Xu Junqing

Peter Krüger

Calogero R Natoli

Kuniko Hayakawa

Wu Ziyu

Keisuke Hatada
email: keisuke.hatada@univ-rennes1.fr

X-ray absorption spectra of graphene and graphene oxide by Full Potential Multiple Scattering calculations with self-consistent charge density

Keywords: numbers: 78.70.Dm, 61.48.Gh, 71.15.Ap

X-ray absorption near edge structure (XANES) of graphene, graphene oxide and diamond are studied by the recently developed real-space full potential multiple scattering (FPMS) theory with space-filling cells. It is shown how accurate potentials for FPMS can be generated from self-consistent charge densities obtained with other schemes, especially the projector augmented wave method. Compared to standard multiple scattering calculations in the muffin-tin approximation, FPMS gives much better agreement with experiment. The effects of various structural modifications on the graphene spectra are well reproduced. (1) Stacking of graphene layers increases the peak intensity in the higher energy region. (2) The spectrum of the C atom located at the edge of graphene sheet shows a prominent pre-edge structure. (3) Adsorption of oxygen gives rise to the so-called interlayer-state peak. Moreover, O K-edge spectra of graphene oxide are calculated for three types of bonding, C-OH, C-O-C and C-O, and the proportions of these bondings at 800 • C are deduced by fitting them to the experimental spectrum.

I. INTRODUCTION

Due to its exceptional electronic structure, mechanical strength and high electrical conductivity [1], graphene is expected to become a key material for future nanotechnologies. Graphene oxide is also widely used for reasons of synthesis and chemical stability. X-ray absorption spectroscopy (XAS) and electron energy loss spectroscopy (EELS) are invaluable tools to study the electronic and atomic structure of defects and adsorbates of graphene layers [2][3][4]. However, because of the existence of various competing structures and the lack of accurate theoretical calculations, the origin of the observed spectral features is generally not well understood yet.

XAS and EELS of graphene has been computed previously using density functional theory (DFT) implemented for ground-state electronic structure calculations [5,6]. These methods usually rely on periodic boundary conditions so that the asymptotic behavior of wavefunction may be not reproduced well for low-dimensional systems in particular directions, and use fixed basis sets whose convergence for high energy states may be problematic. Also, the energy-dependent quasiparticle self-energy is replaced by the DFT ground exchangecorrelation potential (V xc ). Concerning these problems, the multiple scattering method has clear advantages, be-cause the single-particle Schrodinger equation is solved with the exact numerical basis functions for each energy. As a consequence, basis function convergence is not an issue and inclusion of energy dependent self-energies is straightforward. Moreover, the multiple scattering method is easily implemented in reciprocal-space, realspace and mixtures thereof, which makes it a method of choice for low-dimensional and nanostructured systems.

Conventional multiple scattering theory relies on the Muffin-Tin (MT) approximation where the potential is assumed spherically symmetric in each atomic (MT) sphere and constant outside the spheres, that is in the so-called interstitial region. The MT approximation is quite poor for graphene-like systems for the following reasons: (i) The charge density of the C-C σ and π bonding is highly anisotropic. (ii) For light elements such as Carbon, atomic potential scattering is rather weak and thus scattering of the interstitial potential is comparatively strong. Hence full potential (FP) corrections are important. (iii) The available energy range for x-ray absorption fine structure of light element is limited to a few tens of eV. This near-edge region shows rich spectral features due to strong scattering effects and the peaks are sharp because of long core hole lifetimes. Therefore, FP and SCF are necessary to increase the precision of the calculations.

Here we report XANES calculations of ideal and defective graphene, graphene oxide and diamond, using the real-space full potential multiple scattering (FPMS) theory with arbitrarily truncated space-filling cells [7,8].

We also present a method to generate all-electron potentials for FPMS from accurate charge-densities provided by other electronic structure codes, in particular projector augmented wave (PAW) methods [9]. This allows us to access the importance of FP corrections and selfconsistency in multiple scattering calculation of C K-edge XANES. The results are in good agreement with experiment and shed light on the origin of some controversial features of the graphene XANES.

II. THEORY A. FPMS method

There have been many attempts to extend the MS theory to the FP level [10][START_REF] Gonis | Multiple scattering in solids[END_REF][START_REF] Natoli | [END_REF][START_REF] Foulis | Exact distorted-wave approach to multiplescattering theory for general potentials[END_REF][START_REF] Ankudinov | [END_REF][15]. Here we give a brief summary of our FPMS method developed previously by some of us [7]. The real-space FPMS theory with spacefilling cells used here is valid both for continuum and bound states and contains only one truncation parameter -lmax, the maximum angular momentum of spherical wave basis. Space is partitioned by non-overlapping space-filling cells or Voronoi polyhedra. When a Voronoi polyhedron does not contain any atom or is in the interstitial region but still contains charge density, it is called empty cell (EC). Local Schrodinger equation is solved without the limit of the geometrical shape of the potential since the potential is not expanded in spherical harmonics. The absorption cross section is given by

