Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa community acquired pneumonia: Prevalence and locally derived risk factors in a single hospital system.

Objectives: Current American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) community-acquired pneumonia (CAP) guidelines expand the CAP definition to include infections occurring in patients with recent health care exposure. The guidelines now recommend that hospital systems determine their own local prevalence and predictors of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) among patients satisfying this new broader CAP definition. We sought to carry out these recommendations in our system, focusing on the emergency department, where CAP diagnosis and initial empiric antibiotic selection usually ooccur. Methods: We performed a retrospective cohort study of patients admitted with CAP through any of 3 EDs in our hospital system in Northern California between November 2019 and October 2021. Inclusion criteria included an ED admission diagnosis of pneumonia or sepsis, fever or hypothermia, leukocytosis or leukopenia, and consistent chest imaging result. SARS-CoV-2-positive cases were excluded. We abstracted variables historically associated with P. aeruginosa and MRSA. Outcome measures were prevalence of P. aeruginosa and MRSA in the overall clinically defined cohort and among microbiologically confirmed cases and predictors of P. aeruginosa or MRSA isolation, as determined by univariate logistic regression, bootstrapped least absolute shrinkage and selection operator, and random forest analyses. Additionally, we describe the iterative process used and challenges encountered in carrying out the new ATS/IDSA guideline recommendations. Results: There were 1133 unique patients who satisfied our definition of clinically defined CAP, of whom 109 (9.6%) had a bacterial pathogen isolated. There were 24 P. aeruginosa isolates and 11 MRSA isolates in 33 patients. Thus, the prevalence P. aeruginosa and MRSA was 2.9% in the overall CAP cohort, but 30.3% in the microbiologically confirmed cohort. The most important predictors of either P. aeruginosa or MRSA isolation were tracheostomy (odds ratio [OR] 22.08; 95% confidence interval [CI] 10.39-46.96) and gastrostomy tube (OR 14.7; 95% CI 7.14-30.26). Challenges included determining the suspected infection type in patients admitted simply for "sepsis"; interpreting dictated radiology reports; determining functional status, presence of indwelling lines and tubes, and long-term care facility residence from the electronic health record; and correctly attributing culture results to pneumonia. Conclusion: Prevalence of MRSA and P. aeruginosa was low among patients admitted in our medical system with CAP - now broadly defined - but high among those with a microbiologically confirmed bacterial etiology. Our locally derived predictors of MRSA and P. aeruginosa can be used to aid our emergency physicians in empiric antibiotic selection for CAP. Findings from this project might inform efforts at other institutions.

Discriminating Acute Respiratory Distress Syndrome from other forms of respiratory failure via iterative machine learning.

Acute Respiratory Distress Syndrome (ARDS) is associated with high morbidity and mortality. Identification of ARDS enables lung protective strategies, quality improvement interventions, and clinical trial enrolment, but remains challenging particularly in the first 24 hours of mechanical ventilation. To address this we built an algorithm capable of discriminating ARDS from other similarly presenting disorders immediately following mechanical ventilation. Specifically, a clinical team examined medical records from 1263 ICU-admitted, mechanically ventilated patients, retrospectively assigning each patient a diagnosis of "ARDS" or "non-ARDS" (e.g., pulmonary edema). Exploiting data readily available in the clinical setting, including patient demographics, laboratory test results from before the initiation of mechanical ventilation, and features extracted by natural language processing of radiology reports, we applied an iterative pre-processing and machine learning framework. The resulting model successfully discriminated ARDS from non-ARDS causes of respiratory failure (AUC = 0.85) among patients meeting Berlin criteria for severe hypoxia. This analysis also highlighted novel patient variables that were informative for identifying ARDS in ICU settings.

Transfusion-Transmitted Cache Valley Virus Infection in a Kidney Transplant Recipient With Meningoencephalitis.

