Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea.

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5'- and 3'-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

A versatile cis-acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea.

General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.

Insights into the evolutionary conserved regulation of Rio ATPase activity.

Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.

Non-radioactive In Vivo Labeling of RNA with 4-Thiouracil.

RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil , in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii .

In Vivo RNA Chemical Footprinting Analysis in Archaea.

RNA structural conformation and dynamics govern the functional properties of all RNA/RNP. Accordingly, defining changes of RNA structure and dynamics in various conditions may provide detailed insight into how RNA structural properties regulate the function of RNA/RNP. Traditional chemical footprinting analysis using chemical modifiers allows to sample the dynamics and conformation landscape of diverse RNA/RNP. However, many chemical modifiers are limited in their capacity to provide unbiased information reflecting the in vivo RNA/RNP structural landscape. In the recent years, the development of selective-2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology that uses powerful new chemical modifiers has significantly improved in vitro and in vivo structural probing of secondary and tertiary interactions of diverse RNA species at the single nucleotide level.Although the original discovery of Archaea as an independent domain of life is intimately linked to the technological development of RNA analysis, our understanding of in vivo RNA structural conformation and dynamics in this domain of life remains scarce.This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea, Sulfolobus acidocaldarius and Haloferax volcanii.

The Archaeal Proteome Project advances knowledge about archaeal cell biology through comprehensive proteomics.

While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.

Toward Time-Resolved Analysis of RNA Metabolism in Archaea Using 4-Thiouracil.

Archaea are widespread organisms colonizing almost every habitat on Earth. However, the molecular biology of archaea still remains relatively uncharacterized. RNA metabolism is a central cellular process, which has been extensively analyzed in both bacteria and eukarya. In contrast, analysis of RNA metabolism dynamic in archaea has been limited to date. To facilitate analysis of the RNA metabolism dynamic at a system-wide scale in archaea, we have established non-radioactive pulse labeling of RNA, using the nucleotide analog 4-thiouracil (4TU) in two commonly used model archaea: the halophile Euryarchaeota Haloferax volcanii, and the thermo-acidophile Crenarchaeota Sulfolobus acidocaldarius. In this work, we show that 4TU pulse labeling can be efficiently performed in these two organisms in a dose- and time-dependent manner. In addition, our results suggest that uracil prototrophy had no critical impact on the overall 4TU incorporation in RNA molecules. Accordingly, our work suggests that 4TU incorporation can be widely performed in archaea, thereby expanding the molecular toolkit to analyze archaeal gene expression network dynamic in unprecedented detail.

Fetal deaths in the United States. Influence of high-risk conditions and implications for management.

OBJECTIVE: To estimate the effect of specific maternal-fetal high-risk conditions on the risk and timing of fetal death. METHODS: This study examined 10,614,679 non-anomalous singleton pregnancies delivering at or beyond 24 weeks' gestation, derived from the U.S. linked birth/infant death data sets, 1995-1997. Fetal death rates for pregnancies at low risk were compared with pregnancies complicated by chronic hypertension, gestational hypertensive disorders, diabetes, small for gestational age infants, and abruption. Adjusted relative risks as well as population-attributable risks for fetal death were derived by gestational age for each high-risk condition compared with low-risk pregnancies. RESULTS: The fetal death rate for low-risk pregnancies was 1.6 per 1000 births. Adjusted relative risk for fetal death was 9.2 (95% confidence interval [CI] 8.8, 9.7) for abruption, 7.0 (95% CI 6.8, 7.2) for small for gestational age infants, 1.4 (95% CI 1.3, 1.5) for gestational hypertensive disorders, 2.7 (95% CI 2.4, 3.0) for chronic hypertension, and 2.5 (95% CI 2.3, 2.7) for diabetes. Fetal death rates were lowest between 38 and 41 weeks. The fetal death rate (per 1000 births) for these high-risk conditions was 61.4, 9.6, 3.5, 7.6, and 3.9, respectively. Almost two thirds of fetal deaths were attributable to the pregnancy complications examined. CONCLUSION: High-risk conditions in pregnancy are associated with an increased risk for fetal death, particularly in the third trimester. Delivery should be considered at 38 weeks, but no later than 41 weeks, for these pregnancies.