σ(ω)= -8πα ω × mc Im φ c Lc ( r)|ε • r|G( r, r ; E)|ε • r |φ c Lc ( r) d r d r ,
where φ c Lc is the initial core state with L c ≡ (l c , m c ) representing the orbital angular momentum and magnetic quantum number, ω is the frequency of incident light, α is fine structure constant, ε is the electric field direction of incident light and E is the energy of final state. Green's function can be written as

G( r i , r j ; E) = Φ( r i )|([I -GT ] -1 G) ij |Φ( r j ) -δ ij Φ( r < )|Ψ( r > ) ,
where r i is the coordinate with respect to the center of scattering site i. T and G on the right hand side of the equation are the matrix of transition operators of scattering sites and KKR real-space structure factors, respectively. Φ L ≡ L ( Ẽ) -1 LL Φ L where Φ is the solution of local Schrodinger equation which behaves as the first kind of spherical Bessel function at the origin and Ẽ matrix can be computed using values on surface of the cell. Ψ is the solution of local Schrodinger equation irregular at the origin which matches smoothly to spherical Hankel function at the cell boundary. r < (r > ) is the smaller (bigger) one of r and r .

B. FPMS potentials from PAW charge densities

Plane-wave codes using the PAW method are popular because of high accuracy and computational efficiency for total energy DFT calculations. In PAW, the all-electron Kohn-Sham potential is replaced by a pseudo-potential inside the augmentation spheres around the nuclei. This pseudo-potential coincides with the all-electron potential only in the space outside the augmentation sphere. However, for XANES calculations the all-electron potential is required in all space, especially near the nuclei where the core-orbital has a large amplitude. We now present a method for reconstructing the all-electron potential in the augmentation spheres from the all-electron charge density, which is commonly provided in PAW methods, such as the Vienna Ab initio Simulation Package (VASP) [16] that we have used here. As the calculation of a local or semi-local exchange correlation potential from the charge density is straightforward, we only explain how to obtain the all-electron Hartree potential.

The mathematical problem is to find the regular solution of the Poisson equation inside an (augmentation) sphere S of radius r c , with Dirichlet boundary condition on the sphere, i.e., we want to solve

∇ 2 V ( r) = -4πρ( r)
for r < r c , given the charge density ρ for all points inside S, and the potential V on the surface (r = r c ). We introduce the function

W ( r) ≡ S ρ( r ) | r -r | d r ,
which satisfies the Poisson equation, but in general not the boundary condition. W ( r) can easily be calculated in a spherical harmonic basis

W (r, r) = L ω L (r)Y L (r), ω L (r) = rc 0 r l < r l+1 > ρ L (r )4πr 2 dr , ρ L (r) = Y * L (r)ρ(r, r) dr.
where r = r/r. The angular integrals can be performed very accurately on a Lebedev mesh [17]. The general solution of the Poisson equation is the sum of a particular solution, such as W , and the general solution of the corresponding homogeneous (i.e. Laplace) equation. The latter solution is L C L r l Y L (r) for arbitrary coefficients

C L . By choosing C L = [υ L (r c ) -ω L (r c )]/r l c , where υ L (r) = Y * L (r)V (r, r) dr,
the boundary conditions are satisfied, and so the final solution is given by This method has been implemented in a program "VASP2MS" which reads the charge density ρ and the Hartree (pseudo-)potential (for V (r c , r)) provided by the VASP code and calculates the all-electron Hartree potential in all space for use in the FPMS code. In practice the following VASP files are used: AECCAR0,1,2 which contain the core (0), non-SCF valence (1) and SCF valence (2) charge density, respectively. LOCPOT which contains the Hartree pseudopotential inside the atomic spheres and true potential in the rest of the unit cell. The data in these files is stored on a regular 3D grid of the unit cell. Further, RADCHGC and RADCHGV contain the core and valence charge density, expanded in spherical harmonics and stored on radial grids around each nucleus. In "VASP2MS", the VASP Hartree potential (in LOCPOT) is interpolated onto the radial meshes used in FPMS, in the region outside the augmentation spheres. The allelectron charge density ρ is interpolated onto the FPMS mesh everywhere in the cluster. Then, the method described above is applied to find the all-electron Hartree potential inside the augmentation spheres. Finally the exchange-correlation part of the potential is calculated from ρ and added to the Hartree potential.

V (r, r) = L ω L (r) + [υ L (r c ) -ω L (r c )] r l r l c Y L (r) .