BACKGROUND: Cache Valley virus (CVV) is a mosquito-borne virus that is a rare cause of disease in humans. In the fall of 2020, a patient developed encephalitis 6 weeks following kidney transplantation and receipt of multiple blood transfusions. METHODS: After ruling out more common etiologies, metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) was performed. We reviewed the medical histories of the index kidney recipient, organ donor, and recipients of other organs from the same donor and conducted a blood traceback investigation to evaluate blood transfusion as a possible source of infection in the kidney recipient. We tested patient specimens using reverse-transcription polymerase chain reaction (RT-PCR), the plaque reduction neutralization test, cell culture, and whole-genome sequencing. RESULTS: CVV was detected in CSF from the index patient by mNGS, and this result was confirmed by RT-PCR, viral culture, and additional whole-genome sequencing. The organ donor and other organ recipients had no evidence of infection with CVV by molecular or serologic testing. Neutralizing antibodies against CVV were detected in serum from a donor of red blood cells received by the index patient immediately prior to transplant. CVV neutralizing antibodies were also detected in serum from a patient who received the co-component plasma from the same blood donation. CONCLUSIONS: Our investigation demonstrates probable CVV transmission through blood transfusion. Clinicians should consider arboviral infections in unexplained meningoencephalitis after blood transfusion or organ transplantation. The use of mNGS might facilitate detection of rare, unexpected infections, particularly in immunocompromised patients.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Probiotics: Potential novel therapeutics for microbiota-gut-brain axis dysfunction across gender and lifespan.

Probiotics are live microorganisms, which when administered in adequate amounts, present a health benefit for the host. While the beneficial effects of probiotics on gastrointestinal function are generally well recognized, new animal research and clinical studies have found that alterations in gut microbial communities can have a broad range of effects throughout the body. Non-intestinal sites impacted include the immune, endocrine, cardiovascular and the central nervous system (CNS). In particular, there has been a growing interest and appreciation about the role that gut microbiota may play in affecting CNS-related function through the 'microbiota-gut-brain axis'. Emerging evidence suggests potential therapeutic benefits of probiotics in several CNS conditions, such as anxiety, depression, autism spectrum disorders and Parkinson's disease. There may also be some gender-specific variances in terms of probiotic mediated effects, with the gut microbiota shaping and being concurrently molded by the hormonal environment governing differences between the sexes. Probiotics may influence the ability of the gut microbiome to affect a variety of biological processes in the host, including neurotransmitter activity, vagal neurotransmission, generation of neuroactive metabolites and inflammatory response mediators. Some of these may engage in cross talk with host sex hormones, such as estrogens, which could be of relevance in relation to their effects on stress response and cognitive health. This raises the possibility of gender-specific variation with regards to the biological action of probiotics, including that on the endocrine and central nervous systems. In this review we aim to describe the current understanding in relation to the role and use of probiotics in microbiota-gut-brain axis-related dysfunction. Furthermore, we will address the conceptualization and classification of probiotics in the context of gender and lifespan as well as how restoring gut microbiota composition by clinical or dietary intervention can help in supporting health outcomes other than those related to the gastrointestinal tract. We also evaluate how these new learnings may impact industrial effort in probiotic research and the discovery and development of novel and more personalized, condition-specific, beneficial probiotic therapeutic agents.

Transient growth, edge states, and repeller in rotating solid and fluid.

