Apical Loop Research Articles

Dissecting the Conformational Heterogeneity of Stem-Loop Substructures of the Fifth Element in the 5'-Untranslated Region of SARS-CoV-2.

Throughout the family of coronaviruses, structured RNA elements within the 5' region of the genome are highly conserved. The fifth stem-loop element from SARS-CoV-2 (5_SL5) represents an example of an RNA structural element, repeatedly occurring in coronaviruses. It contains a conserved, repetitive fold within its substructures SL5a and SL5b. We herein report the detailed characterization of the structure and dynamics of elements SL5a and SL5b that are located immediately upstream of the SARS-CoV-2 ORF1a/b start codon. Exploiting the unique ability of solution NMR methods, we show that the structures of both apical loops are modulated by structural differences in the remote parts located in their stem regions. We further integrated our high-resolution models of SL5a/b into the context of full-length 5_SL5 structures by combining different structural biology methods. Finally, we evaluated the impact of the two most common VoC mutations within 5_SL5 with respect to individual base-pair stability.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.

The utility of USB microscope for assessment of Raynaud’s phenomenon patients in rheumatology practice

The aim of the study was to assess the utility of USB microscope (DinoLite) for evaluation of patients with primary and secondary Raynaud’s phenomenon (RP) in rheumatic diseases, , using magnification 200x.
 Methods: The study represents retrospective analysis of capillaroscopic images of 53 patients with RP – primary and secondary in the context of systemic sclerosis (SSc) or other rheumatic diseases i.e., undifferentiated connective tissue disease (UCTD), prescleroderma and systemic lupus erythematosus (SLE). Capillaroscopic images are obtained from 8 fingers (II÷V bilaterally) using USB microscope (DinoLite) at magnification 200x. Capillary diameters were measured (arterial, venous and apical loop) as well as the number of capillaries per millimeter and capillary length. The capillaroscopic images were categorized into the following groups i.e., 1. absence of microangiopathy: i) normal pattern, ii) nonspecific changes; 2. presence of microangiopathy i.e., “scleroderma/ scleroderma-like” pattern.
 Results: Images suitable for analysis with good visibility that permit analysis of the major capillaroscopic parameters were available in all patients (100%). Among 53 included patients, 14 patients were with SSc, 12 cases with primary RP, and 27 patients with secondary RP in other connective tissue diseases (22 patients with UCTD, 1 – with prescleroderma and 4 patients with SLE). „Scleroderma“ pattern was detected in 11 patients with SSc and in all these cases the capillaroscopic images were classifiable into one of the three distinct validated phases i.e., “early”, ”active” and ”late”. Presence of microvascular changes type “scleroderma-like” pattern was detected also in 17 among 27 patients with other connective tissue diseases. In primary RP patients, capillaroscopy revealed either normal pattern or nonspecific findings, but without features of microangiopathy.
 Conclusion: Capillaroscopic images of good quality, which could be analyzed and interpreted, are usually obtained using USB microscope. This permits evaluation of the major capillaroscopic parameters including quantitative measurement of the capillary diameters, capillary density and length. Assessment of the degree of microvascular changes i.e., staging of microangiopathy is also applicable and was possible in all cases with microangiopathy. The available software although less sophisticated vs those of videocapillaroscopes, providess the opportunity for adequate analysis of capillaroscopic parameters. The images obtained via USB microscope using magnification 200x are easily classified into “scleroderma”/“scleroderma-like” pattern, non-specific changes and normal findings using also the software of the device. Thus, USB microscopes using standard, 200x magnification could be suggested as an alternative of videocapillaroscopes in every day rheumatology practice especially in low-budget cases.

Structural atlas of human primary microRNAs generated by SHAPE-MaP

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

Substrate promiscuity of Dicer toward precursors of the let-7 family and their 3′-end modifications