C. Computational details

The C K-edge XANES spectra are calculated with the real-space multiple scattering method using the following forms of the potential. 1) non-SCF-FP: a full-potential calculation without self-consistency. The potential is calculated directly in the FPMS code and corresponds to the superposition of atomic charge densities. 2) SCF-FP: a self-consistent FP calculation, where the potential has been reconstructed from the charge density obtained by VASP code with the PAW [9] method. 3) SCF-MT: the same as SCF-FP except that the muffin-tin (MT) approximation is applied. The graphene layer is taken as the xy plane and the x axis along a carbon-carbon bond. The absorption cross section for light polarized along a = x, y, z is denoted σ a , σ=(σ z +σ x +σ y )/3 is the unpolarized spectrum, and we define σ x-y =(σ x +σ y )/2 as the in-plane (z=0) cross section. For systems with C3v point symmetry or higher, such as core-excited graphene which has D3h point symmetry, we have σ x = σ y . For an angle α between electric field and xy plane, the absorption cross-section is σ α =σ z sin 2 α+σ x cos 2 α.

Table I lists positions and spherical radii of atoms and ECs in the unit cell of graphene and diamond. Figure 1 is a schematic representation of the space partitioning used for graphene. The number of ECs is sufficient as we have checked by adding two more EC layers which did not change the XANES results. For the optical potential we take the Hedin-Lundqvist (HL) form [18] whose energydependent imaginary part accounts for damping effects through plasmon loss, with an additional constant imaginary potential accounting for the finite core-hole lifetime and leading to Lorentzian broadening. The lifetime broadening is 0.1 eV [19,20] for C K-edge and 0.15 eV for O K-edge [20,21], but in case of graphene, the value 0.2 eV was also reported [22,23]. In order to better see the spectral fine structure at high energy, the imaginary part of the HL potential has been omitted in a few cases (Figs. 5, 8 and 16). Moreover, the spectra are Gaussian broadened by 0.4 eV (graphene) and 0.3 eV (diamond) in order to account for experimental effects such as instrumental errors, structural uncertainties, vibration, etc. From the convergence test, we take lmax = 3.

In Fig. 2 the effect of self-consistency on the groundstate charge density of graphene is shown for the z = 0 plane. Differences between SCF and non-SCF calculations are clearly visible, but the charge redistribution appears to be rather small.

Core-hole effects are treated using the so-called finalstate rule (FSR), that is, in the final state the orbitals are relaxed around the full core-hole. In non-SCF-FP, this relaxation is calculated self-consistently but only at the atomic level. In SCF-FP, orbital relaxation effects beyond the absorbing atom are also taken into account through a VASP supercell calculation which yields the self-consistent FSR charge density and electrostatic potential for the bulk material. In this case, the core electron is put into the lowest unoccupied molecular orbital and the frozen-core approximation is applied, i.e., only valence electrons are relaxed. In order to make the interaction between periodic images of the core-hole negligible, a large supercell (SC) must be used. The converged size of SC is found to be 4×4×1 for graphene and few-layer graphene (FLG), and 4×4×4 for diamond. However, for safety, bigger SCs are used in this work as shown in Table II.

Figure 3 shows the distribution of the differential allelectron valence charge densities in xy plane, which are the differences between the densities of a 5×5×1 SC of graphene with a core-hole, and that of the ground-state graphene. It is seen that charge density redistribution is negligible for points further than 2.5 Å from the corehole.

In the DFT VASP calculation, the gradient corrected exchange-correlation functional PW91 [24] has been used but the local density approximation gives virtually identical results as we have checked. K-point sampling in the Brillouin zone and plane wave cut-off energy (Ecut) are given in Table II. For the SC dimension perpendicular to the layer, we took 15 Å such that the interaction between periodic images of the graphene layer is negligible. When the VASP potentials are imported to the real-space cluster calculation, the final state potential is used only for atoms not further away from the absorbing site than some distance R, of the order of half the SC dimension. For atoms beyond R, the ground-state VASP potential is used. In this way the periodic repetition of core-holes, which is an artefact of k-space methods (such as VASP) is avoided and convergence of the final results as a function of VASP SC size is very efficient.

III. XANES OF GRAPHENE

From Ref. [25] and [26], we have summarized the representative peaks of graphene and FLG in Table III. The calculated values correspond to SCF-FP calculation with a cluster size of 30 Å. The theoretical energy scale has been rigidly shifted such as to align peak A with experiment (285.5 eV). Peaks A, B and B2 were identified as π * resonance, σ * resonance and excitonic-state [27,28] peak, respectively.

In Fig. 4 the experimental spectra [25,26] for two light polarizations are shown along with the present calculations done in different potential approximations. It is obvious that the FP calculations (non-SCF-FP or SCF-FP) agree much better with experiment than the muffintin calculation (SCF-MT). The differences between non-SCF-FP and SCF-FP spectra are small, indicating that self-consistency affects XANES much less than FP cor- rections. The peak positions of non-SCF-FP and SCF-FP spectra differ by about 1 eV and SCF-FP shows better agreement with experiment. Indeed, for out-of-plane polarization (α=74 • , Fig. 4(a)) the SCF-FP calculation agrees very well with the data. However, a peak near 307.5 eV, labeled as "F+G", appears in the SCF-FP result but it is absent or faint in experiment, see Fig. 4(a).

