Flexible pri-miRNA structures enable tunable production of 5’ isomiRs

SUMMARYDrosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5’ isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that, in addition to previously known features, the overall structural flexibility of pri-miRNA impacts Drosha cleavage fidelity. Internal loops and nearby G·U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5’ isomiR production. By analyzing patient data deposited in The Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5’ isomiR biogenesis in tumors.HIGHLIGHTSFlexible pri-miRNA structures lead to 5’ isomiR productionInternal loops and G·U pairs of pri-miRNA contribute to alternative Drosha cleavagesAlternative Drosha cleavage results in 5’ isomiRs from both strands of pre-miRNAs5’ isomiR production is upregulated by pri-miRNA-associated RBPs in cancersGRAPHICAL ABSTRACT

Investigation of the Time Resolution Set Up Used to Compute the Full Load Vortex Rope in a Francis Turbine

The flow in a Francis turbine at full load is characterised by the development of an axial vortex rope in the draft tube. The vortex rope often promotes cavitation if the turbine is operated at a sufficiently low Thoma number. Furthermore, the vortex rope can evolve from a stable to an unstable behaviour. For CFD, such a flow is a challenge since it requires solving an unsteady cavitating flow including rotor/stator interfaces. Usually, the numerical investigations focus on the cavitation model or the turbulence model. In the present works, attention is paid to the strategy used for the time integration. The vortex rope considered is an unstable cavitating one that develops downstream the runner. The vortex rope shows a periodic behaviour characterized by the development of the vortex rope followed by a strong collapse leading to the shedding of bubbles from the runner area. Three unsteady RANS simulations are performed using the ANSYS CFX 17.2 software. The turbulence and cavitation models are, respectively, the SST and Zwart models. Regarding the time integration, a second order backward scheme is used excepted for the transport equation for the liquid volume fraction, for which a first order backward scheme is used. The simulations differ by the time step and the number of internal loops per time step. One simulation is carried out with a time step equal to one degree of revolution per time step and five internal loops. A second simulation used the same time step but 15 internal loops. The third simulations used three internal loops and an adaptive time step computed based on a maximum CFL lower than 2. The results show an influence of the time integration strategy on the cavitation volume time history both in the runner and in the draft tube with a risk of divergence of the solution if a standard set up is used.

Telomere damage induces internal loops that generate telomeric circles

Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops) that occur in the proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion.

Telomere damage induces internal loops that generate telomeric circles

Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops), that occur in proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage with the accumulation of extrachromosomal telomeric circles and telomere erosion.

The internal loops in the lower stem of primary microRNA transcripts facilitate single cleavage of human Microprocessor

The human Microprocessor complex cleaves primary microRNA (miRNA) transcripts (pri-miRNAs) to initiate miRNA synthesis. Microprocessor consists of DROSHA (an RNase III enzyme), and DGCR8. DROSHA contains two RNase III domains, RIIIDa and RIIIDb, which simultaneously cleave the 3p- and 5p-strands of pri-miRNAs, respectively. In this study, we show that the internal loop located in the lower stem of numerous pri-miRNAs selectively inhibits the cleavage of Microprocessor on their 3p-strand, thereby, facilitating the single cleavage on their 5p-strand. This single cleavage does not lead to the production of miRNA but instead, it downregulates miRNA expression. We also demonstrate that by manipulating the size of the internal loop in the lower stem of pri-miRNAs, we can alter the ratio of single-cut to double-cut products resulted from the catalysis of Microprocessor, thus changing miRNA production in the in vitro pri-miRNA processing assays and in human cells. Therefore, the oscillating level of the single cleavage suggests another way of regulation of miRNA expression and offers an alternative approach to miRNA knockdown.

Floating-point Semantics of Analyzed Programs

This chapter focuses on floating-point semantics. It first outlines these semantics. The chapter then revisits previous results and adapts them to account for floating-point computations, assuming a bound on the rounding error is provided. A last part focuses on the approaches to bound these imprecisions, over-approximating the floating-point errors. Here, provided bounds on each variable, computing the floating-point error can be performed with classical interval-based analysis. Kleene-based iterations with interval abstract domain provide the appropriate framework to compute such bounds. This is even simpler in this setting because of the focus on bounding the floating-point error on a single call of the dynamic system transition function, that is, a single loop body execution without internal loops.