The human let-7 miRNA family consists of thirteen members that play critical roles in many biological processes, including development timing and tumor suppression, and their levels are disrupted in several diseases. Dicer is the endoribonuclease responsible for processing the precursor miRNA (pre-miRNA) to yield the mature miRNA, and thereby plays a crucial role in controlling the cellular levels of let-7 miRNAs. It is well established that the sequence and structural features of pre-miRNA hairpins such as the 5′-phosphate, the apical loop, and the 2-nt 3′-overhang are important for the processing activity of Dicer. Exceptionally, nine precursors of the let-7 family (pre-let-7) contain a 1-nt 3′-overhang and get mono-uridylated in vivo, presumably to allow efficient processing by Dicer. Pre-let-7 are also oligo-uridylated in vivo to promote their degradation and likely prevent their efficient processing by Dicer. In this study, we systematically investigated the impact of sequence and structural features of all human let-7 pre-miRNAs, including their 3′-end modifications, on Dicer binding and processing. Through the combination of SHAPE structural probing, in vitro binding and kinetic studies using purified human Dicer, we show that despite structural discrepancies among pre-let-7 RNAs, Dicer exhibits remarkable promiscuity in binding and cleaving these substrates. Moreover, the 1- or 2-nt 3′-overhang, 3′-mono-uridylation, and 3′-oligo-uridylation of pre-let-7 substrates appear to have little effect on Dicer binding and cleavage rates. Thus, this study extends current knowledge regarding the broad substrate specificity of Dicer and provides novel insight regarding the effect of 3′-modifications on binding and cleavage by Dicer.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

AAACH is a conserved motif in a cis-acting replication element that is artificially inserted into Senecavirus A genome

Cis-acting replication element (cre) is required for generating a diuridylylated VPg that acts as a protein primer to initiate the synthesis of picornaviral genome or antigenome. The cre is a stem–loop structure, dependent of different picornaviruses, located in different genomic regions. The AAACA motif is highly conserved in the apical loop of cre among several picornaviral members, and plays a key role in synthesizing a diuridylylated VPg. We previously demonstrated that senecavirus A (SVA) also possesses an AAACA-containing cre in its genome. Its natural cre (Nc), if functionally inactivated through site-directed mutagenesis (SDM), would confer a lethal impact on virus recovery, whereas an artificial cre (Ac) is able to compensate for the Nc-caused functional inactivation, leading to successful rescue of a viable SVA. In this study, we constructed a set of SVA cDNA clones. Each of them contained one functionally inactivated Nc, and an extra SDM-modified Ac. Every cDNA clone had a unique SDM-modified Ac. The test of virus recovery showed that only two SVAs were rescued from their individual cDNA clones. They were AAACU- and AAACC-containing Ac genotypes. Both viruses were serially passaged in vitro for analyzing their viral characteristics. The results showed that both AAACU and AAACC genotypes were genetically stable during twenty passages, implying when the Nc was functionally inactivated, SVA could still use an AAACH-containing Ac to complete its own replication cycle.

An intronic RNA element modulates Factor VIII exon-16 splicing.

Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure-function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3'-end of intron-15 (TWJ-3-15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3-15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3-15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing.

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble.

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 μs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds.

High-resolution structure of stem-loop 4 from the 5'-UTR of SARS-CoV-2 solved by solution state NMR.

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonancespectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Boththe tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.

Structure of pre-miR-31 reveals an active role in Dicer–TRBP complex processing

As an essential posttranscriptional regulator of gene expression, microRNA (miRNA) levels must be strictly maintained. The biogenesis of many miRNAs is mediated by trans-acting protein partners through a variety of mechanisms, including remodeling of the RNA structure. miR-31 functions as an oncogene in numerous cancers, and interestingly, its biogenesis is not known to be regulated by protein-binding partners. Therefore, the intrinsic structural properties of the precursor element of miR-31 (pre-miR-31) can provide a mechanism by which its biogenesis is regulated. We determined the solution structure of pre-miR-31 to investigate the role of distinct structural elements in regulating processing by the Dicer-TRBP complex. We found that the presence or absence of mismatches within the helical stem does not strongly influence Dicer-TRBP processing of the pre-miRNAs. However, both the apical loop size and structure at the Dicing site are key elements for discrimination by the Dicer-TRBP complex. Interestingly, our NMR-derived structure reveals the presence of a triplet of base pairs that link the Dicer cleavage site and the apical loop. Mutational analysis in this region suggests that the stability of the junction region strongly influences processing by the Dicer-TRBP complex. Our results enrich our understanding of the active role that RNA structure plays in regulating miRNA biogenesis, which has direct implications for the control of gene expression.

Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis.