For in-plane polarization (α=0 • , Fig. 4(b)) there is some systematic disagreement between experiment and all calculations, namely overestimation of peak B intensity and absence of peaks A and B2. The presence of peak A in the experimental data of Fig. 4(b) is very surprising, since peak A was identified in the σ z spectrum (Fig. 4(a)) as the π * resonance, which should exactly vanish for in-plane (α=0 • ) polarization (as it does in the calculations). This observation strongly suggests that some out-of-plane signal (σ z ) is mixed into the experimental spectra of Fig. 4(b). The reasons may be a misalignment in the experiment, or more likely, the fact that the graphene layer is not perfectly flat [29], such that locally the electric field cannot be parallel to the graphene plane everywhere.

The leading low energy peaks of the calculated spectra (peak A in Fig. 4(a) and peak B in Fig. 4(b)) are too intense, which indicates that charge relaxation around the core-hole is overestimated. This might be due to the residual self-interaction in DFT [30]. Further, the excitonic-state -peak B2 separated from peak B by about 1.0 eV in the experiment is not reproduced in the calculations even if the gaussian broadening is reduced to 0.2 eV. The theoretical problems with the leading peaks (B, B2) are likely related to the frozen-core approximation and to dynamical screening effects, i.e., the energy dependence of the complex self-energy [31,32]. The nonlocality of the particle-hole exchange interaction [33] is a further issue. It should be noted that the poor descrip- tion of the leading peaks is a common problem for K-edge XANES of light elements and could so far not be cured by any ab initio method we are aware of, including the Bethe-Salpeter-Equation approach [34].

Furthermore, in the experiment in Fig. 4(a), two weak peaks, a pre-edge one -A2 and peak K, were observed near 284 and 288 eV, respectively. In Ref. [25], peak A2 was attributed to a state about 0.8 eV below π * , which was mentioned in a rather old density of state (DOS) calculation of graphene [35]. However, in more recent DOS calculations [36][37][38], there does not exist any unoccupied state lower than π * state. We therefore believe that there is no such state in perfect graphene, and that the observation of peak A2 in Ref. [25] was due to a structural imperfection. In Sec. III C we show that edge atoms can produce a feature similar to peak A2.

The origin of peak K has been contentious, and alternative descriptions ascribe it to residual functionalization [39,40], especially the bonding between carbon and oxygen, or a free-electron-like interlayer-state [25,41]. In our SCF-FP result of XANES of the graphene cluster of radius 30 Å, for out-of-plane polarization (α=74 • , Fig. 4(a)), there does not exist any distinct peak near 288 eV. Therefore, peak K probably will not arise in XANES of ideal graphene. Possible origins are discussed at the end of this section and Sec. IV A.

A. Cluster size dependence

In the following, the theoretical spectra correspond to the SCF-FP method, unless stated otherwise. Figure 5 shows the cluster size dependence of the calculated spectra. Good convergence is achieved for a cluster radius of about 20 Å, except for some very fine features. Convergence is faster on the high energy side of the spectrum, as it is usually observed in real-space calculations for states above Fermi level.

We have found that the pre-edge peak -A2 and the oscillatory features between peaks A and B, can appear for small clusters, but as the cluster size increases, they become weaker. For a cluster radius of 30 Å, the features between peak A and B are gone. Therefore, finite size and vacancies in the graphene sheet may contribute to peak A2 and K. However, we can expect these peaks to be negligible for graphene of high structural quality, that is to say, perfectly flat sheets of several µm size or bigger with low defect concentration.

B. Layer effects

In this section, XANES calculations of 2-layer graphene (2LG), 3-layer graphene (3LG) and graphite are reported. The clusters radius is 15 Å and the distance between layers is 3.4 Å. We consider Bernal stacking whose order is 1212... [1] as shown in the left panel of Fig. 6. Each layer has two types of carbon atoms -A and B, giving a total of two types in both 2LG and graphite and four types in 3LG. The spectra of the different types are summed for comparison with experiment. The cluster potential is constructed using the strategy outlined above. Explicitly, for sites not further than 6 Å from the absorber and in the same layer, the core-excited (supercell) graphene VASP potential is used. For all other sites, the graphite VASP ground-state potential is used.

In order to check that this way to construct the cluster potential is reliable, we have compared it with another strategy, where both VASP potentials of core-excited (SC) 3LG and ground-state 3LG are used. We find that both strategies give very similar results (not shown) which indicates that charge density of a graphene layer is not much affected by the presence of other layers. Consistently, the spectral differences (not shown) between the inequivalent sites A, B is also small.

