Uveal Melanoma: a miR-16 disease?

Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 years. Despite the discovery of gene signatures, the molecular actors driving the cancer aggressiveness are not fully understood and UM is still associated to a dismal overall survival at metastatic stage. Here, we showed that microRNA-16 (miR-16) is involved in uveal melanoma by an unexpected mechanism. By defining the miR-16-interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels (sponge RNAs). Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes such as cyclin D1 and D3, is impaired. This miR-16 non-canonical base-pairing to sponge RNAs can explain both the derepression of miR-16 targets and the promotion of oncogenes expression observed for patients with poor overall survival in two cohorts. miR-16 activity assessment using our sponge-signature discriminates the patients overall survival as efficiently as the current method based on copy number variations and driver mutations detection. To conclude, miRNA loss of function due to miRNA sequestration seems to promote cancer burden by two combined events; loss of brake and an acceleration. Our results highlight the oncogenic role of the non-canonical base-pairing between miRNAs/mRNAs in uveal melanoma.

Nucleobase clustering contributes to the formation and hollowing of repeat-expansion RNA condensate

RNA molecules with repeat expansion sequences can phase separate into gel-like condensate, and this process may lead to neurodegenerative diseases. Here we report that in the presence of Mg2+ ion, RNA molecules containing 20CAG repeats coacervate into filled droplets or hollowed condensate. Using hyperspectral stimulated Raman spectroscopy, we show that RNA coacervation is accompanied by the stacking and clustering of nucleobases, while forfeiting the canonical base-paired structure. At an increasing RNA/Mg2+ ratio, the RNA droplets first expand in sizes, and then shrink and adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like droplets, the nucleobase-clustered structure is more prominent at the rim than at the center, accounting for the rigidification of RNA droplets. Thus, our finding has broad implications for the general aging processes of RNA-containing membrane-less organelles.

N6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5

Alkbh5 catalyzes demethylation of the N6-methyladenosine (m6A), an epigenetic mark that controls several physiological processes including carcinogenesis and stem cell differentiation. The activity of Alkbh5 comprises two coupled reactions. The first reaction involves decarboxylation of α-ketoglutarate (αKG) and formation of a Fe4+═O species. This oxyferryl intermediate oxidizes the m6A to reestablish the canonical base. Despite coupling between the two reactions being required for the correct Alkbh5 functioning, the mechanisms linking dioxygen activation to m6A binding are not fully understood. Here, we use solution NMR to investigate the structure and dynamics of apo and holo Alkbh5. We show that binding of m6A to Alkbh5 induces a metal-centered rearrangement of αKG that increases the exposed area of the metal, making it available for binding O2. Our study reveals the molecular mechanisms underlying activation of Alkbh5, therefore opening new perspectives for the design of novel strategies to control gene expression and cancer progression.

Finding recurrent RNA structural networks with fast maximal common subgraphs of edge-colored graphs

RNA tertiary structure is crucial to its many non-coding molecular functions. RNA architecture is shaped by its secondary structure composed of stems, stacked canonical base pairs, enclosing loops. While stems are precisely captured by free-energy models, loops composed of non-canonical base pairs are not. Nor are distant interactions linking together those secondary structure elements (SSEs). Databases of conserved 3D geometries (a.k.a. modules) not captured by energetic models are leveraged for structure prediction and design, but the computational complexity has limited their study to local elements, loops. Representing the RNA structure as a graph has recently allowed to expend this work to pairs of SSEs, uncovering a hierarchical organization of these 3D modules, at great computational cost. Systematically capturing recurrent patterns on a large scale is a main challenge in the study of RNA structures. In this paper, we present an efficient algorithm to compute maximal isomorphisms in edge colored graphs. We extend this algorithm to a framework well suited to identify RNA modules, and fast enough to considerably generalize previous approaches. To exhibit the versatility of our framework, we first reproduce results identifying all common modules spanning more than 2 SSEs, in a few hours instead of weeks. The efficiency of our new algorithm is demonstrated by computing the maximal modules between any pair of entire RNA in the non-redundant corpus of known RNA 3D structures. We observe that the biggest modules our method uncovers compose large shared sub-structure spanning hundreds of nucleotides and base pairs between the ribosomes of Thermus thermophilus, Escherichia Coli, and Pseudomonas aeruginosa.

Identification of weak non-canonical base pairs around riboswitch-ligand recognition sites by solid-state NMR exchange spectroscopy

Base pairs are fundamental building blocks of RNA structures, and their stability and open-close equilibrium constitutes the dynamic picture. Weak base pairs, which feature the characteristics of low stability and rapid base pair opening, often play a critical role in RNA functions. However, site-specific identification of weak base pairs in RNA is challenging. Here, we report a solid-state NMR (SSNMR)-based two-dimensional proton-detected water–RNA exchange spectroscopy (WaterREXSY) to address this challenge. The approach uses the chemical exchange between hydrogen-bonded imino protons within the base pair and excited water molecules to polarize the imino protons for SSNMR observation. This process takes advantages that the imino protons within weak pairs undergo fast exchange rates with water, enabling a quick build-up and efficient detection. This method is used to characterize the weak pair in the riboA71–adenine complex (i.e., the 71nt-aptamer domain of the add adenine riboswitch from Vibrio vulnificus). We identify U47•U51, a weak non-canonical base pair that constitutes the U47•U51•(adenine-U74) base tetrad around the ligand-binding pocket. This result suggests that the breakage of U47•U51 may be the early stage in the process of ligand release.

