabidopsis thaliana.

Defects in RNA maturation and RNA decay factors may generate substrates for the RNA interference machinery. This phenomenon was observed in plants where mutations in some RNA-related factors lead to the production of RNA-quality control small interfering RNAs and several mutants show enhanced silencing of reporter transgenes. To assess the potential of RNAi activation on endogenous transcripts, we sequenced small RNAs from a set of Arabidopsis thaliana mutants with defects in various RNA metabolism pathways. We observed a global production of siRNAs caused by inefficient pre-mRNA cleavage and polyadenylation leading to read-through transcription into downstream antisense genes. In addition, in the lsm1a lsm1b double mutant, we identified NIA1, SMXL5, and several miRNA-targeted mRNAs as producing siRNAs, a group of transcripts suggested being especially sensitive to deficiencies in RNA metabolism. However, in most cases, RNA metabolism perturbations do not lead to the widespread production of siRNA derived from mRNA molecules. This observation is contrary to multiple studies based on reporter transgenes and suggests that only a very high accumulation of defective mRNA species caused by specific mutations or substantial RNA processing defects trigger RNAi pathways.

Arabidopsi s Spliceosome Factor SmD3 Modulates Immunity to Pseudomonas syringae Infection.

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of major biotic stress response factors were also altered upon treatment with Pseudomonas effectors. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst, verified by northern and RT-qPCR, showed that lack of SmD3-b protein deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Importantly, we show that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. We propose that it is the malfunction of the stomata that is the primary cause of an altered mutant response to the pathogen. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

Decreased Incidence of Infections Caused by Pathogens Transmitted Commonly Through Food During the COVID-19 Pandemic - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2017-2020.

Foodborne illnesses are a substantial and largely preventable public health problem; before 2020 the incidence of most infections transmitted commonly through food had not declined for many years. To evaluate progress toward prevention of foodborne illnesses in the United States, the Foodborne Diseases Active Surveillance Network (FoodNet) of CDC's Emerging Infections Program monitors the incidence of laboratory-diagnosed infections caused by eight pathogens transmitted commonly through food reported by 10 U.S. sites.* FoodNet is a collaboration among CDC, 10 state health departments, the U.S. Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS), and the Food and Drug Administration. This report summarizes preliminary 2020 data and describes changes in incidence with those during 2017-2019. During 2020, observed incidences of infections caused by enteric pathogens decreased 26% compared with 2017-2019; infections associated with international travel decreased markedly. The extent to which these reductions reflect actual decreases in illness or decreases in case detection is unknown. On March 13, 2020, the United States declared a national emergency in response to the COVID-19 pandemic. After the declaration, state and local officials implemented stay-at-home orders, restaurant closures, school and child care center closures, and other public health interventions to slow the spread of SARS-CoV-2, the virus that causes COVID-19 (1). Federal travel restrictions were declared (1). These widespread interventions as well as other changes to daily life and hygiene behaviors, including increased handwashing, have likely changed exposures to foodborne pathogens. Other factors, such as changes in health care delivery, health care-seeking behaviors, and laboratory testing practices, might have decreased the detection of enteric infections. As the pandemic continues, surveillance of illness combined with data from other sources might help to elucidate the factors that led to the large changes in 2020; this understanding could lead to improved strategies to prevent illness. To reduce the incidence of these infections concerted efforts are needed, from farm to processing plant to restaurants and homes. Consumers can reduce their risk of foodborne illness by following safe food-handling and preparation recommendations.

Recency-Weighted Statistical Modeling Approach to Attribute Illnesses Caused by 4 Pathogens to Food Sources Using Outbreak Data, United States.

Foodborne illness source attribution is foundational to a risk-based food safety system. We describe a method for attributing US foodborne illnesses caused by nontyphoidal Salmonella enterica, Escherichia coli O157, Listeria monocytogenes, and Campylobacter to 17 food categories using statistical modeling of outbreak data. This method adjusts for epidemiologic factors associated with outbreak size, down-weights older outbreaks, and estimates credibility intervals. On the basis of 952 reported outbreaks and 32,802 illnesses during 1998-2012, we attribute 77% of foodborne Salmonella illnesses to 7 food categories (seeded vegetables, eggs, chicken, other produce, pork, beef, and fruits), 82% of E. coli O157 illnesses to beef and vegetable row crops, 81% of L. monocytogenes illnesses to fruits and dairy, and 74% of Campylobacter illnesses to dairy and chicken. However, because Campylobacter outbreaks probably overrepresent dairy as a source of nonoutbreak campylobacteriosis, we caution against using these Campylobacter attribution estimates without further adjustment.