Apart from Bernal stacking, other stackings have been reported for multilayer graphene, most importantly rhombohedral stacking whose order is 123123... [1] (see FIG. 7. Comparisons between calculated polarized C K-edge XANES of 3LG with a cluster whose size is 15 Å and the experiment [25]. The angle between electric field and xy plane is also 74 deg. To achieve better agreement with the experiment, we used 0.7 eV Gaussian broadening. the right panel of Fig. 6). We have calculated the C K-edge XANES of 3LG in rhombohedral stacking, but found only tiny differences from Bernal stacking. The differences appear to be too small for an experimental determination of the stacking sequence from the XANES results.

The XANES of 3LG are shown in Fig. 7. The calculated peak intensities are systematically larger than in the data, but the positions of the peaks and their relative intensities are very well reproduced. In the calculated spectrum, a weak feature can be seen at 289 eV, close to the peak K energy. We note that the peak in the calculation is due to the finite cluster radius (15 Å) in the xyplane. In Fig. 8, graphene, 2LG, 3LG and graphite are compared. When the number of layers goes up, peaks G, H and J [42] increase quite strongly and peak C slightly, whereas peak A2 decreases somewhat. These tendencies agree well with experiment [25,26]. Moreover, the oscillatory features near 288.5 eV remain unchanged, which indicates that the effects of stacking of graphene layers on peak K is weak.

C. Edge effects

One-dimensional structures such as graphene ribbons are interesting for potential applications. The reduced dimensionality and the presence of the edge atoms can strongly modify the electronic and transport properties of graphene. The local electronic structure at the edge of a graphene has been measured by X-ray absorption microscopy [43] and EELS [44,45]. Here we study XANES of a zigzag-type edge terminated by hydrogen, and focus on several particular kinds of carbon atoms which are colored pink and labelled by numbers as shown in Fig. 9. The spectra are calculated using a cluster of the semi- [44]. Each number corresponds to the C atom which is labelled by the same number as in Fig. 9.

infinite graphene zigzag edge of radius of 25 Å around the absorber. Atomic positions at the edge are relaxed in a VASP calculation with a 1×22 SC containing 1 hydrogen and 22 carbon atoms along the axis perpendicular to the edge. The final state potential is generated as follows. Atomic and EC sites far from the edge by about 12 Å, the graphene ground-state potential is used. For sites near the edge, FSR potentials have been generated using a 4×16 SC terminated by hydrogen on both sides ( with 8 H and 64 C atoms) and one core-hole on the absorbing site.

Figure 10 shows calculated unpolarized C K-edge XANES of individual C atoms at or near the graphene zigzag edge, in comparison with calculated XANES of infinite graphene and experimental EELS spectrum of the individual outermost C atom at the edge taken from Ref. [44]. Our calculated spectra of edge carbon atoms are similar to calculated results in Ref. [44] and [45]. We found that XANES of the C atom labelled by "6" is quite similar to XANES of infinite graphene, except that the whole spectrum is shifted a little to lower energy. However, supercell calculations with vacuum space can not reproduce the absolute value of potential with respect to the vacuum level precisely for low dimensional systems, so that the accuracy of chemical shift between different structures is not ensured.

The overall spectral shape of the experimental spectrum, and in particular the edge-induced low-energy peak, are reproduced by the calculation. The main difference with the spectra of infinite graphene is the appearance of a new strong low-energy peak at 283.8 eV, which fits well the pre-edge peak (A2) position observed in some XANES experiments of graphene (see Table III and Fig. 4(a)), and has been attributed to a localized state at the Fermi level at the zigzag edge [46]. Experimentally, Ref. [43] and [47] have found that the more edge C atoms are probed in XAS, the higher the intensity of the pre-edge peak. Moreover, Other kinds of structure modulations like charged-specie adsorption may also create such a pre-edge peak [43,48,49]. From all the experimental and theoretical evidences, it seems that the A2 peak is largely due to edge states or other structural modifications rather than being an intrinsic peak of perfect graphene.

There are some discrepancies of the peak positions between calculated and experimental spectrum of the outermost edge C atom (labelled by "1"), which is also true for another experiment [45]. The disagreement can be attributed to the following points: (i) Core-hole effects are not considered well as described in Sec. III. (ii) The potential generated by VASP code may lead to some deviations, e.g., of vacuum level, since the asymptotic behavior may be not reproduced well for low-dimensional systems in particular directions by the plane-wave method. (iii) The energy resolution of the experiment is 0.4 eV [44], which is not very fine. (iv) The absolute energy of cal-FIG. 13. Calculation of polarization dependent C K-edge XANES of carbon atoms directly bonded to oxygen, for three types of graphene oxide. culated spectrum is aligned according to experimental XANES of graphene from Ref. [25]. It is not surprising that there is ambiguity in the absolute energies of different experiments. For example, in Ref. [45], the energy position of peak π * is fixed as 285.0 eV which is different from 285.5 eV [25] used in this work. (v) In reality the edge structure is not as perfectly ordered as of calculation, it can be distorted or not flat, so that the spectrum is modified. Moreover, the edge structure may be unstable under the incident electron beam [44]. (vi) The contribution of neighboring C atoms may be mixed in [45].

