Storm surges cause coastal inundation due to setup of the water surface resulting from atmospheric pressure, surface winds and breaking waves. Here we show that during Typhoon Haiyan, the setup generated by breaking waves near the fringing-reef-protected town of Hernani, the Philippines, oscillated with the incidence of large and small wave groups, and steepened into a tsunami-like wave that caused extensive damage and casualties. Though fringing reefs usually protect coastal communities from moderate storms, they can exacerbate flooding during strong events with energetic waves. Typical for reef-type bathymetries, a very short wave-breaking zone over the steep reef face facilitates the freeing of infragravity-period fluctuations (surf beat) with little energy loss. Since coastal flood planning relies on phase-averaged wave modelling, infragravity surges are not being accounted for. This highlights the necessity for a policy change and the adoption of phase-resolving wave models for hazard assessment in regions with fringing reefs. The failure of a broad fringing coral reef to protect the village of Hemani from a tsunami-like wave during Typhoon Haiyan came as a destructive surprise. Here, the authors present results from a phase-resolving wave model and show that the steep reef face facilitated the release of energetic infragravity waves. The tsu−1 (refs c=(gh)1/2, where g is the acceleration due to gravity and h is the local water depth. This would have required a water depth on the order of 20 m or less for the storm to have been able to keep up with the free long wave generated. However, the water depth offshore of Hernani quickly drops to kilometres  together with the Delft-3D/SWAN16−1  at the lPreviously, Nakaza and Hino2018t=625 s in To recreate the tsunami-like wave that struck Hernani, the offshore SWAN wave spectrum was input to the two-dimensional (plan view) Boussinesq-type phase-resolving wave model BOSZ24f=0.0035 Hz (frequency f is the inverse of period T) as illustrated in f=0.004 Hz. The hypothetical case of ‘no reef' in f=0.002 Hz, which corresponds to the reef's resonant frequency. Resonance has a much larger effect here than in the real bathymetry case because the high seawall limits the domain to the exact size of the quarter-wave oscillator, whereas in the real reef case the domain is lengthened by the run-up of the surf beat on land. The case of a steep ‘drop-off' immediately offshore of the beach has a peak at the wave group frequency of 0.004 Hz of slightly larger magnitude than that of the ‘reef'. Since the reef face bathymetry is identical for both ‘reef' and ‘drop-off' cases, the similarity of the wave spectra between these two scenarios indicates that the mechanism responsible for the tsunami-like wave of To determine the relative contribution of offshore wave groups and reef resonance to amplification of the surf beat, BOSZ was evaluated in one-dimension along the transect shown in Interestingly, the wave that struck Hernani was not a worst-case scenario: if a similar storm to Typhoon Haiyan was to strike a site fronted by a narrower reef with an equally steep reef face, resonance over the reef would become significant, with the potential of forming an even larger surf beat and subsequently more intense flooding. This is illustrated in F on buildings near the beach. F  on the beach for the ‘reef' and ‘no-reef' cases. For storms of up to about 50% the strength of Typhoon Haiyan, the reef protects the shore from incident waves. During larger storms, however, it elevates the potential for damage due to an increasing hydrodynamic force in the infragravity frequency band.The effect of the presence of the reef on damage onshore was further investigated using one-dimensional BOSZ simulations. Storms of various intensities were modelled by scaling down the central pressure and maximum wind speed of Typhoon Haiyan . For eacThe tsunami-like wave that struck Hernani was the result of abrupt breaking of waves over the steep reef face generating an energetic surf beat. Had the surf beat been in resonance with the reef flat15Land surface and coral reef elevations were measured from 25 to 27 May 2014 via walking and driving surveys with two Ashtech ProMark 100 differential global positioning system (DGPS) units, in base/rover differential configuration, and post-processed for vertical accuracy with GNSS Solutions software. The water depth seaward of the coral reef was measured from a hired fishing canoe, using a Hondex PS-7 handheld digital sounder, which has an operational range of up to 80 m. Sounder readings were photographed with a Canon PowerShot D20 global positioning system (GPS) camera to record the geographic location of each measurement. All elevation and depth measurements were referenced to MSL using the TPXO global tide modelSince the elevation of the coral reef flat in Hernani was not measured during the 25–26 May field campaign in Hernani, the reef flat elevation of a similar reef stretch in Guiuan  had a resolution of 0.01°(about 1 km), and the small domain  had a resolution of 0.0004°(about 40 m). Coarse bathymetry data was taken from GEBCOSWAN output was produced for various storm intensities to quantify the effect of the overall wave energy on the formation of the bore that struck Hernani. In f. To avoid any artificial wave group generation, the SWAN spectra were resampled to a logarithmic spacing with a smallest interval of df=1/3,600 s, which excludes repetitions of the input signal over the 1-h model run. Furthermore, to ensure quality of the BOSZ results, the high frequency tail of the SWAN spectra were truncated at 0.13 Hz and proportionally redistributed over the remaining frequency bins according to the constraints of the dispersion approximations in the model's governing equations, as described in Li et al.To generate the input wave time series for the phase-resolving BOSZ wave model24et al.et al.The wavemaker source function used in BOSZ24n of 0.035 s m−1/3  as well as along a one-dimensional transect passing through the location of the house seen washed away in Gensis' film1/3 ref.  was usedhttp://hydraulic.lab.irides.tohoku.ac.jp/.As no measurements of actual wave heights are available from the site, the phase-resolving depth-resolving Volume-of-Fluid model OpenFOAMThe model results can be understood through comparison with analytical oscillation periods of the water surface. In the incident wave time series, the minimum wave group return period is given by μ0=m0 and μ2=m2−m12/m0. The spectral moments mr are given by equation (2)where f is the frequency (Hz), E is the spectral energy density (m2 Hz−1) and r is an integer. The actual wave group return period can be longer than Tenvelope,min. For Typhoon Haiyan Tenvelope,min is 230 s, as represented by the 100% storm of where The resonant period of a quarter-wave oscillator such as a reef flat is given by equation (3)Tresonance is the resonant period, L is the cross-shore length of the reef, g is the acceleration due to gravity, h is the mean water depth over the reef and n is a positive integer defining the amount of nodes. This type of oscillation consists of a node at the reef face and an antinode at the beach. Assuming L=750 m , h=4 m (the mean water depth over the reef) and n=1 , the reef resonant period is 480 s (corresponding to a frequency f=0.0021 Hz).where The period of a basin seiche, a numerical artifact of the closed model domain, is given by equation (4)n=1) seiche period is the time it takes a long wave to propagate from the left wall of the domain, reflect from the reef face .with the same variable definitions as in x=2,700 m from the start of the transect. Landward of this location, energy in the gravity wave range is low, but a non-negligible amount of energy exists even beyond the seawall at x=3,314 m, indicating ocean waves propagate atop the surf beat onto land. At infragravity frequencies, the largest energy is near f=0.0035 Hz, which is the incident wave group period. This is strongest over the reef, but also energetic landward of the seawall. Energy is also substantial over the reef at its resonant frequency at f=0.0021 Hz, though this band is not as strong in the area between the beach and the house location. Energy at low frequencies f<0.001 Hz indicate the static setup induced by waves breaking over the reef. Energy in deep water at f=0.0021 Hz and f=0.0035 Hz is a result of reflection of the surf beat from the shore back into deep water as a free wavef=0.006 Hz and is an artifact of the closed model domain. Consequently, energy at f=0.012 Hz is the first superharmonic of the basin seiche. The basin seiche shoals between the foot of the shelf slope at x=1,500 m and the reef crest; shoaling transfers energy of a wave to its superharmonicsx=3,200 m. Almost no energy in the gravity wave range exists landward of the seawall at x=3,314 m. At infragravity frequencies, the same basin seiches as in the ‘reef' case are present, though they contain no energy landward of the seawall. Significant energy near the wave group frequency f=0.0035 Hz does exist landward of the seawall; however, the amount of energy is smaller than in the real reef case. The only other infragravity energy present is at a very low frequency, representing the static setup.For the ‘no-reef' case, gravity wave energy dissipates gradually between the reef crest and the beach at x=2,700 m (offshore extent of the surf zone) and x=3,200 m (beach), the ‘no-reef' case displays more energy than the ‘reef' scenario. However, landward of the beach, much more infragravity energy exists with the ‘reef' bathymetry. Schäffer2 Hz−1 present over a frequency range of 0.002 Hz in et al.et al.equation (5)van Dongeren hx is the bed slope, ω is the radial frequency of the infragravity wave, g is the acceleration due to gravity and hb is the depth of wave breaking. Their et al.where hx=0.2 and the slope of the beach is hx=0.017 , infragravity bore formation is similar, but short-period gravity waves are less damped than in the case with the reef.In the case of Hernani, the slope of the reef face is hx=0.017 . The offhx=0.017 , the watutations  show thaThough The hydrodynamic force on a buildingequation (6)ρ is the water density, CD is an empirical drag coefficient, B is the width of an exposed building in the cross-flow direction, h is the flow depth and u is the flow speed.where How to cite this article: Roeber, V. & Bricker, J .D Destructive tsunami-like wave generated by surf beat over a coral reef during Typhoon Haiyan. Nat. Commun. 6:7854 doi: 10.1038/ncomms8854 (2015).Animation of the water surface over the entire BOSZ model domain. The black triangle is the location of the house in Fig. 2. Grayscale indicates topography. Offshore and lateral boundary wavemakers and sponge layers are visible.Animation of flow depth and velocity vectors during the incidence of a large wave set into Hernani. The black triangle is the location of the house in Fig. 2. White lines are sea bottom elevation contours beginning at mean sea level (MSL) and continuing downward at 1 m intervals. Black lines are land elevation contours beginning at 1 m above MSL and continuing upward at 1 m intervals to 10 m above MSL (higher elevations omitted).