Noncoding 6S RNAs regulate transcription by binding to the active site of bacterial RNA polymerase holoenzymes. Processing and decay of 6S-1 and 6S-2 RNA were investigated in Bacillus subtilis by northern blot and RNA-seq analyses using different RNase knockout strains, as well as by in vitro processing assays. For both 6S RNA paralogs, we identified a key-but mechanistically different-role of RNase J1. RNase J1 catalyzes 5'-end maturation of 6S-1 RNA, yet relatively inefficient and possibly via the enzyme's "sliding endonuclease" activity. 5'-end maturation has no detectable effect on 6S-1 RNA function, but rather regulates its decay: The generated 5'-monophosphate on matured 6S-1 RNA propels endonucleolytic cleavage in its apical loop region. The major 6S-2 RNA degradation pathway is initiated by endonucleolytic cleavage in the 5'-central bubble to trigger 5'-to-3'-exoribonucleolytic degradation of the downstream fragment by RNase J1. The four 3'-exonucleases of B. subtilis-RNase R, PNPase, YhaM, and particularly RNase PH-are involved in 3'-end trimming of both 6S RNAs, degradation of 6S-1 RNA fragments, and decay of abortive transcripts (so-called product RNAs, ∼14 nt in length) synthesized on 6S-1 RNA during outgrowth from stationary phase. In the case of the growth-retarded RNase Y deletion strain, we were unable to infer a specific role of RNase Y in 6S RNA decay. Yet, a participation of RNase Y in 6S RNA decay still remains possible, as evidence for such a function may have been obscured by overlapping substrate specificities of RNase Y, RNase J1, and RNase J2.

Nailfold Videocapillaroscopy for Non-Invasive Assessment of Microcirculation and Prognostic Correlation with Endothelial Dysfunction, Cardiovascular Risk Factors, and Non-HLA Antibodies in Heart Transplant Recipients: A Pilot Study.

Early identification of allograft vasculopathy and the concomitant elimination of adverse risk factors is essential for improving the long-term prognosis of heart transplant (HTx) recipients with underlying cardiovascular disease (CVD). The major aim of this pilot study was to conduct a non-invasive imaging evaluation of the HTx patient microcirculation by employing nailfold video-capillaroscopy (NVC) in a well-characterized patient and control cohort, and to correlate these data with endothelial cell function, accompanied by studies of traditional cardiovascular risk factors and non-HLA antibodies in HTx recipients. Ten patients undergoing HTx (mean age of 38 ± 14 years) were recruited for the study and compared to a control group of 12 well-matched healthy volunteers (mean age 35 ± 5 years) with normal body mass index (BMI). Detailed medical records were collected from all individuals. NVC was performed using CapillaryScope 200 MEDL4N microscope. For functional readout and correlation analysis, endothelial cell network formation in conjunction with measurements of patient serum levels of vascular endothelial growth factor (VEGF) and non-HLA autoantibodies directed against the angiotensin II type-1-receptor (anti-AT1R-Ab), endothelin-1 type-A-receptor (anti-ETAR-Ab), protease-activated receptor-1 (anti-PAR-1-Ab), and VEGF-A (anti-VEGF-A-Ab) were studied. Our NVC analysis found that the average apical loop diameter of nailfold capillaries was significantly increased in HTx recipients (p = 0.001). In addition, HTx patients with more prominent changes in capillaroscopic patterns were characterized by the presence of traditional cardiovascular risk factors, and HTx patients had increased levels of anti-AT1R-ab, anti-ETAR-ab, and anti-VEGF-A-Ab (p = 0.017, p = 0.025, and p = 0.003, respectively). Capillary diameters most strongly correlated with elevated serum levels of troponin T and triglycerides (R = 0.69, p = 0.028 and R = 0.81, p = 0.004, respectively). In conclusion, we found that an abnormal NVC pattern in HTx patients is associated with traditional CVD risk factors and that NVC is a useful non-invasive tool to conveniently monitor changes in the microvasculature of HTx patients.

Conserved Structure Associated with Different 3'CITEs Is Important for Translation of Umbraviruses.

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.