Prenatal care and black-white fetal death disparity in the United States: heterogeneity by high-risk conditions.

OBJECTIVE: To determine the impact of prenatal care in the United States on the fetal death rate in the presence and absence of obstetric and medical high-risk conditions, and to explore the role of these high risk conditions in contributing to the black-white disparity. METHODS: This is a population-based, retrospective cohort study using the national perinatal mortality data for 1995-1997 assembled by the National Center for Health Statistics. Fetal death rate (per 1000 births) and adjusted relative risks were derived from multivariable logistic regression models. RESULTS: Of 10,560,077 singleton births, 29,469 (2.8 per 1000) resulted in fetal death. Fetal death rates were higher for blacks than whites in the presence (4.2 versus 2.4 per 1000) and absence (17.2 versus 2.5 per 1000) of prenatal care. Lack of prenatal care increased the (adjusted) relative risk for fetal death 2.9-fold in blacks and 3.4-fold in whites. Blacks were 3.3 times more likely to have no prenatal care compared with whites. Over 20% of all fetal deaths were associated with growth restriction and placental abruption, both in the presence and absence of prenatal care. Lack of prenatal care was associated with increased fetal death rates for both blacks and whites in the presence and absence of high-risk conditions. CONCLUSION: In the Unites States, strategies to increase prenatal care participation, especially among blacks, are expected to decrease fetal death rates.

Second-trimester genetic sonography in patients with advanced maternal age and normal triple screen.

OBJECTIVE: To estimate the value of second-trimester genetic sonography in detecting fetal Down syndrome in patients with advanced maternal age (at least 35 years) and normal triple screen. METHODS: Since July 1999, a prospective collection and recording of all individual triple screen risks for fetal Down syndrome was initiated for all patients with advanced maternal age presenting in our ultrasound unit for second-trimester genetic sonography. Genetic sonography evaluated the presence or absence of multiple aneuploidy markers. Outcome information included the results of genetic amniocentesis, if performed, and the results of pediatric assessment and follow-up after birth. RESULTS: By June 2001, 959 patients with advanced maternal age and normal triple screen were identified. Outcome information was obtained in 768 patients. The median risk for fetal Down syndrome based on maternal age was 1:213 (range 1:37-1:274). The median risk for fetal Down syndrome based on triple screen results was 1:1069 (range 1:275-1:40,000). A total of 673 patients had normal genetic sonography, and none (0%) had Down syndrome; 95 had one or more aneuploidy markers present, and four (4.2%) had fetuses with Down syndrome. The triple screen risks for these four fetuses ranged from 1:319 to 1:833. CONCLUSION: This study suggests that patients with advanced maternal age and normal genetic sonography carried very little risk for Down syndrome. The use of genetic sonography may increase the detection rate of fetal Down syndrome in this group of pregnant women.

Infertility case management: improving both short-term and long-term outcomes while reducing costs.

PURPOSE/OBJECTIVES: Infertility is a growing medical condition as more women are desirous of having children at an older age. It is estimated to be a $3 billion business, and, while infertility treatment is a for-profit commercial endeavor, the product is noncommercial (baby or babies). The treatment process may be complicated with overutilization, drug wastage, and adverse outcomes. High-order multiple gestations may result in preterm births, chronic adult diseases, and lifelong neurological impairments (such as cerebral palsy). The total national cost of infertility treatment unfortunately equals the cost of providing care to these babies in the nursery and neonatal intensive care unit. This article explores the potential benefit of the integration of information technology with clinical case management to reduce overall cost and improve provider accuracy. PRIMARY PRACTICE SETTING(S): Office-based telephonic nurse case management and pharmacology management practice. FINDINGS/CONCLUSIONS: The article demonstrates that the challenging integration of information technology with clinical case management is very effective and improves provider accuracy, resulting in the best transfer of real-time information. The case management program at Women's Integrated Network Healthcare has been shown to lower infertility treatment costs by 30% to 40% and lower the numbers of high-order multiple gestations. IMPLICATIONS FOR CASE MANAGEMENT PRACTICE: Eighty-one percent of the cost reduction is related directly to case management, not reduction in physician fees or unit pharmaceutical costs. Case management can improve effectiveness and quality of conception, and there is a reduction in high-order multiple gestations. It was also found that, by expanding infertility benefits and including case management as the pivotal element, payers and employers could recognize significant savings and, more importantly, the women and families would benefit.