IV. OXYGEN ADSORPTION ON GRAPHENE

Among the various graphene derivatives, graphene oxide is the most important one. Oxygen can be bound in different ways to C, on the basal plane, on edge or near vacancies, which makes this system quite complex. For simplicity, we focus on three types of basal-plane oxygen species -C-O-C (COC), C-OH (COH) and C=O (CO). This is suitable if the basal plane is complete so that there are few vacancies and the contribution of edge oxygen species can be negligible which is the case for large samples.

We compare two structural models -STR1 and STR2, corresponding to low and high oxygen density, respectively. In STR1, a single oxygen is put on the center of large graphene cluster. In STR2, oxygen atoms are put in a periodic array on graphene, with 4 O atoms per 6×6×1 SC, corresponding to an oxygen density of 5.3%, see Fig 12. The potential is constructed in the same fashion as before, e.g., for STR1, using FSR potentials from a 4×4×1 SC (see Fig. 11) VASP calculation for sites closer than 5 Å from the absorber and ground-state potential of the infinite graphene sheet for further away atoms. In both structural models, the cluster size of the XANES calculation is 20 Å, and the computational parameters are similar to the graphene case.

A. C K-edge XANES

In this section, we consider the low-density structure (STR1) and focus on the XANES spectrum of the C atom directly bonded to oxygen. Firstly, we want to stress that SCF is important in this case, since in non-SCF-FP calculations ( not shown), the main peaks of σ z of COC and COH are shifted to higher energies by about 1.5 eV compared with SCF-FP results. In Fig. 13, σ z of COC and COH have a big peak near 288 and 288.5 eV, respectively, which fits the position of peak K. Combining the previous discussions in Sec. III, we make the following conclusions: (i) Since peak K does not arise for ideal graphene and is only weakly affected by stacking of graphene layers, the interpretation using interlayerstate [25,41] is probably not suitable. (ii) Finite size and vacancies in the graphene sheet can lead to some oscillations between the two leading peaks -A and B, which may contribute to peak K. However, for the high-quality graphene sample, this kind of contribution is expected to be quite weak. (iii) Two typical oxygen species of GO adsorbed on the basal plane, C-O-C and C-OH, give rise to peak K, so that they are two possible origins of peak K in some experimental XANES of GO.

However, in cases where proportion of oxygen is bigger, other structure modulations can play important roles. In a considerable number of experiments [37,[50][51][52][53], near 288 eV, we can observe a very pronounced and sharp peak at a higher energy and one or two broader peaks at lower energies. The broader peaks can be attributed to COC and COH bondings on the basal plane, while the sharp one is normally attributed to the carboxylic acid group (-COOH) on the edge or near vacancies.

B. O K-edge XANES

In the MT approximation, the potential in the interstitial region is approximated by a constant. The choice of this parameter leads to an uncertainty in the energy scale and thus in chemical shifts. This ambiguity is absent in FPMS with space-filling cells, and as a result, chemical shifts become predictive and much more accurate than in the MT approximation. Further, since the structures of infinite graphene and graphene oxide studied in our work are similar, and we used vacuum spaces of the same size for the supercell calculations, the chemical shift can be accurate.

Figure 14 shows polarized XANES of COC, COH and CO for the two structural models calculated in SCF-FP and for STR1 also in non-SCF-FP. The peak positions change quite strongly between SCF-FP and non-SCF-FP, which means that the use of SCF potential is important in this case. There are also clear differences between the two structural models, indicating substantial interaction between adsorbed oxygen species in the high-density case (STR2) which leads to extra spectra fine structure.

Next, we have tried to simulate the experimental O Kedge XANES of GO in 800 • C in Ref. [50] shown in Fig. 15(c). This experiment was performed at 90 • incidence of the linearly polarized X-rays so that σ z should not contributed to the observed XANES. However, in can be seen in the left picture of Fig. 7 of this same Ref. [50] that the 1s -π * peak is fairly intense, which implies a nonnegligible σ z contribution. By fitting to the experimental spectrum, we find the weight of σ z of about 12%.

Cross sections of three oxygen species of two structure models are shown in Fig. 15(a) and 15(b). We have fitted the proportions of COC, COH and CO to the experiment, and obtain 44%, 56% and 0% for STR1, and 33%, 67% and 0% for STR2. The final XANES of both structural models are shown in Fig. 15(c). Since the concentration of oxygen is very low, there is an ambiguity of the intensity of the experimental spectra. Therefore, we have done the fittings to its derivative. Both calculations are qualitatively in accordance with the experiment. Moreover, the differences between two calculated results are small. This implies that an oxygen concentration change of several percent will not affect the spectrum much.

V. COMPARISONS WITH OTHER METHODS

Here we compare the present FPMS results with three other theoretical methods.