For the classical problem of the rotation of a solid, we show a somehow surprising behavior involving large transient growth of perturbation energy that occurs when the moment of inertia associated to the unstable axis approaches the moment of inertia of one of the two stable axes. In that case, small but finite perturbations around this stable axis may induce a total transfer of energy to the unstable axis, leading to relaxation oscillations where the stable and unstable manifolds of the unstable axis play the role of a separatrix, an edge state. For a fluid in solid-body rotation, a similar linear and nonlinear dynamics apply to the transfer of energy between three inertial waves respecting the triadic resonance condition. We show that the existence of large transient energy growth and of relaxation oscillations may be physically interpreted as in the case of a solid by the existence of two quadratic invariants, the energy and the helicity in the case of a rotating fluid. They occur when two waves of the triad have helicities that tend towards each other, when their amplitudes are set such that they have the same energy. We show that this happens when the third wave has a vanishing frequency which corresponds to a nearly horizontal wave vector. An inertial wave, perturbed by a small-amplitude wave with a nearly horizontal wave vector, will then be periodically destroyed, its energy being transferred entirely to the unstable wave, although this perturbation is linearly stable, resulting in relaxation oscillations of wave amplitudes. In the general case we show that the dynamics described for particular triads of inertial waves is valid for a class of triadic interactions of waves in other physical problems, where the physical energy is conserved and is linked to the classical conservation of the so-called pseudomomentum, which singles out the role of waves with vanishing frequency.

Differential contribution of transcriptomic regulatory layers in the definition of neuronal identity.

Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.

Time of day is an inaccurate surrogate marker for sleep-wake state in an epilepsy monitoring unit.

OBJECTIVE: To assess the correlation between time of day and sleep-wake state prior to seizure onset for seizures recorded in an inpatient epilepsy monitoring unit. METHODS: We prospectively enrolled a consecutive series of patients undergoing inpatient epilepsy monitoring. For each epileptic seizure recorded, continuous EEG data preceding seizure onset was reviewed and scored as W, N1-3, or REM in ten 30-second epochs. Time of day was divided into four 6-h phases (0600-1159, 1200-1759, 1800-2359, 0000-0559). The preictal sleep-wake state was then correlated to nocturnal (0000-0559) versus diurnal (0600-2359) times of day. RESULTS: A total of 102 seizures from 42 patients met enrollment criteria over a period of 19 months. Eighty-five seizures occurred during the diurnal phase, and 17 seizures occurred during the nocturnal phase. Thirty-six percent of all seizures (n = 37) were preceded by at least 1 epoch of sleep. The proportion of patients sleeping prior to a seizure within each 6-h phase varied significantly from the overall distribution only during nocturnal phase. Seventy-six percent of nocturnal seizures and 28 % of diurnal seizures were preceded by sleep. Therefore, the nocturnal time window from 0000-0559 had a sensitivity of 0.65 (95 % confidence interval 0.48-0.78), a specificity of 0.06 (0.02-0.15), a positive predictive value of 0.28 (0.20-0.39), and a negative predictive value of 0.24 (0.10-0.39) in association with sleep-onset seizures. SIGNIFICANCE: The time of day is an inaccurate surrogate for preictal sleep-wake state in the epilepsy monitoring unit despite a correlation between nocturnal period and sleep. Diurnal sleep is a common phenomenon in the inpatient unit.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation.

BACKGROUND: RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. RESULTS: We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. CONCLUSIONS: SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications.

A Case of Tick-Borne Paralysis in a Traveling Patient.

BACKGROUND: Tick paralysis is a neurotoxic tick-borne illness that causes ascending paralysis and may lead to respiratory failure. Patients often undergo extensive testing and prolonged hospitalization before the proper diagnosis is reached. CASE PRESENTATION: An 88-year-old man with dementia and dyslipidemia presented with new onset gait instability and was admitted for suspected cerebellar stroke. Exam was significant for the inability to perform tandem gait. Investigations included comprehensive metabolic panel, complete blood count, and noncontrast CT scan; none of them found any evidence of acute pathology. Two days into admission, a tick with surrounding erythema was found on the patient's left lateral chest during bathing. Dramatic improvement in truncal ataxia was noted following tick extraction. DISCUSSION: Clinical suspicion of tick paralysis is often low due to the rarity of the condition. Although it is imperative to rule out acute cerebral or cerebellar pathology, a thorough skin examination should be performed on admission in any patient with new onset ataxia and ascending paralysis. This can lead to early diagnosis, conservation of resources, and the avoidance of subjecting patients to invasive testing.