RNA Chemical Labeling with Site-Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin-Sensing Riboswitch Aptamer Domain.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

61 The nailfold videocapillaroscopy in pediatric Behçet’s disease: a multi-center study

BackgroundBehçet's disease (BD) is a chronic inflammatory disease characterized by recurrent oral aphthous and genital ulcers accompanied by eye, joint, skin, gastrointestinal and central nervous system involvement. The vascular involvement may affect both the arterial and venous systems. Nailfold videocapillaroscopy (NVC) is an easy and non-invasive method used in the evaluation of microcirculation. With this study, we aimed to find the characteristics and prevalence of nailfold capillary alterations in patients with juvenile BD and to analyze their possible relationship between clinical characteristics and activity of the disease.MethodsPatients aged 5–21 years with a diagnosis of juvenile BD and followed up for at least six months were included in the study. Demographic and clinical characteristics of the patients were recorded. NVC was performed on 8 fingers of both hands, excluding the thumbs, and four consecutive non overlapping fields for each of fingers were evaluated (32 fields per patient). Capillary density, capillary width (arterial width, venous width, apical loop), capillary morphology and the presence of meandering capillary, micro hemorrhage, avascular area, neoangiogenesis, capillary ramification were evaluated from the images. Capillary morphology were evaluated by classifying them into four groups as normal, minor abnormalities, major abnormalities and scleroderma pattern. The presence of abnormilities in at least two fingers were recorded as capillary abnormality. The semi quantitative rating score 1–3 was applied for each capillaroscopic alteration.Results37 patients from 6 pediatric rheumatology centers were included in the study. The mean age of patients was 17 years (IOR 13–19) and 20 (54.1%) of them were girls. The patients were evaluated in four clusters according to their clinical presentations. Nineteen patients had mucocutaneous involvement, 9 patients had uveitis, 8 patients had vascular and neurological involvement, and 4 patients had gastrointestinal system involvement. During the follow-up period, genital ulcers developed in 22 patients, erythema nodosum in 9 patients, pseudofolliculitis in 18 patients, uveitis in 10 patients, vascular involvement in 8 patients, and neurological involvement in 5 patients. Anterior uveitis was present in five, posterior uveitis in three, panuveitis in one, and retinal vasculitis in three of the patients with ocular involvement. Four patients had lower extremity venous thrombosis, three patients had central nervous system (CNS) thrombosis, and one patient had both lower extremity and CNS thrombosis.When capillary morphology was evaluated; normal morphology was present in 16 patients, minor abnormality in 13 patients, and major abnormality in 8 patients. Median capillary density was 8, capillary length was 325 µm, arterial width was 12 µm, venous width was 16 µm, apical loop width was 18 µm, capillary width was 39 µm, and intercapillary distance was 107 µm. Neoagiogenesis was seen in 13 patients, enlarged capillaries in 12 patients, capillary meandering in 9 patients, bushy capillaries in 5 patients, bizarre capillaries in 4 patients, and microhemorrhage in 3 patients. Neoangiogenesis was found to be significantly more common in the NVC evaluation of patients with lower haemoglobin values at the time of diagnosis (p = 0.014).ConclusionNVC is an in vivo, non-invasive, and inexpensive imaging technique that allows the direct observation of the capillary network in living tissue throughout the skin and it may be preferred in juvenile BD for evaluating microvascular involvement. 61 Figure 1Capillaroscopic alterations of patients

Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes

HIV-1 RNA dimerization is a critical step in viral life cycle. It is a prerequisite for genome packaging and plays an important role in reverse transcription and recombination. Dimerization is promoted by the DIS (dimerization initiation site) hairpin located in the 5′ leader of HIV-1 genome. Despite the high genetic diversity in HIV-1 group M, only five apical loops (AAGCGCGCA, AAGUGCGCA, AAGUGCACA, AGGUGCACA and AGUGCAC) are commonly found in DIS hairpins. We refer to the parent DISes with these apical loops as DISLai, DISTrans, DISF, DISMal, and DISC, respectively. Based on identity or similarity of DIS hairpins to parent DISes, we distributed HIV-1 M genomes into five dimerization groups. Comparison of the primary and secondary structures of DIS, SD and Psi hairpins in about 3000 HIV-1 M genomes showed that the mutation frequencies at particular nucleotide positions of these hairpins differ among the dimerization groups, and DISF may be an origin of other parent DISes. We found that DIS, SD and Psi hairpins have hundreds of variants, only some of them occurring rather frequently. The lower part of DIS hairpin with G x AGG internal loop is highly conserved in both HIV-1 and SIV genomes. We supposed that the G-quadruplex, located 56 nts downstream of the Gag start codon, may participate in switching of HIV-1 leader RNA from BMH (branched multiple hairpins) to LDI (long distance interaction) conformation.

Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors

Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base–base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3′ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.