Preliminary Incidence and Trends of Infections with Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2016-2019.

To evaluate progress toward prevention of enteric illnesses, the Foodborne Diseases Active Surveillance Network (FoodNet) of CDC's Emerging Infections Program monitors the incidence of laboratory-diagnosed infections caused by eight pathogens transmitted commonly through food at 10 U.S. sites.* This report summarizes preliminary 2019 data and describes changes in incidence compared with that during 2016-2018. The incidence of enteric infections caused by these eight pathogens reported by FoodNet sites in 2019 continued to increase or remained unchanged, indicating progress in controlling major foodborne pathogens in the United States has stalled. Campylobacter and Salmonella caused the largest proportion of illnesses; trends in incidence varied by Salmonella serotype. Widespread adoption of whole genome sequencing (WGS) of bacteria has improved the ability to identify outbreaks, emerging strains, and sources of pathogens. To maximize the potential of WGS to link illnesses to particular sources, testing of isolates by clinical and public health laboratories is needed. Reductions in Salmonella serotype Typhimurium suggest that targeted interventions (e.g., vaccinating chickens and other food animals) might decrease human infections. Reducing contamination during food production, processing, and preparation will require more widespread implementation of known prevention measures and of new strategies that target particular pathogens and serotypes.

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA.

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

RNA Helicases from the DEA(D/H)-Box Family Contribute to Plant NMD Efficiency.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry. Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporter mRNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient overexpression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.

A novel 5'-hydroxyl dinucleotide hydrolase activity for the DXO/Rai1 family of enzymes.

Modifications at the 5'-end of RNAs play a pivotal role in determining their fate. In eukaryotes, the DXO/Rai1 family of enzymes removes numerous 5'-end RNA modifications, thereby regulating RNA turnover. Mouse DXO catalyzes the elimination of incomplete 5'-end caps (including pyrophosphate) and the non-canonical NAD+ cap on mRNAs, and possesses distributive 5'-3' exoribonuclease activity toward 5'-monophosphate (5'-PO4) RNA. Here, we demonstrate that DXO also catalyzes the hydrolysis of RNAs bearing a 5'-hydroxyl group (5'-OH RNA). The crystal structure of DXO in complex with a 5'-OH RNA substrate mimic at 2.0 Å resolution provides elegant insight into the molecular mechanism of this activity. More importantly, the structure predicts that DXO first removes a dinucleotide from 5'-OH RNA. Our nuclease assays confirm this prediction and demonstrate that this 5'-hydroxyl dinucleotide hydrolase (HDH) activity for DXO is higher than the subsequent 5'-3' exoribonuclease activity for selected substrates. Fission yeast Rai1 also has HDH activity although it does not have 5'-3' exonuclease activity, and the Rat1-Rai1 complex can completely degrade 5'-OH RNA. An Arabidopsis DXO1 variant is active toward 5'-OH RNA but prefers 5'-PO4 RNA. Collectively, these studies demonstrate the diverse activities of DXO/Rai1 and expands the collection of RNA substrates that can undergo 5'-3' mediated decay.

Non-canonical translation initiation in yeast generates a cryptic pool of mitochondrial proteins.

Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria. Using bioinformatics tools, we provide compelling evidence that in yeast the potential for producing alternative protein isoforms by non-AUG translation initiation is much more prevalent than previously anticipated and may apply to as many as a few thousand proteins. Several hundreds of candidates are predicted to gain a mitochondrial targeting signal (MTS), generating an unrecognized pool of mitochondrial proteins. We confirmed mitochondrial localization of a subset of proteins previously not identified as mitochondrial, whose standard forms do not carry an MTS. Our data highlight the potential of non-canonical translation initiation in expanding the capacity of the mitochondrial proteome and possibly also other cellular features.

Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity.

The DXO family of proteins participates in eukaryotic mRNA 5'-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5'-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties.

Small Nucleolar RNAs Tell a Different Tale.

Transcribing RNA Polymerase II interacts with multiple factors that orchestrate maturation and stabilisation of messenger RNA. For the majority of noncoding RNAs, the polymerase complex employs entirely different strategies, which usually direct the nascent transcript to ribonucleolytic degradation. However, some noncoding RNA classes use endo- and exonucleases to achieve functionality. Here we review processing of small nucleolar RNAs that are transcribed by RNA Polymerase II as precursors, and whose 5' and 3' ends undergo processing to release mature, functional molecules. The maturation strategies of these noncoding RNAs in various organisms follow a similar pattern but employ different factors and are strictly correlated with genomic organisation of their genes.