The impact of prenatal care on postneonatal deaths in the presence and absence of antenatal high-risk conditions.

OBJECTIVE: This study was undertaken to determine the association, if any, between prenatal care and postneonatal death in the presence and absence of high-risk pregnancy conditions. STUDY DESIGN: Data were derived from the national linked birth/infant death data set for the years 1995 to 1997 provided by the National Center for Health Statistics. Analyses were restricted to singleton live births that occurred after 23 completed weeks of gestation. Multiple births, congenital malformations, chromosomal abnormalities, missing data on gestational age, and birth weight less than 500 g were excluded. Multivariable logistic regression analyses were used to adjust for various antenatal high-risk conditions, maternal age, gravidity, gestational age at delivery, birth weight, maternal education, marital status, smoking, and alcohol use. Postneonatal death rate was defined as the number of deaths between 28 and 365 days of life per 1,000 neonatal survivors. RESULTS: For 10,512,269 singleton live births analyzed, 21,962 (2.1 per 1,000) resulted in postneonatal death. Postneonatal death rates were higher for African American women than white women in the presence (3.8 vs 1.7 per 1,000) and absence (11.2 vs 5.3 per 1,000) of prenatal care. Lack of prenatal care was associated with increased relative risk (RR) for postneonatal death, 1.8-fold in African American women and 1.6-fold in white women. Lack of prenatal care was associated with increased postneonatal death rates to a similar degree for the individual high-risk pregnancy conditions for both African American and white women. Lack of prenatal care was associated with increased postneonatal death rates, especially in the presence of postterm pregnancy (RR 2.3, 95% CI 1.6, 3.1), pregnancy-induced hypertension (RR 2.2, 95% CI 1.5, 3.4), intrapartum fever (RR 2.1, 95% CI 1.2, 3.5), and small-for-gestational-age infant (RR 1.6, 95% CI 1.3, 2.0). CONCLUSION: Lack of prenatal care should be considered as a high-risk factor for postneonatal death for both African American and white women, especially if the pregnancy has been complicated by postdates, pregnancy-induced hypertension, intrapartum fever or small-for-gestational-age infant.

Down syndrome risk estimation after normal genetic sonography.

OBJECTIVE: The objective of this study was to determine whether there are any indication-specific variations in risk reduction for fetal Down syndrome after a normal genetic sonogram. STUDY DESIGN: A second-trimester genetic sonogram was offered to all pregnant women who were at increased risk for fetal Down syndrome (>/=1:274) because of either advanced maternal age (>/=35 years), an abnormal triple screen, or both. Outcome information included the results of genetic amniocentesis (if performed), the results of pediatric assessment, and follow-up after birth. Normal genetic sonography was defined as the absence of all ultrasound aneuploidy markers. RESULTS: The overall prevalence of fetal Down syndrome in the tested population was 1.41% (53/3,753 pregnancies); however, in the presence of normal genetic sonography, the overall prevalence of fetal Down syndrome was 0.21% (7/3,291 pregnancies). The overall risk reduction for fetal Down syndrome in the presence of normal genetic sonography was 6.64-fold (95% CI, 3.01-14.62); the overall negative likelihood ratio was 0.15 (95% CI, 0.07-0.33). In the presence of normal genetic sonography, the risk for fetal Down syndrome was reduced by 83% in patients with advanced maternal age, 88% in patients with abnormal triple screen, 89% in patients with abnormal triple screen who were <35 years old, and 84% in patients who had both abnormal triple screen and advanced maternal age. CONCLUSION: There were no significant variations in the risk reduction for fetal Down syndrome in the presence of normal genetic sonography. Regardless of the indication for testing, the likelihood for fetal Down syndrome was reduced by 83% to 89%. This information will be useful in counseling pregnant women who are at high risk for fetal Down syndrome and who prefer to undergo genetic sonography before deciding about genetic amniocentesis.