Formamide chemistry potentially satisfies all of the steps from the very initial precursors to RNA. Water chemistry enters the scene in RNA non-enzymatic synthesis and recombination.Reactions of formamide (NH Top-down reasoning is the most direct, logical approach to biological problems related to systems and processes that are not occurring anymore. This is especially true when one deals with origin of life matters. In reasoning and experimenting about RNA origins, two different, but potentially converging, approaches are used: the pragmatic and the philological. in vitro molecular evolution studies  to H2NCOH, all the way to a large set of prebiotic compounds of (pre)genetic and (pre)metabolic relevance appears to be, at the moment, an essentially continuous path, devoid of interruptions or thermodynamically or kinetically insurmountable gaps. Naturally, this is not to say that the scenario is totally clear or complete, especially in the absence of an agreed-upon definition of the ur-environment. The route from formamide to acyclonucleosides and to at least one extant nucleoside is well-understood and continuous, recently gaining consensus [In summary, the route from [HCN + Honsensus ,27. In fonsensus ,29,30,31Polymerization of 3',5'-cAMP and of 3',5'-cGMP has been reported . PolymerBased on the measured stacking of the 3',5'-cyclic monomers , the acta posteriori intercalation of the fluorescent dye, SYBR Gold. The detection of polymerization by this additional and different technique validates the reaction.This polymerization reaction has been confirmed by Dieter Braun’s group , showingPolymerization of 3',5'-cGMP is more efficient under dry conditions than in water; the difference consisting in (i) faster kinetics and (ii) a lower temperature requirement for the transphosphorylation step under dry conditions (to be detailed elsewhere). Enhancement of polymerization by decreasing water concentrations coheres with the Gibbs free-energy paradox, which states that reactions that proceed through the release of water are hampered by the presence of water ,37.The comparison of the polymerization of 3’,5’-cGMP under dry conditions and in formamide  shows thAgain, this is not to say that RNA non-fastidiously and easily emerged out of a formamide solution. However, the observations that: (i) nucleoside synthesis is streamlined when initiated by formamide even though under high-energy proton beams  mimickinIn thermodynamical terms, RNA polymerization is a form of stabilization, as detailed by the comparative analysis of the stability of the weakest bonds in RNA precursor monomers or in RNA ,39   as an “aThe prebiotically relevant point here is that this reaction occurs by the action of spontaneously-generated polymers derived from abiotically plausible precursors, enhancing the information content of a polymeric mixture. This mechanism potentially represents a plausible means to generate RNA sequence complexity and to approach the questions: how could the extremely complex ribosomal machinery  start toSummarizing these studies, RNA molecules derived from the interactions of non-enzymatically polymerized oligoG with oligonucleotides containing a sufficiently long complementary sequence are active ribozymes, their activity consisting of the nucleophilic attack of an acceptor 3'-hydroxyl group on the phosphorus of a donor 3',5' phosphodiester bond. The location of the donor phosphate, the orientation and the structure of the surrounding sequences determine the result, consisting of a single cleavage, leading to a one-step chain elongation event.The cleavage by the 3' OH of the acceptor molecules preferentially occurs on an XpX step located one step 3' distal from the 5' extremity, in an unpaired structure, at the tip of the presumptive sequence complementarity-determined double-stranded structure , presumaThe fact that the interaction between simple sequence oligonucleotides leads to further transformation of the sequence information provides the proof-of-principle that the nonenzymatic generation of oligoG RNA molecules from the prebiotically plausible 3',5'-cyclic nucleotides is the first step of a potentially (pre)genetic process.No matter how complex the variety of extant ribozyme activities are, they necessarily evolved from initially simple sequences ,63 endowWe have here concisely described that all of the steps leading from formamide to short RNA sequences endowed with simple ribozyme activity are experimentally plausible. Life is a robust phenomenon, and its origin most likely depended on robust processes, easily available starting materials and thermodynamically sound reactions. Formamide chemistry might have played a non-secondary role in origin-of-life processes: its abundance in space has been recently reported ; the num


Studying the factors that control gene expression is of substantial importance for rheumatic diseases with poorly understood etiopathogenesis. In the past, gene expression microarrays have been used to measure transcript abundance on a genome-wide scale in a particular cell, tissue or organ. Microarray analysis has led to gene signatures that differentiate rheumatic diseases, and stages of a disease, as well as response to treatments. Nowadays, however, with the advent of next-generation sequencing methods, massive parallel sequencing of RNA tends to be the technology of choice for gene expression profiling, due to several advantages over microarrays, as well as for the detection of non-coding transcripts and alternative splicing events. In this review, we describe how RNA sequencing enables unbiased interrogation of the abundance and complexity of the transcriptome, and present a typical experimental workflow and bioinformatics tools that are often used for RNA sequencing analysis. We also discuss different uses of this next-generation sequencing technology to evaluate rheumatic disease patients and investigate the pathogenesis of rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis and Sjögren’s syndrome. Gene expression profiling is the analysis of the expression or activity of genes, in order to understand how genes contribute to certain biological functions, and to elucidate the pathogenic mechanisms of diseases and disorders. In order to acquire new information about the role of genes under various conditions, gene expression is typically measured in different conditions or tissues, such as at different time points, or between normal and cancer/disease cells. Differential expression (DE) analysis between conditions produces gene signatures that are characteristic of the condition or disease being studied. For example, a gene signature in rheumatoid arthritis (RA) is a collection of genes that are either up- or down-regulated when compared with normal cells . In the field of rheumatic diseases, gene expression profiling studies have used DNA microarrays extensively –3. DNA mSuch limitations are largely absent in RNA sequencing (RNA-seq), a next-generation sequencing (NGS) method largely used for the genome-wide measurement of RNA abundance and the detection of alternative splicing events , 6. CompIn this review, we present a typical RNA-seq workflow, experimental choices and data analysis pipelines. We also discuss recent published studies Table , as wellIn the past few years, sequencing technologies and chemistries have been advancing at a rapid pace. Several companies offer NGS platforms, with Illumina’s HiSeq and MiSeq , and LifIndependently of the technology of choice, a typical RNA-seq workflow is a multi-step process that includes RNA and library preparation, sequencing, and data analysis Fig. . During Researchers face several challenges when designing an RNA-seq experiment regarding decisions that can affect both the cost and the accuracy of the experiment. Such challenges involve selecting the optimal number of replicates and the sequencing depth required to achieve reliable detection power of DE. As far as replicate choice is concerned, several studies –19, as wAnother decision that researchers have to make before sending their samples to a sequencing facility for library preparation is whether to choose SRs or PE reads. As mentioned above, SRs refer to fragments that are read by the sequencer from only one end to the other, generating the sequence of base pairs, while for PE reads the sequencer starts at one read end, finishes this direction at the specified read length and then starts another round of reading from the opposite end of the fragment. PE reads is a necessary configuration in an RNA-seq experiment when increased mapping accuracy is important in order to assess genome rearrangements, identify fusion genes and detect alternative splicing events. Although SRs are usually preferred as an RNA-seq strategy for measuring transcript abundance due to lower cost, it is recommended to perform PE sequencing whenever possible , 26.Finally, multiplexing is a method that allows pooling libraries from multiple