(1) Multiple Scattering in the MT approximation (MT-MS). Following common practice, we have used slightly overlapping spheres in the MT calculation. Here the diameter of the spheres is taken 15% larger than the nearest neighbor distance, i.e., the overlap factor (ovlp) is 1.15. Note that in FPMS, overlap between atomic cells is strictly zero, and when an overlap factor is indicated, it refers to the spheres in which the atomic cells are enclosed.

The spectra in Fig. 16, are plotted on the photoelectron final energy scale, without alignment to experiment, in order to see the peak shifts between different approximations. The SCF-FP calculation with space filling (bottom curve) gives by far the best results (see also Fig. 4). All other schemes, including SCF-FP without ECs, lack most of the fine structure in the energy range 5-20 eV. Therefore, both FP treatment inside each atomic cell and filling of interstitial space by empty cells are necessary for accurate XANES. Compared with SCF-FP, all other spectra are shifted, especially those without ECs. This implies that for MS calculations without space-filling, large errors in the absolute energy levels must be expected. Moreover, we have noticed that the results obtained in the MT approximation are very sensitive to the constant interstitial potential and charge, which are usually treated as free parameters.

(2) DFT. In Ref. [5] and [56], Augmented Plane Wave method and pseudopotential plane wave method were used to calculate EELS of graphene, respectively. Their results are similar to ours. In Ref. [57], the authors have done DFT calculations in low energy region with linear combination of atomic orbitals (LCAO) method to obtain initial and final state wavefunctions to compute XAS of graphene. There are some differences between their and our results, e.g. related to layer effects. A drawback of the LCAO method is that uniform convergence of the types and amount of basis functions are not ensured [58,59] over the XANES energy range. This may lead to difficulties for studying XAS in graphene-related systems.

3) PAW Green's function method (PAW-GF). In Ref. [54], a scheme for XANES has been devised based on the recursion method. An advantage of this method is that the Hamiltonian needs to be diagonalized only for occupied states while the core-level spectrum is computed as a continued fraction. In Fig. 17 conditions from a k-to a real-space cluster method, do not reduce the accuracy of the underlying PAW electronic structure calculation. This is an important check of our new method. While XANES calculations are numerically lighter in PAW-GF, a main advantage of SCF-FPMS is that it can directly be used for a variety of other electron spectroscopies (EXAXS, UPS, XPS, etc) while this remains to be shown for PAW-GF. Further, when looking at the spectra of Fig. 17 in more detail, the SCF-FP spectrum compares slightly better with the experimental one beyond 302 eV. This might be due to the fact that in the PAW method the partial waves and projectors are fixed and adapted to the energy interval of the occupied valence electrons. This is not an issue in SCF-FPMS, since the all-electron potential is used for solving the Schrödinger equation of the continuum states.

VI. CONCLUSION

We have presented full-potential MS calculations for XANES of graphene and related systems. The potentials were generated from self-consistent charge densities obtained through plane-wave PAW calculations with the VASP code. With this new scheme C and O Kedge XANES have been studied for graphene, few-layer graphene, graphite, three types of graphene oxide, and zigzag edge of graphene, and results have been compared with experiment and several other theoretical methods.

With the present full-potential method, good agreement with experiment has been obtained for all systems, while conventional MT calculations give poor results. The main variations of the XANES spectra as a function of the number of graphene layers has been successfully reproduced. The analysis of C K-edge XANES in graphene edge atoms and that of graphene oxide has helped to clarify the origin of several controversial peaks in the graphene spectrum, especially the pre-edge shoulder (A2) and the so-called interlayer-state peak (K). By calculating the O K-edge XANES of three types of graphene oxide, we showed that the proportion of different oxygen species can be estimated by a fit to the experimental spectrum.

FIG. 1 .

 1 FIG. 1. (a) Positions of carbon atoms (blue ball) and ECs -empty cells (green ball) for graphene. (b) and (c) show schematic diagrams of the graphene cluster with space filling cells viewed along z and y axis, respectively. The blue cell represents a carbon atom, and the green cell is EC.

FIG. 2 .

 2 FIG. 2. Charge density in the xy-plane (z=0) of the graphene unit cell from the non-SCF FPMS (left) calculation and the SCF (VASP) calculation (right). The unit of charge density is Å-3 . Contour lines are plotted for values Fi=5 (-1+0.1i) Å-3 , where i=0,1. . . 20, from the corner to the region around carbon atoms.

FIG. 4 .

 4 FIG. 4. Comparison between calculated polarized C K-edge XANES of graphene with a cluster of radius 30 Å and experiments [25, 26]. α is the angle between electric field and xy plane. Since α=74 deg in left panel, σ ≈ 0.924 σz+0.076 σx (see Sec. II C). The reason why peak A arises in right panel despite the fact that α=0 deg is discussed in the text of Sec. III. Dashed vertical lines show the peak positions of experiments. SCF-FP, non-SCF-FP and SCF-MT are defined in Sec. II C. (non-)SCF represents using (non-)self-consistent potential. While FP and MT represent full potential and Muffin-Tin version of multiple scattering theory, respectively.

FIG. 5 .