The impact of prenatal care in the United States on preterm births in the presence and absence of antenatal high-risk conditions.

OBJECTIVE: This study was undertaken to determine the association between prenatal care in the United States and preterm birth rate in the presence, as well as absence, of high-risk pregnancy conditions for African American and white women. STUDY DESIGN: Data were derived from the natality data set for the years 1995 to 1998 provided by the National Center for Health Statistics. Analyses were restricted to singleton live births that occurred at >/=20 weeks' gestation. Multiple births, fetal deaths, congenital malformations, chromosomal abnormalities, missing data on gestational age, and birth weight less than 500 g were excluded. Multivariable logistic regression analyses were used to adjust for the presence or absence of various antenatal high-risk conditions, maternal age, gravidity, marital status, smoking, alcohol, and education. Prenatal care was considered present if there was one or more prenatal visits. Preterm delivery was defined as delivery at less than 37 completed weeks of gestation. RESULTS: For 14,071,757 births analyzed, 1,348,643 (9.6%) resulted in preterm birth. Preterm birth rates were higher for African American women than white women in the presence (15.1% vs 8.3%) and absence (34.9% vs 21.9%) of prenatal care. The absence of prenatal care increased the relative risk for preterm birth 2.8-fold in both African American and white women. There was an inverse dose-response relationship between the number of prenatal visits and the gestational age at delivery both among African American and white women. Lack of prenatal care was associated with increased preterm birth rates to a similar degree in the presence of pregnancy complications for both African American and white women, ranging from 1.6-fold to 5.5-fold for the various antenatal high-risk conditions. CONCLUSION: In the United States, prenatal care is associated with fewer preterm births in the presence, as well as absence of high-risk conditions for both African American and white women. Strategies to increase prenatal care participation may decrease preterm birth rates.

The impact of prenatal care on neonatal deaths in the presence and absence of antenatal high-risk conditions.

OBJECTIVE: The purpose of this study was to determine the association between prenatal care in the United States and the neonatal death rate in the presence and absence of antenatal high-risk conditions. STUDY DESIGN: Data were derived from the national perinatal mortality data sets for the years 1995 through 1997, which were provided by the National Center for Health Statistics. Analyses were restricted to singleton live births that occurred after 23 completed weeks of gestation. Multivariable logistic regression analyses were used to adjust for the presence or absence of various antenatal high-risk conditions, maternal age, gestational age at delivery, and birth weight. RESULTS: Of 10,530,608 singleton live births, 18,339 (1.7/1000 births) resulted in neonatal death. Neonatal death rates (per 1000 live births) were higher for African American infants compared with white infants in the presence (2.7 vs 1.5, respectively) and absence (10.7 vs 7.9, respectively) of prenatal care. Lack of prenatal care was associated with an increase in neonatal deaths, which was greater for infants born at > or =36 weeks of gestation (relative risk, 2.1; 95% CI, 1.8, 2.4). Lack of prenatal care was also associated with increased neonatal death rates in the presence of preterm premature rupture of the membranes (relative risk, 1.3; 95% CI, 1.1, 1.5), placenta previa (relative risk, 1.9; 95% CI, 1.2, 2.9), fetal growth restriction (relative risk, 1.7; 95% CI, 1.2, 1.6), and postterm pregnancy (relative risk, 1.4; 95% CI, 1.0, 2.9). CONCLUSION: In the United States, prenatal care is associated with fewer neonatal deaths in black and white infants. This beneficial effect was more pronounced for births that occurred at > or =36 weeks of gestation and in the presence of preterm premature rupture of the membranes, placenta previa, fetal growth restriction, and postterm pregnancy.