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

Metazoan pri-miRNAs and pre-miRNAs fold into characteristic hairpins that are recognized by the processing machinery. Essential to the recognition of these miR-precursors are their apical junctions where double-stranded stems meet single-stranded hairpin loops. Little is known about how apical junctions and loops fold in three-dimensional space. Here we developed a scaffold-directed crystallography method and determined the structures of eight human miR-precursor apical junctions and loops. Six structures contain non-canonical base pairs stacking on top of the hairpin stem. U-U pair contributes to thermodynamic stability in solution and is highly enriched at human miR-precursor apical junctions. Our systematic mutagenesis shows that U-U is among the most efficiently processed variants. The RNA-binding heme domain of pri-miRNA-processing protein DGCR8 binds longer loops more tightly and non-canonical pairs at the junction appear to modulate loop length. Our study provides structural and biochemical bases for understanding miR-precursors and molecular mechanisms of microRNA maturation.

The complete mitochondrial genomes of four Baikal molluscs from the endemic family Baicaliidae (Caenogastropoda: Truncatelloida)

Here, we present the complete mitochondrial (mt) genomes of four members of the Baicaliidae Fisher, 1885, a truncatelloidean family that is endemic to Lake Baikal (East Siberia). The mt genomes are those of Korotnewia korotnevi (15,171 bp), Godlewskia godlewskii (15,224 bp), Baicalia turriformis (15,127) and Maackia herderiana (15,154 bp). All these mt genomes contain 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 22 transfer RNA (tRNA) genes. We detected non-canonical base pairs in some of the tRNA genes and variable numbers of non-coding spacers; some tRNAs do not have a TψC loop. We found gene order to be highly conserved in these Lake Baikal species and similar to the majority of caenogastropod mt genomes available on GenBank. A position of the putative control region is delimited to the non-coding region between trnF and the cox3 gene. It contains the ‘GAA(A)nT’ motif at the 3′ end and is similar to the replication origin found in most Caenogastropoda studied to date. We also compared the evolutionary rates of different genes to evaluate their use in different kinds of population or phylogenetic studies of this group of gastropods.

Melting Temperature Measurement and Mesoscopic Evaluation of Single, Double and Triple DNA Mismatches

Unlike the canonical base pairs AT and GC, the molecular properties of mismatches such as hydrogen bondings and stacking interactions are strongly dependent on the identity of the neighbouring base pairs. As a result, due to the sheer number of possible combinations of mismatches and flanking base pairs, only a fraction of these have been studied in varying experiments or theoretical models. Here, we report on the melting temperature measurement and mesoscopic analysis of contiguous DNA mismatches in nearest-neighbours and next-nearest neighbour contexts. A total of 4032 different mismatch combinations, including single, double and triple mismatches were covered. These were compared with 64 sequences containing all combination of canonical base pairs in the same location under the same conditions. The mesoscopic calculation, using the Peyrard-Bishop model, was performed on the set of 4096 sequences, and resulted in estimates of on-site and nearest-neighbour interactions that can be correlated to hydrogen bonding and base stacking. Our results confirm many of the known properties of mismatches, including the peculiar sheared stacking of tandem GA mismatches. More intriguingly, it also reveals that a number of mismatches present strong hydrogen bonding when flanked on both sites by other mismatches.

Melting Temperature Measurement and Mesoscopic Evaluation of Single, Double and Triple DNA Mismatches

Unlike the canonical base pairs AT and GC, the molecular properties of mismatches such as hydrogen bondings and stacking interactions are strongly dependent on the identity of the neighbouring base pairs. As a result, due to the sheer number of possible combinations of mismatches and flanking base pairs, only a fraction of these have been studied in varying experiments or theoretical models. Here, we report on the melting temperature measurement and mesoscopic analysis of contiguous DNA mismatches in nearest-neighbours and next-nearest neighbour contexts. A total of 4032 different mismatch combinations, including single, double and triple mismatches were covered. These were compared with 64 sequences containing all combination of canonical base pairs in the same location under the same conditions. The mesoscopic calculation, using the Peyrard-Bishop model, was performed on the set of 4096 sequences, and resulted in estimates of on-site and nearest-neighbour interactions that can be correlated to hydrogen bonding and base stacking. Our results confirm many of the known properties of mismatches, including the peculiar sheared stacking of tandem GA mismatches. More intriguingly, it also reveals that a number of mismatches present strong hydrogen bonding when flanked on both sites by other mismatches.

Finding recurrent RNA structural networks with fast maximal common subgraphs of edge-colored graphs

MotivationsRNA tertiary structure is crucial to its many non-coding molecular functions. RNA architecture is shaped by its secondary structure composed of stems, stacked canonical base pairs, enclosing loops. While stems are captured by free-energy models, loops composed of non-canonical base pairs are not. Nor are distant interactions linking together those secondary structure elements (SSEs). Databases of conserved 3D geometries (a.k.a. modules) not captured by energetic models are lever-aged for structure prediction and design, but the computational complexity has limited their study to local elements, loops, and recently to those covering pairs of SSEs. Systematically capturing recurrent patterns on a large scale is a main challenge in the study of RNA structures.ResultsIn this paper, we present an efficient algorithm to compute maximal isomorphisms in edge colored graphs. This framework is well suited to RNA structures and allows us to generalize previous approaches. In particular, we apply our techniques to find for the first time modules spanning more than 2 SSEs, while improving speed a hundredfold. We extract all recurrent base pair networks among all non-redundant RNA tertiary structures and identify a module connecting 36 different SSEs common to the 23S ribosome of E. Coli and Thermus thermophilus. We organize this information as a hierarchy of modules sharing similarities in their structure, which can serve as a basis for future research on the emergence of structural patterns.Availabilityhttp://csb.cs.mcgill.ca/carnaval2