samples into a single sequencing reaction. In order to identify the ‘origin’ of sequenced reads, a short nucleotide sequence (approximately six to seven nucleotides), called the barcode or index, is attached to each cDNA fragment during library preparation. The barcodes are read during sequencing, allowing the reads to be traced back to their original samples. Choosing to perform RNA-seq multiplexing can reduce the cost of an experiment, but will also produce smaller number of reads per sample. For example, a single flow cell lane from an Illumina HiSeq 2500 platform routinely produces approximately 200 million SRs for one sample without multiplexing. If four samples are multiplexed, then approximately 50 million SRs per sample will be produced at a much lower sequencing cost per sample.de novo assembly of RNA-seq data is also commonly used for studying the transcriptome of species without reference genomes, such as non-model organisms in microbiome and metagenomics studies; however, discussion of RNA-seq as a de novo transcript assembly tool is beyond the scope of this review.From the data analysis perspective, RNA-seq includes the following steps: assessment of the quality of the sequenced reads (using tools such as FastQC ), removaRead alignment remains one of the most computationally intensive steps in the entire process, since it requires the alignment of tens or hundreds of millions of reads to multiple gigabases for a typical mammalian genome. Some of the most popular alignment programs used for RNA-seq include Bowtie/TopHat , BWA 2828, and SThe next step involves estimating the abundance of known genes or transcripts. Frequently, this involves determining the number of reads that map to known genes or transcripts, also known as read counts. In this analysis, the number of reads supporting each gene or transcript according to gene annotation  are determined using programs such as HTSeq . AlthougHowever, it has been discussed in some studies that RPKMs/FPKMs have certain limitations that can bias estimates of DE , 32 and For the identification of non-coding RNA, such as miRNA and lncRNA, the data analysis pipelines differ from the ones used for DE analysis of genes/transcripts. After the reads are aligned against the reference genome, non-coding annotations are used, such as the miRBase (database of known miRNAs), lncRNAdb (database of lncRNAs), ncRNAdb (database of non-coding regulatory RNAs) and others. Related third party analysis tools for this purpose include mirRanalyzer , miRToolVisualization of the mapped reads  in a genome browser, such as the UCSC Genome Browser  or the IRA is a chronic systemic autoimmune disorder that primarily affects the joints and ultimately leads to their destruction . It affeBRCA1 in DNA damage response, were found to potentially contribute to the pathogenesis of RA. Overall, this study shows the first complete transcriptome analysis of SFs from patients with RA using RNA-seq and reveals a complete repertoire of active molecules, networks and pathways of differentially expressed genes and their isoforms in RASFs. As suggested by the authors, follow-up analyses using a larger number of patient samples will be necessary to validate the alterations in transcriptional regulation reported in this study and provide the resources necessary to elucidate the molecular mechanisms underlying the role of SFs in the pathogenesis of RA.In this study , severalThe study by Shi and colleagues  used RNAOverall, this study  showed tIL-17 were reported as 2-month predictors of a 6-month clinical response, although their baseline levels of transcripts did not predict efficacy. In contrast, IL6R was a good baseline predictor of efficacy but its expression did not change from baseline to 2 months. The results that are briefly presented in this study [Abatacept (CTLA4Ig) belongs to the biologic class of drugs, which means that it works similarly to natural substances in the immune system and is used to decrease inflammation in RA . Althougis study  demonstrP = 0.001, fold change = −3.73 and −4.74) and one lncRNA at 5q33.3 , with yet unclear biological functions. Overall, the authors of this abstract used RNA-seq to create gene signatures of different disease states in JIA, but also to detect novel lncRNAs that may have functional consequences in JIA.A recent study  that is SRP14, UQCRB and ATP5I were the most statistically differentially expressed protein-coding transcripts between SS and control. Further investigation is required to study the biological functions of these genes and their potential role in SS. DE analysis of non-coding transcripts revealed a lncRNA at 2p25.1, a region found to be associated with transcription factor binding sites. This RNA-seq study [The study described in   is a transcription factor that regulates the expression of pro-inflammatory cytokines and type I IFNs and is believed to be involved in the pathogenesis of SLE. Genetic variants of the onocytes . It has lleagues  used RNAIRF5 transcript variants from purified monocytes of three healthy donors and six SLE patients. RNA-seq was subsequently used in order to obtain a more accurate and in-depth estimate of the differences between IRF5 transcript expression in primary immune cells of healthy donors and SLE patients. The most important finding of this study is that RNA-seq results  correlated with cloning and gave similar abundance rankings in SLE patients. This indicates the power of RNA-seq to identify and quantify spliced transcripts of a single gene at a greater depth compared with molecular cloning. Moreover, the authors of the study provide evidence that SLE patients express a different IRF5 transcript signature from healthy donors and that the IRF5-SLE risk haplotype is among the top four most abundant IRF5 transcripts expressed in SLE patients. Finally, this study suggests that RNA-seq of mammalian transcriptomes can provide a wealth of information on transcript assembly and abundance estimates and, because of its unbiased nature, it can be useful for de novo junction discovery.Using standard molecular cloning techniques, the authors first identified and isolated 14 new differentially spliced High-throughput NGS has marked the new age of biomedical research, since it offers the ability to sequence entire genomes or transcriptomes within days and to mine for previously unknown sequences in an unbiased manner. NGS methods have already been used to study a variety of biological systems and have been valuable tools in identifying markers for activity and progression in a variety of diseases. The advantages of RNA-seq in particular allow us to illustrate and study the complexity of transcriptomes more comprehensively.IRF5 transcript signature of SLE patients that is distinct from healthy donors and an IRF5-SLE risk haplotype in the top four most abundant IRF5 transcripts expressed in SLE patients and not in healthy donors. Although these studies involved only a small number of patient samples, they all show the potential of RNA-seq as a tool to assess and study different rheumatic diseases.In this review, we present studies based on the RNA-seq transcriptome analysis of patients with RA, SLE and SS that aim to shed light on the mechanisms of these rheumatic diseases. Importantly, we show the variety of RNA-seq applications and their flexibility to provide both quantitative and qualitative characterization of the transcriptomes under study. Gene expression profiling of RA patients was used to study the role of SFs in the pathogenesis of the disease, and gene signatures of the monocyte transcriptome in SLE patients showed globally dysregulated gene expression. Novel lncRNAs were identified from PBMCs of patients with JIA, as well as from SS patients, but further studies are required to elucidate whether these have functional consequences in these diseases. The discovery of novel miRNAs and disease biomarkers from minor salivary glands of patients with SS was also feasible with RNA-seq. In-depth single gene profiling was achieved by RNA-seq, revealing an As already discussed, the advantages and applications of RNA-seq are multifold. To our understanding, the main challenges of RNA-seq originate from the large amounts of data generated and involve the computational complexities associated with data analysis. As RNA-seq is becoming more affordable for research labs, the only daunting challenge is to select the most appropriate programs and tools for a specific RNA-seq application and to be able to understand and control the algorithmic parameters. Aside from these informatics challenges, which are steadily being overcome as more user-friendly and fast programs become available, RNA-seq is a particularly advantageous technology that embraces the complexity of the transcriptome and provides a mechanism to understand the underlying regulatory code.New technologies’. Other articles in this series can be found at [This article is part of the series ‘found at .


RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally.Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1 minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5 minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequence features.By improving 4-thiouracil labeling of RNA in Our analysis of non-coding RNAs reveals a highly significant association between non-coding RNA stability, transcript length and predicted secondary structure. Our quantitative analysis of the kinetics of pre-mRNA splicing in yeast reveals that ribosomal protein transcripts are more efficiently spliced if they contain intron secondary structures that are predicted to be less stable. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing.The online version of this article (doi:10.1186/s13059-015-0848-1) contains supplementary material, which is available to authorized users. The RNA levels detected in cells at steady state are the consequence of multiple dynamic processes within the cell. Eukaryotic genomes are pervasively transcribed, but the accumulation of many transcripts is limited by processes that regulate their synthesis and decay . In addiIn the case of intron-containing genes, the level of mature transcripts is influenced by splicing, as well as by synthesis and decay. Splicing of pre-mRNAs (precursors of messenger RNAs)  occurs iA diverse range of methods has been used to measure the kinetics of RNA processing. One approach involves inhibiting RNA polymerase II (Pol II) activity with a transcription inhibitor such as 5,6-dichloro-1-β-D-ribobenzimidazole, then removing this inhibition to promote synchronized transcription. For example, measuring the delay after Pol II reaches the 3′ ends of introns before the spliced flanking exons can be detected provides an estimate of the time for splicing . SplicinMetabolic labeling is a way of proportionally enriching classes of RNA that are difficult to detect in wild-type cells at steady state. For example, 4-thiouridine (4sU) has been used to label newly synthesized transcripts in human cells  and buddLabeling with 4sU has been used to measure RNA synthesis, decay and splicing rates in human and yeast; however, the shortest labeling time was 3 min, by which time a substantial fraction of the newly transcribed RNA was already spliced or degraded , 15. TheSaccharomyces cerevisiae, we incubated cells with 4tU for very short periods, extracted the RNA, and treated it with a thio-reactive reagent to biotinylate the newly synthesized transcripts that contain thiol moieties . Substituting the observed values for α0 and α1, we obtained the 9.1 % upper bound as reported in the main text.Therefore, the proportion of background reads over the total was bounded by . All raw read count and feature data can be found in Additional file No ethical approval was required for this study.


Here, we review the current state (and gaps) of knowledge on N2 fixation associated with coral reef organisms and their ecosystems. By summarizing the existing literature, we show that benthic N2 fixation is an omnipresent process in tropical reef environments. Highest N2 fixation rates are detected in reef-associated cyanobacterial mats and sea grass meadows, clearly showing the significance of these functional groups, if present, to the input of new N in reef ecosystems. Nonetheless, key benthic organisms such as hard corals also importantly contribute to benthic N2 fixation in the reef. Given the usually high coral coverage of healthy reef systems, these results indicate that benthic symbiotic associations may be more important than previously thought. In fact, mutualisms between carbon (C) and N2 fixers have likely evolved that may enable reef communities to mitigate N limitation. We then explore the potential effects of the increasing human interferences on the process of benthic reef N2 fixation via changes in diazotrophic populations, enzymatic activities, or availability of benthic substrates favorable to these microorganisms. Current knowledge indicates positive effects of ocean acidification, warming, and deoxygenation and negative effects of increased ultraviolet radiation on the amount of N fixed in coral reefs. Eutrophication may either boost or suppress N2 fixation, depending on the nutrient becoming limiting. As N2 fixation appears to play a fundamental role in nutrient-limited reef ecosystems, these assumptions need to be expanded and confirmed by future research efforts addressing the knowledge gaps identified in this review.Tropical coral reefs are among the most productive and diverse ecosystems, despite being surrounded by ocean waters where nutrients are in short supply. Benthic dinitrogen (N In the decades to come, life in the ocean will be confronted with a series of environmental conditions that have no parallel in human history  from the burning of fossil fuels is dramatically changing the world's climate (IPCC The marine nitrogen (N) cycle is one of the most important of all biogeochemical cycles, as N is an essential building block in all life forms. The N cycle significantly influences the cycles of other elements and particularly the carbon (C) cycle Fig. , becauseate IPCC .2) fixation, the conversion of N2 to organic N) and N loss . These are particularly important because of the inability of most marine organisms to use elemental N2 . As a consequence, their balance determines the net biologically available N for the biosphere and therefore marine productivity (Arp −1) in the oceanic N2 fixation rate compared to N loss  found N2-fixing microorganisms throughout all marine environments, ranging from deep-sea vents to highly productive shelf areas  compared to the amount of N that is fixed annually by benthic N2 fixers (Gruber μm cell diameter) are likely an equally important source of new N in the open ocean  studies have recently shown that assimilation of both organic and inorganic N sources resulted in rapid incorporation of nitrogen into uric acid crystals, forming temporary N storage sites within the dinoflagellate endosymbionts   associations in coral reef ecosystems is not debated, and we have only begun to investigate the distribution and diversity of diazotrophic populations associated with benthic reef organisms and substrates. Furthermore, the role of diazotrophs in contributing to primary production and growth and their potential susceptibility to climate change still needs to be resolved. As mutualisms  bind different species to a common fate, their breakdown as a result of climate change may enhance biodiversity loss and ecosystem disruption  produced by burning of fossil fuels and deforestation  are rare in warm tropical oceans. It has been suggested that the reduced gas solubility and increased respiration rates in warmer waters make the possession of heterocysts under such conditions disadvantageous  are widespread in benthic environments, so that increases in N2 fixation are likely to appear here as well. Nevertheless, heterocystous cyanobacteria form mats in warm coral reef environments   resulting from global climate change have the potential to increase the amount of N fixed globally  are lost  and seawater pH  resulting in bleaching and productivity loss  remains uncertain  consumption and protecting the enzyme nitrogenase from inactivation  significantly increased since the 1920s, most likely due to the increased input of river-borne nutrients   and dissolved organic matter (DOM) as well as trace metals , are mostly limiting N2 fixation and uptake of organic or inorganic N compounds), their ability to fix N2 is advantageous over non-N2-fixing phytoplankton species, especially under N-limiting conditions  can significantly alter the competition within the phytoplankton community, thereby further threatening the stability and functioning of the whole ecosystem  often fix Nd Capone ). Nevertd Capone , additiop et al.  suggestevel Fig. . Therefo2 content in the World's oceans  in the early afternoon  levels dropped to hypoxic conditions as a consequence of coral spawning events , the microbial community tends to be dominated by SRBs, particularly when nitrate is exhausted  reaching the biosphere  on corals and other reef-associated biota  and UVB (290–320 nm), can alter photosynthesis and growth in cyanobacteria  to investigate the potential effects of global and local environmental stressors on the metabolic activity and stability of these partnerships; and (4) to scale the effects of environmental stressors on the input of fixed N to the entire reef ecosystem. Moreover, careful site selection should be considered, where we can utilize natural habitats which currently undergo similar climate-driven stressor(s) expected for the future, for example, CO2 seeps.This article highlights the paramount role of N2 fixation rates as well as ecological and biogeochemical approaches are necessary for understanding the functioning of such a complex ecosystem.Given the difficulty in isolation and cultivation of microorganisms, including symbiotically associated types, several culture-independent approaches Fig.  are usef


Accurate and precise quantification of aortic regurgitation (AVR) severity by cardiovascular magnetic resonance is essential for the clinical decission-making and timing of surgery. The regurgitant flow volume (RV) can be measured directly by 2D phase contrast (PC) velocity measurements. The velocity encoding parameter, venc, has been identified by others as an important factor for accurate and precise determination of RV. For large vessel and high signal to noise measurements, though, integration of the measured blood flow velocities over the vessel lumen and over the cardiac phases for calculation of RV should average out variations in the measured velocities and enable high precision estimates of RV independently of venc. Furthermore, application of a correction method that effectively reduces the background velocity offset in the PC image to a sufficiently low value should enable accurate estimation of RV and remove the venc dependency. The aim of the study was to demonstrate venc insensitivity in the estimated RV with effectively background offset corrected PC velocity measurements.Measurements with high and low venc  were performed at the sinotubular junction on patients  and volunteers  using a 1.5 T scanner. Corrections for background offsets were automatically performed by the scanner and post-acquisition by means of adaptive image filtering. The mean background offset, standard deviation (sd) and coefficient of variation (cv) of repeated measurements were determined for the whole cohort. The mean RV, and sd and cv of RV were determined for patients and volunteers separately. For comparison, Wilcoxon signed-rank test was performed at a significance level of p<0.05.A background offset was present in all measurements. After correction, the offset was small Fig  and did We have demonstrated venc insensitivity in both the accuracy and precision of RV using background offset corrected PC velocity measurements. Without the need for adjustment of venc to the blood flow velocity, the time for the examination will be substantially reduced.This study was funded by a project grant from the Health & Medical Care Committee of the Regional Executive Board, Västra Götaland Region, Sweden.


Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant  Early development is controlled by maternally deposited mRNAs and the RNA-binding proteins (RBPs) that regulate them. Here the authors describe the identification of a large number of RBPs bound to polyadenylated RNAs in Drosophila embryos before and after the maternal-to-zygotic transition, revealing changes in RBPs activity during development. Drosophila, the zygote's DNA remains mostly transcriptionally silent for hours after pronuclear fusion, and early development is controlled by maternal messenger RNAs (mRNAs) and the RNA-binding proteins (RBPs) that regulate them. RBPs constitute a heterogeneous class of proteins that control all aspects of an mRNA's lifecycle. A dynamic set of RBPs associates with and accompanies nascent transcripts throughout maturation, localization, translation and decay. Previous attempts to create a census of RBPs of a cell relied on homology searches and in vitro approaches. While computational analyses contributed to annotate the compendium of RBPs harbouring classical RNA-binding domains, their capacity to discover unorthodox RBPs was limitedIn vitro protein–RNA screens reported RNA binding not only of canonical RBPs, but also of proteins previously unrelated to RNA biology5in vitro conditions calls for validation of these unconventional RBPs in a physiological environment.The maternal-to-zygotic transition (MZT) occurs in animal embryos during the first stages of development. In this process maternally deposited molecules are degraded and gradually replaced by products of the zygotic genome12C. elegansRecently, a proteome-wide method for RBP identification was developed and applied successfully to different cell lines7810Drosophila embryo. Beyond providing a comprehensive compendium of Drosophila melanogaster RBPs, we have applied this method to embryos at two different developmental stages employing a multiplexed proteomic approach, generating, to our knowledge, the first dynamic RNA interactome until now. Comparison of samples prepared from early and late embryos reveals that the RNA interactome undergoes important changes during development. This plasticity of the RNA-bound proteome can be explained either by changes in overall protein abundance or modulation of the RNA-binding activity of RBPs. Our analysis provides new insight into the molecular features of early development by highlighting proteins that may link development to RNA metabolism.We have further developed RNA interactome capture to enable identification of the RBPs functioning in a living multicellular organism, the Drosophila embryo we further developed the RNA interactome capture method established by Castello et al.8in vivo cross-linking of protein–RNA complexes and subsequent capture on oligo(dT) magnetic beads. Irradiation with 254 nm ultraviolet light activates the nucleotide bases that then rapidly form stable covalent bonds with some amino acid residues, preferentially nucleophilic and aromatic, placed at ‘zero distance'in vivo available to us. Capture of poly(A)+ RNA by hybridization with oligo(dT) probes is compatible with the use of high salt concentrations and ionic detergents for efficient removal of non-covalently bound proteins. The resulting samples are compatible with tandem mass tag (TMT) labelling for multiplexed quantitative proteomics using Orbitrap MS combined with rigorous statistical methods to catalogue proteins as high-confidence RBPs -bound fractions recovered from ultraviolet-irradiated, cross-linked (CL) embryos contained a complex protein mixture distinct from the total lysate, whereas the non-cross-linked (noCL) controls were essentially devoid of proteins . SpecifiDrosophila, we applied RNA interactome capture to early, pre-MZT (0–1 h post-fertilization) and late (4.5–5.5 h), post-MZT embryos  term enrichment analysis of the RNA interactome, as compared to the total lysate, revealed enrichment of biological processes in which RNA has a key role, such as translation and RNP structure organization . We counDrosophila embryoset al. (476) is comparable to the number we identified and also includes many proteins not previously shown to bind RNA (99).A parallel study reports the RNA interactome of early bona fide RBPs. Compared to the total embryo lysate, our dataset is significantly enriched in canonical RBDs such as RNA recognition motif (RRM), ribosomal and K-homology (KH) domains  previously known and 37 (of 236) newly discovered RBPs contain repetitive motifs in intrinsically disordered regions, which are protein segments lacking a stable three-dimensional structure, typically of low amino acid complexity  of proteins listed as enzymes ( http://enzyme.expasy.org/). Whereas in yeast all steps of the glycolytic pathway can be catalyzed by enzymes identified as RNA binders, some of them binding mRNAs encoding glycolytic enzymes10Drosophila only two glycolytic enzymes—phosphofructokinase and phosphoglyceromutase—are found in the RNA interactome , and the protein–RNA complexes were visualized by immunoblotting.We employed flies expressing transgenic EB1-GFP, endogenously tagged GFP-CycB or free GFP. After ultraviolet crosslinking of live embryos, each candidate protein was immunoprecipitated and purified under stringent conditions using the anti-GFP nanobodybona fide RBPs. Immunoblotting confirmed the efficient immunoprecipitation of both GFP-CycB and EB1-GFP, as well as smaller products, presumably proteolytic fragments . Enrichment rates of alternatively spliced mRNAs among those coding for RBPs and RBP candidates were similar in the three aforementioned dynamic classes  than in the exons used at the late stage   . When co.23e−02) .D. melanogaster at different developmental stages obtained in an unbiased proteome-wide experiment. Selection of the RNA-bound protein fraction was achieved through cross-linking of RBPs to RNA by ultraviolet light and specific precipitation of poly(A)+ RNA on oligo(dT) magnetic beads under stringent conditions. We improved the RNA capture procedure, that was initially optimized for cell linesThis resource presents the first, to our knowledge, comprehensive list of RBPs in Drosophila, despite the fact that enrichment of some proteins could not be quantified due to missing intensity values in the control noCL sample. Although this technical issue made data analysis challenging, it signified that our efforts in reducing protein background resulted in generation of extremely high quality RBP specimens, where background signal in the negative control was close to zero for most of the proteins quantified in the ultraviolet-irradiated samples. Our data show that TMT MS3 allows efficient reduction of experimental noise, but challenge the current data analysis pipelines where the existence of an intensity ratio between the two conditions is required to quantify a proteinAnalysis of captured samples by the recently developed TMT MS3 methodDrosophila RBPs so far by applying the optimized RNA interactome capture method to embryos at two distinct developmental stages—before and after the MZT. Our analysis confirms RNA-binding ability of hundreds of previously known RBPs and provides new links between RNA metabolism and development, including new evidence for RBP involvement in regulation of cell differentiationWe generated the most comprehensive list of high-confidence Drosophila interactome with data available for four other species reveals that proteins that are conserved and appear in all interactomes are predominantly previously known RBPs involved in core processes of RNA metabolism, such as translation and processing, whereas species-specific proteins are enriched for novel RBPs. This also highlights the utility of an experimental technique such as interactome capture for discovery of novel, species-specific RBPs.Our comparison of the Drosophila810Drosophila is particularly interesting. It was previously shown that 37 mRNAs encoding metabolic enzymes are bound and possibly downregulated by a key MZT driver, SmaugDrosophila than in other organisms for which RNA interactomes are available. Why this might be, as well as the roles of metabolic enzymes in RNA metabolism and regulation, should be clarified by future studies.In light of evidence indicating that regulation of metabolism might be tightly bound to the MZT in We confirmed the ability of two newly discovered RBPs, CycB and EB1, to bind RNA directly by a reciprocal approach that involves precipitation of an individual protein and detection of the bound RNA by radioactive labelling. In addition, based on the labelling patterns of presumable degradation products of EB1-GFP, we propose that its APC binding domain harbours RNA-binding activity, not excluding, however, that the