Efficient and Accurate Quantitative Profiling of Alternative Splicing Patterns of Any Complexity on a Laptop.

Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly-with hardware requirements compatible with a laptop-models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.

QAPA: a new method for the systematic analysis of alternative polyadenylation from RNA-seq data.

Alternative polyadenylation (APA) affects most mammalian genes. The genome-wide investigation of APA has been hampered by an inability to reliably profile it using conventional RNA-seq. We describe 'Quantification of APA' (QAPA), a method that infers APA from conventional RNA-seq data. QAPA is faster and more sensitive than other methods. Application of QAPA reveals discrete, temporally coordinated APA programs during neurogenesis and that there is little overlap between genes regulated by alternative splicing and those by APA. Modeling of these data uncovers an APA sequence code. QAPA thus enables the discovery and characterization of programs of regulated APA using conventional RNA-seq.

Cell-type specific potent Wnt signaling blockade by bispecific antibody.

Cell signaling pathways are often shared between normal and diseased cells. How to achieve cell type-specific, potent inhibition of signaling pathways is a major challenge with implications for therapeutic development. Using the Wnt/ß-catenin signaling pathway as a model system, we report here a novel and generally applicable method to achieve cell type-selective signaling blockade. We constructed a bispecific antibody targeting the Wnt co-receptor LRP6 (the effector antigen) and a cell type-associated antigen (the guide antigen) that provides the targeting specificity. We found that the bispecific antibody inhibits Wnt-induced reporter activities with over one hundred-fold enhancement in potency, and in a cell type-selective manner. Potency enhancement is dependent on the expression level of the guide antigen on the target cell surface and the apparent affinity of the anti-guide antibody. Both internalizing and non-internalizing guide antigens can be used, with internalizing bispecific antibody being able to block signaling by all ligands binding to the target receptor due to its removal from the cell surface. It is thus feasible to develop bispecific-based therapeutic strategies that potently and selectively inhibit signaling pathways in a cell type-selective manner, creating opportunity for therapeutic targeting.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.

An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

Oncogenic Activation of the RNA Binding Protein NELFE and MYC Signaling in Hepatocellular Carcinoma.

Global transcriptomic imbalance is a ubiquitous feature associated with cancer, including hepatocellular carcinoma (HCC). Analyses of 1,225 clinical HCC samples revealed that a large numbers of RNA binding proteins (RBPs) are dysregulated and that RBP dysregulation is associated with poor prognosis. We further identified that oncogenic activation of a top candidate RBP, negative elongation factor E (NELFE), via somatic copy-number alterations enhanced MYC signaling and promoted HCC progression. Interestingly, NELFE induces a unique tumor transcriptome by selectively regulating MYC-associated genes. Thus, our results revealed NELFE as an oncogenic protein that may contribute to transcriptome imbalance in HCC through the regulation of MYC signaling.

RNAcompete-S: Combined RNA sequence/structure preferences for RNA binding proteins derived from a single-step in vitro selection.

RNA-binding proteins recognize RNA sequences and structures, but there is currently no systematic and accurate method to derive large (>12base) motifs de novo that reflect a combination of intrinsic preference to both sequence and structure. To address this absence, we introduce RNAcompete-S, which couples a single-step competitive binding reaction with an excess of random RNA 40-mers to a custom computational pipeline for interrogation of the bound RNA sequences and derivation of SSMs (Sequence and Structure Models). RNAcompete-S confirms that HuR, QKI, and SRSF1 prefer binding sites that are single stranded, and recapitulates known 8-10bp sequence and structure preferences for Vts1p and RBMY. We also derive an 18-base long SSM for Drosophila SLBP, which to our knowledge has not been previously determined by selections from pure random sequence, and accurately discriminates human replication-dependent histone mRNAs. Thus, RNAcompete-S enables accurate identification of large, intrinsic sequence-structure specificities with a uniform assay.