N-terminal microtubule-binding domain of EB1 may also contribute to RNA binding. In contrast, only the full-length version of GFP-CycB was radioactively labelled, suggesting that either the full-length or the C-terminal fragment of CycB mediates RNA binding.We propose that RNA binding might have a role in CycB regulation or targeting: for example, localized RNAs might tether CycB to the loci where its activity is required, such as centrosomesDrosophila embryos provided in this study reveals, expectedly, high dynamicity of the RNA-bound fraction during the MZT and confirms deeper involvement of RBPs in the early, pre-MZT embryo that is transcriptionally inactive. Comparison of protein abundance changes in the RNA-bound fraction and in the total proteome allowed their separation into three dynamic classes, which indicates that various mechanisms might regulate RNA binding, including active regulation of an RBP's affinity for RNA and isoform exchange. Possible active regulation mechanisms include, but are not limited to allosteric regulation by trans-acting factors such as proteins, RNAs or small molecules, competitive binding of trans-acting factors at the RNA-binding site and post-translational modifications. Some RBPs might also appear to bind RNA more efficiently at one of the stages if the mRNAs that they bind have undergone a change in polyadenylation status. Observed dynamic behaviours of some of the known RBPs such as splicing factors indicate that they may have other functions in addition to previously characterized ones. The fact that these splicing factors preferentially bind RNA in the early, pre-MZT embryo is surprising for two reasons: first, transcription, and accordingly, pre-mRNA processing is generally low in the rapidly dividing nuclei of embryos at the early, syncytial stageAnalysis of the RNA interactomes of pre- and post-MZT By analyzing protein features encoded by exons that are differentially expressed during the MZT we determined that alternative splicing indeed has the potential to affect the architecture of an RBP and therefore can impact on the biochemical properties of the protein. The fact that inclusion of some RBDs occurs preferentially in the early, transcriptionally quiescent stage aligns well with the special importance of RBPs in the post-transcriptional control of maternal mRNAs.et al.Differential exon expression analysis also revealed that disordered regions are alternatively spliced at both stages, in accordance with previous reports844D. melanogaster Oregon-R flies were used to obtain embryos for RNA interactome capture (Bloomington Stock 5). RNA-binding activity of CycB and EB1 was validated using the endogenously tagged w1118; cycB FlyTrap line , and flp16 ref. , respect−1 Li-dodecylsulphate, 12.5 mM dithiothreitol—DTT). The other half was irradiated with 1.0 J cm−2 254 nm ultraviolet light  on ice under a layer of ice-cold phosphate buffered saline supplemented with 0.1% Tween-20, collected on a sieve, then homogenized in lysis buffer. After lysis embryo extracts were cleared by centrifugation at 15,000g for 30 min, diluted to total protein concentration 0.1 mg ml−1, incubated at 60 °C for 15′ and cooled to room temperature.Zero-to-one hour-old embryos collected from ten population cages each containing ∼ 10,000 flies were either processed immediately  or incubated for 4.5 h and then processed . After collection on a 0.18 mm mesh sieve, embryos were rinsed with distilled water, weighed and the sample was split into two. One half  was homogenized in lysis buffer .Poly(A)+ RNA was captured according to the protocol of ref. To obtain optimal yields of poly(A)+ RNA, each lysate was put through the RNA interactome capture procedure four times. Eluates from subsequent rounds of RNA interactome capture were pooled and concentrated using Amicon filter units with a molecular weight cut-off of 3,000 Da.−1 4′,6-diamidino-2-phenylindole (DAPI) and visualized on a confocal fluorescent microscope.An aliquot of embryos used for RNA interactome capture was dechorionated by bleach treatment (30 s incubation in 5% NaClO), rinsed with distilled water and fixed in a 1:1 mixture of heptane and methanol. After gradual rehydration, embryos were stained with 0.1 μg mlTo control the quality of the captured RNA, an aliquot of each eluate was treated with protease K and subsequently purified as in ref. gapdh1 and ts mRNAs, we performed reverse transcription followed by RT-qPCR. RT was performed using the SuperScript III first-strand synthesis system (Thermo Fischer Scientific), equal amounts of total and oligo(dT) captured RNA were used. Dilutions of first-strand DNA libraries prepared from total noCL RNA were used as standards. Amplification was performed in the presence of 1 mM of each oligonucleotide and 1 × SYBR Green RCR Master Mix (Applied Biosystems). Relative amounts of targets were calculated using StepOne software. Sequences of oligonucleotides used for quantification of 18S rRNA were taken from ref. gapdh1 and ts mRNAs were designed with Primer3 software51For quantification of 18S rRNA and −1 of RNases A and T1. After 30 min incubation at 37 °C an aliquot of proteins was supplemented with LiDS loading buffer (Thermo Fischer Scientific) containing 20 mM DTT, separated on 4–12% polyacrylamide gels  and either stained with silver using Pierce Silver Stain Kit (Thermo Fischer Scientific) or electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes.Eluates were supplemented with RNase buffer to a final concentration 20 mM Tris–HCl pH 7.5, 150 mM NaCl and 200 U ml−1 milk powder. Membranes were then hybridized with antibodies diluted in PBST-milk that are listed in PVDF membranes were blocked in phosphate buffered saline supplemented with 0.1% Tween-20 (PBST) and containing 100 g LCaptured proteins were digested with Lys-C and labelled with 6plex TMT as previously describedw1118 embryos were collected and irradiated with 1 J cm−2 ultraviolet light (254 nm) and lysed in 10 × RIPA buffer  and cleared by 10 min centrifugation at 15,000g. Lysates containing GFP were diluted with w1118 lysates to reduce GFP concentration to levels comparable with those of GFP-CycB and EB1-GFP. Lysates were diluted with RIPA buffer to 10 mg ml−1 total protein, then diluted 1:10 with low salt buffer  to reduce the concentration of detergents. A total of 10 μl of suspension of magnetic beads coated with Lama alpaca anti-GFP antibody (Chromotek GFP-Trap_M) were added to 1 ml of each lysate and incubated at 4 °C for 2 h. Beads were washed six times with 1 ml high salt buffer . Washes were performed at room temperature to avoid SDS precipitation. Beads were then washed twice with 1 × T4 PNK buffer B (Thermo Fischer Scientific) and incubated for 15 min in 10 ul of reaction mix containing 1 × T4 PNK buffer B, 2 μl T4 PNK and 1 mCi γ-32P-adenosyntriphosphate (γ-ATP) . Beads were then separated from the reaction mix and washed 5 times with low salt buffer supplemented with 0.1% Triton-X100. Labelled complexes of GFP proteins and RNA were eluted by incubation in 1 × LiDS buffer (Thermo Fischer Scientific), separated on polyacrylamide gels and transferred to PVDF membranes as described above. Autoradiography was recorded using phosphor screens and GE Typhoon 7000 scanner, and GFP proteins were detected as described above.Zero-to-two hour-old embryos expressing GFP, GFP-CycB or EB1-GFP and Drosophila reference genome version R6.07 using STAR. Differential exon usage was called using the R/Bioconductor package DEXSeq.To determine the total transcriptome, poly(A)+ RNA from 0–1 h and 4.5–5.5 h-old embryos was purified as described above, treated by Turbo DNase I (Thermo Fischer Scientific) according to manufacturer's instructions and purified by phenol–chloroform extraction and ethanol precipitation. cDNA libraries were prepared form the resulting samples using a SENSE mRNA-Seq Library Prep Kit V2 (Lexogen) and analyzed by single-end 50 sequencing on an Illumina HiSeq2000. Reads were aligned to the The MS proteomics data that support the findings of this study have been deposited in the ProteomeXchange Consortium via the PRIDE partner repositoryHow to cite this article: Sysoev, V. O. et al. Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Nat. Commun. 7:12128 doi: 10.1038/ncomms12128 (2016).Supplementary Figures 1-4, Supplementary Tables 1-2, Supplementary Notes 1-3 and Supplementary ReferencesThe RNA interactome of the Drosophila embryoComparison of the Drosophila RNA interactome and RNA interactomes of other speciesComparison of the Drosophila RNA interactome and RNA interactomes of other species with regard to enzymesComparison of RNA interactomes and total proteomes of early Drosophila embryos before and after the maternal-to-zygotic transition.


We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.How the essential pre-mRNA splicing factor U2AF The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations.The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts65 is essential for viability in vertebrates and other model organisms  and ultimately dissociates from the active spliceosome.The early-stage pre-mRNA splicing factor U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract1265 RRM1 and RRM2 connected by a shortened inter-RRM linker  detailed a subset of nucleotide interactions with the individual U2AF65 RRMs1465 RRM1 and RRM2 connected by a linker of natural length , yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition.Biochemical characterizations of U2AF651,2 domain comprising the core RRM1–RRM2 folds  is relatively weak compared with full-length U2AF65  nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures  are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures.We tested the contribution of the U2AFaffinity . Mutagen65 RRM1 and RRM2 directly contact the bound Py tract. Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site , mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold  bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated  with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker . Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein . We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L , which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229  for RNA binding  comprising a weak  and strong  polypyrimidine tracts preceding two alternative splice sites65, the PY splice site is used and the remaining transcript remains unspliced. When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documentedPY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site . Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65  measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations25651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution.The direct interface between U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2  residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances  that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å)65 conformations that lack inter-RRM contacts.The inter-RRM dynamics of U2AFand RRM2 , Methods651,2 was modified with equimolar amount of Cy3 and Cy5. Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. Hence, molecules that were conjugated to two donor or two acceptor fluorophores were excluded from analysis.Double-cysteine variant of U2AF65 in the absence of RNA  protein was tethered to a slide via biotin-NTA/Ni+2 resin. Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4  to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347)65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA.We first characterized the conformational dynamics spectrum of U2AFe of RNA . The dou of ∼0.4 . Approxi of ∼0.4 . Despite of ∼0.4 . Approxi of ∼0.4 . We cann65 with the AdML Py-tract prototype. Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state  to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin .We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AFET state . To asseTA resin . A 0.45 651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts65 is bound to interrupted or continuous Py tract.We examined the effect on U2AF651,2L bound to Py-tract oligonucleotides. It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations651,2L.The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value  bound to the slide-tethered RNA showed fluctuations between distinct FRET values. The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value  of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (65 conformation) is complemented by ‘induced fit' , as discussed below.Importantly, the majority of traces (∼70%) of U2AFET value . Hidden T traces  suggests65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. Notably, a triple mutation of three residues  in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the The U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit —possibly due to the selective versus nearly universal requirements of these factors for splicing. A confirmed somatic mutation of U2AF65 in patients with MDS, L187V651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se.Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS)65–RNA association  experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces  bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value  indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association.The finding that U2AF651,2L (residues 141–342 of NCBI RefSeq NP_009210) was expressed in Escherichia coli strain BL21 Rosetta-2 as a GST-fusion protein in the vector pGEX6P-2 and purified by glutathione affinity, followed by anion exchange and gel filtration chromatography. The GST-tagged protein was bound to a GSTrap column  in 1 M NaCl, 25 mM HEPES, pH 7.4 and eluted using 150 mM NaCl, 100 mM Tris, pH 8 containing 10 mM glutathione. The GST tag was cleaved from the protein by treatment with PreScission Protease during dialysis against a buffer containing 100 mM NaCl, 25 mM HEPES, pH 8, 5% (v/v) glycerol, 5 mM DTT, 0.25 mM EDTA and 0.1 mM PMSF. Cleaved GST was separated from the U2AF651,2L by subtractive glutathione affinity chromatography in 100 mM NaCl, 25 mM Tris, pH 8, 0.2 mM TCEP followed by subtractive anion-exchange chromatography with a HiTrap Q column . The final purification step was size-exclusion chromatography on a Superdex-75 prep-grade column  that had been previously equilibrated with 100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM tris(2-carboxy-ethyl)phosphine (TCEP). The purified U2AF651,2L was concentrated using a Vivaspin 15 R (Sartorius) centrifugal concentrator with 10 kDa MWCO, and the protein concentration was estimated using the calculated extinction coefficient of 8,940 M−1cm−1 and absorbance at 280 nm. Shorter constructs    and indi65 (residues 1–475) and the U2AF35-UHM  initially were expressed and purified separately as GST fusion proteins. Following GST cleavage and ion-exchange chromatography , U2AF65 was combined with slight excess U2AF35-UHM (in stoichiometric ratio of 1:1.2) and dialysed overnight. The final U2AF heterodimer was purified by size-exclusion chromatography using a Superdex-200 prep-grade column  pre-equilibrated with 150 mM NaCl, 25 mM HEPES, pH 6.8, 0.2 mM TCEP. Representative purified U2AF651,2L and U2AF65–U2AF35-UHM proteins are shown in For comparative RNA-binding experiments, full-length human U2AFHigh-performance liquid chromatography-purified oligonucleotides (sequences shown in −1 Superase-In (Ambion Life Technologies)). The final RNA concentration in the cuvette was 30 nM. Volume changes during addition of the protein were <10% to minimize dilution effects. The fluorescence anisotropy changes during titration were measured using a FluoroMax-3 spectrophotometer temperature controlled by a circulating water bath at 23 °C. Samples were excited at 490 nm and emission intensities recorded at 520 nm with a slit width of 5 nm. The titrations were repeated three times in succession. Each titration was fit with Graphpad Prism v4.0 to obtain the apparent equilibrium dissociation constant (KD)KA) are the reciprocal of the KD. The average KD's or KA's and s.e.m. among the three replicates were calculated using Excel and are reported in P values from a two-tailed unpaired t-test were calculated using Graphpad Prism v4.0.For RNA-binding experiments, purified proteins and RNA were diluted separately >100-fold in binding buffer  was used (WT U2AF65) and in parallel mutated to encode the Q147A/R227A/V254P triple-mutant protein (Mut U2AF65). The pyPY minigene was a gift from M. Carmo-Fonseca 5 cells per well) and grown as monolayers in MEM (Gibco Life Technologies) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin. After 1 day, the cells were transiently transfected with either 0.5 μg of pyPY plasmid or a mixture of 0.5 μg of U2AF65 variant and 0.5 μg of pyPY plasmid per well using appropriately adjusted Lipofectamine 2000 (Invitrogen Life Technologies) ratio according to the manufacturer's instructions.For transfection experiments, the full-length human U2AF65 and Mut U2AF65 expression levels  and detected using SuperSignal WestPico chemi-luminescent substrate (Pierce Thermo Scientific Inc.). Blots were imaged using a IS4000MM system . For size analysis, fluorescent images of the BioRad Precision Plus Dual Color Standards were overlaid directly.For immunoblots of WT U2AFn levels , transfe and reverse (5′-ATGGCATGGACTGTGGTCATGAGT-3′) primers for a GAPDH control. The RT-PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The percentages of splice site use were calculated from the background corrected intensities I using the formula 100% × I(py)/[I(py)+I(PY)+I(unspliced)] for py spliced (I(PY)/[I(py)+I(PY)+I(unspliced)] for PY spliced (For reverse transcription PCR (RT-PCR), the total RNA was isolated 2 days post transfection using the Cells-to-cDNA II kit (Ambion Life Technologies). The RT-PCR reaction comprised 35 cycles (94 °C per 60 s—60 °C per 50 s—72 °C per 60 s) with forward (5′-TGAGGGGAGGTGAATGAGGAG-3′) and reverse (5′-TCCACTGGAAAGACCGCGAAG-3′) primers for the  spliced  or 100%  spliced . The ban651,2L and given oligonucleotide were mixed to achieve respective final concentrations of 1.0 and 1.1 mM and incubated on ice for 20–30 min. For each oligonucleotide, sparse matrix screens of the Jancarik and Kim Crystal ScreenBefore crystallization, the purified U2AF651,2-binding sites for seven nucleotides, the new terminal residues of the U2AF651,2L construct were presumed to contact an additional nucleotide and the crystal packing of a central nucleotide between the RRM1/RRM2 of dU2AF651,2 was presumed to represent one nucleotide. Also considering the known proclivity for deoxy(d)U to co-crystallize with dU2AF651,2  . For comparison, searches with the NMR structure (PDB ID 2YH1) as a search model failed to find a solution. The initial structure revealed a greater number of central nucleotide-binding sites than expected. The oligonucleotide binding register had slipped to place the BrdU in the preferred site, leave the 5′ terminal-binding sites empty, and the terminal nucleotide unbound and disordered. Subsequent oligonucleotides were designed to place BrdU in the preferred site, fill the unoccupied 5′ terminal sites, capture rU at the central sites, and compare rC at the terminal site.The oligonucleotide sequences were optimized and the structures were determined as follows: in addition to the previously characterized dU2AF1,2 ref.  and for 1,2 ref. , we init651,2L protein co-crystals with oligonucleotide 5′-phosphorylated (P)-rUrUdUdUrUdU(BrdU)dU were obtained using a reservoir of 200 mM LiCl, 100 mM sodium citrate pH 4.0, 8% (w/v) polyethylene glycol (PEG) 6,000, 10% (v/v) PEG 300, 10% (v/v) dioxane with 0.1 μl of N,N-bis[3-(D-gluconamido)propyl]deoxy-cholamide (deoxy-BigCHAP) (14 mM) added to the hanging drop and cryoprotected by sequential layering with reservoir solution supplemented with increasing PEG 300 to a final concentration of 26%. Co-crystals with either 5′-(P)rUrUdUrUrU(BrdU)dUdU or 5′-(P)rUrUrUdUrUrU(BrdU)dUrC were obtained from 1 M succinate, 100 mM HEPES, pH 7.0, 1–3% (w/v) PEG monomethylether 2,000. The former was cryoprotected by coating with a 1:1 (v/v) mixture of silicon oil and Paratone-N and the latter by sequential transfer to 21% (v/v) glycerol. Data sets for flash-cooled crystals were collected at 100 K using remote access to SSRL BL12-2. Structures were determined by molecular replacement using the initial U2AF651,2L/rUrUrUdUdU(BrdU)dUrUrU structure as a search model. Consistent sets of free-R reflections were maintained . Models were built using COOTThe U2AF651,2LFRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF65 residues 113–343. The single cysteine of human U2AF65 was replaced by alanine (C305A), which is a natural amino-acid variation among U2AF65 homologues. Single A181C and Q324C mutations were introduced in each RRM for fluorophore attachment at residues that were carefully selected to meet experimental criteria described in the Results. The U2AF651,2LFRET was purified by the same method as described above for U2AF651,2L protein and binds RNA with similar affinity as U2AF651,2L phosphine (TCEP)) using Zeba Spin Desalting Columns 7K MWCO . To initiate the labelling reaction, 4 μl each of cyanine (Cy)3-Maleimide and Cy5-Maleimide  stock solutions (10 mM in DMSO) were pre-mixed  and then added to 200 μl of 20 μM protein . The labelling reaction was incubated at room temperature in the dark for 2 h and then quenched by the addition of 10 mM DTT. The labelled protein was separated from excess dye using a Zeba Spin Desalting Column followed by size exclusion chromatography using a pre-packed Superdex-75 10/300 GL  column in Labelling Buffer. Our previous experience of conjugating cysteines with maleimide derivatives of fluorophores and suggests that nonspecific modification of aminogroups of proteins with fluorescent dyes under the employed experimental conditions is negligible. Consistent with specific labelling of A181C and Q324C, the labelling efficiencies were ∼60% each for Cy3 and Cy5 as estimated using the dye extinction coefficients  and the calculated extinction coefficient of the U2AF651,2LFRET protein , and correcting for the absorbance (A) of the dyes at 280 nm :The U2AFAF651,2L . Before 651,2L affinity for a purine-interrupted Py tract comprising an rUrUrUrUrU tract that is expected to bind U2AF65 RRM2/inter-RRM linker, a central rArA and an rUrUrUrCrC tract that is expected to bind RRM1. The tandem purines represent a compromise between significant inhibition of U2AF65 binding by longer A interruptionsFor smFRET experiments with a ‘strong', homogeneous Py tract, we used the prototypical AdML sequence (5′-CCUUUUUUUUCC-3′). To investigate the inter-RRM separation in the presence of a ‘weak' Py tract interrupted by purines, we compared the U2AFFor the reversed immobilization of RNA via a complementary biotinyl-DNA primer experiment, the AdML Py-tract RNA was extended to include the DNA counterpart of downstream AdML intron/exon sequences that were complementary to the biotinyl-DNA primer. To increase separation from the slide surface, a hexaethylene glycol linker (18PEG) was inserted between the AdML Py-tract RNA and the tethered DNA duplex. The tethered oligonucleotide sequences included: 5′-rCrCrUrUrUrUrUrUrUrUrCrC/18PEG/dAdCdAdGdCdTdCdGdCdG-dGdTdTdGdAdGdGdAdCdAdA-3′ annealed to 5′-biotinyl-dTdTdGdTdCdCdTdCdAdA-dCdCdGdCdGdAdGdCdTdGdT-3' (purchased with high-performance liquid chromatography purification from Integrated DNA Technologies).−1 glucose oxidase, 0.625% glucose, 0.02 mg ml−1 catalase), 1.5 mM Trolox (used to eliminate Cy5 blinking) and 6 mM β-mercaptoethanol. The sample chamber was assembled from quartz microscope slides and glass cover slips coated with a mixture of m-PEG and biotin-PEG and pre-treated with neutravidin (0.2 mg ml−1). Surface tethering of doubly labelled U2AF651,2LFRET(Cy3/Cy5) via its His-tag (4) for 20 min before addition of 5 nM U2AF651,2LFRET(Cy3/Cy5). After 10 min, unbound sample was removed by washing the sample chamber with imaging buffer. The AdML RNA ligand was added to the imaging buffer at a concentration of 5 μM , whereas the rArA-interrupted RNA was added at a concentration of 10 μM. Alternatively, to detect binding of doubly labelled U2AF651,2LFRET(Cy3/Cy5) to surface-tethered RNA ligand  was then added to the imaging buffer.The smFRET measurements were carried out at room temperature in 50 mM HEPES, pH 7.4, 100 mM NaCl. The imaging buffer also contained an oxygen-scavenging system , with the exposure time set at 100 ms. We typically took up to five 5-minute-long movies while imaging different sections of the slide for each sample. Before each measurement, we checked for non-specific binding by adding doubly-labeled U2Fret to the slide in the absence of neutravidin and imaging the slide. Non-specific binding was virtually absent.Single-molecule FRET measurements were taken as previously described28Eapp) were calculated from the emission intensities of donor (ICy3) and acceptor (ICy5) as follows: Eapp=ICy5/(ICy5+ICy3). The FRET distribution histograms were built from traces that showed single-step photobleaching in both Cy3 and Cy5 signals using a Matlab script generously provided by Prof. Peter Cornish . Anti-correlated changes in donor and acceptor intensities with constant sum of intensities indicated the presence of an energy transfer in single molecules labelled with one donor and one acceptor dye. All histograms were smoothed with a five-point window and plotted using Origin software (Origin Lab Co). Idealization of FRET trajectories was done using the hidden Markov model algorithms via HaMMy software (http://bio.physics.illinois.edu/HaMMy.asp)x-axis) to ending FRET efficiency value (y-axis) was represented by a heat map. The range of FRET efficiencies from 0 to 1 was separated in 200 bins. The resulting heat map was normalized to the most populated bin in the plot; the lower- and upper-bound thresholds were set to 20% and 100% of the most populated bin, respectively.Collected data sets were processed with IDL and Matlab softwares, using scripts downloaded from a freely available source: physics.illinois.edu/cplc/software. Apparent FRET efficiencies  was separated into 100 bins. The FRET efficiency range  was separated into 100 bins. A heat map is used to represent the frequency of sampling of each FRET state over time; frequency in each bin was normalized to the most populated bin in the plot with lower- and upper-bound thresholds set at 10% and 80% of the most populated bin, respectively.The surface contour plots were generated as follows: the individual single-molecule FRET traces –(iv).How to cite this article: Agrawal, A. A. et al. An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal. Nat. Commun. 7:10950 doi: 10.1038/ncomms10950 (2016).Supplementary Figures 1-8, Supplementary Discussion and Supplementary References.Overall structure and central nucleotide interactions.