 5 FIG.5. Calculated XANES in SCF-FP case with different sizes -radii of clusters whose center is the absorbing atom. σ z(x) is polarized absorption cross section with electric field along z(x) axis.

FIG. 6 .

 6 FIG. 6. Schematic diagram of Bernal (left) and rhombohedral (right) stacking of graphene layers.

FIG. 8 .

 8 FIG.8. Calculated XANES of graphene, 2LG, 3LG and Graphite. σ z(x) represents σ z(x) . Arrow represents the variation trend of peak intensity as layer number increases.

FIG. 9 .

 9 FIG. 9. Schematic diagram of the graphene zigzag edge.

FIG. 11 .

 11 FIG.11. Supercells of three types of GO, gray -carbon, red -oxygen, white -hydrogen.

FIG. 14 .

 14 FIG. 14. Calculated O K-edge XANES of GO with different structure models in SCF-FP and non-SCF-FP, STR1 -the structure model containing only one oxygen atom, STR2 -the structure model containing oxygen about 5.3%. Details about structure models are described in Sec. IV.

FIG. 15 .

 15 FIG. 15. Calculated O K-edge polarized XANES of GO where the direction of electric field is the same as the experiment [50] and the experiment shown in (b). (a) Using structure mode STR1 containing only one oxygen atom. (b) Using STR2 containing oxygen about 5.3%. (c) The weighted average of XANES of three types of GO using STR1 and STR2. The proportions of COC, COH and CO obtained by fitting them to the experiment are 44%, 56% and 0% for STR1, and 33%, 67% and 0% for STR2.

  FIG. 16. Calculated XANES of graphene in SCF-FP and SCF-MT cases. The cluster size is always 25 Å. ovlp is overlap factor of spherical radii. (no EC) EC represents that ECs are (not) added.

TABLE I .

 I Cartesian coordinates and radius of polyhedra of atoms and ECs in the unitcell of graphene and diamond. All data are in the unit of Å.

	System and type	x	y	z	r
	1. Graphene
	Carbon	1.424	0	0	0.95
	Carbon	2.848	0	0	0.95
	EC	0	0	±0.7	1.05
	EC	1.068	±0.6166101	1.2	0.9
	EC	1.068	±0.6166101	-1.2	0.9
	EC	2.136	0	±1.2	0.9
	2. Diamond
	Carbon	±0.445875 ±0.445875 ±0.445875 0.89175
	EC	±1.337625 ±1.337625 ±1.337625 0.89175

TABLE II

 II

. Parameters used in VASP calculations.

TABLE III

 III

		σz			σx
	energy (eV) cal exp	symbol	energy (eV) cal exp	symbol
	283.7	283.7	A2	-	291.7	B2
	285.5	285.5	A	292.5	292.6	B
	-	288.2	K	297.1	297.8	D
	294.5	294.0	C	301.5	301.8	E
	307.7	308.0	G	307.0	306.8	F
	-	315.5	H	327.0	327.5	I

. Peak positions of calculated XANES of graphene with 30 Å size in SCF-FP case and experimental XANES of graphene and FLG. Peak H only exists in the experiment of FLG. "-" represents not clear or not being reproduced in calculations.

ACKNOWLEDGMENTS

We would like to thank T. Konishi and W. Huang for illuminating discussions. Computational resources of USTC supercomputing center are gratefully acknowledged. Parts of this work have been funded by European FP7 MSNano network under Grant Agreement No. PIRSES-GA-2012-317554, by FP7 MS-BEEM under Grant Agreement No. FP7-PEOPLE-2013-IEF-625388, by COST Action MP1306 EUSpec and by JSPS KAK-ENHI Grant Number 25887008.


Lucie Pessel-Vivares

Laurent Houzet

Sébastien Lainé

Marylène Mougel

Insights into the nuclear export of murine leukemia virus intron-containing RNA

Keywords: RNA export, nucleocytoplasmic trafficking, intron-containing RNA, intron retention, cis

The retroviral genome consists of an intron-containing transcript that has essential cytoplasmic functions in the infected cell. This viral transcript can escape splicing, circumvent the nuclear checkpoint mechanisms and be transported to the cytoplasm by hijacking the host machinery. Once in the cytoplasm, viral unspliced RNA acts as mRNA to be translated and as genomic RNA to be packaged into nascent viruses. The murine leukemia virus (MLV) is among the first retroviruses discovered and is classified as simple Retroviridae due to its minimal encoding capacity. The oncogenic and transduction abilities of MLV are extensively studied, whereas surprisingly the crucial step of its nuclear export has remained unsolved until 2014. Recent work has revealed the recruitment by MLV of the cellular NXF1/Tap-dependent pathway for export. Unconventionally, MLV uses of Tap to export both spliced and unspliced viral RNAs. Unlike other retroviruses, MLV does not harbor a unique RNA signal for export. Indeed, multiple sequences throughout the MLV genome appear to promote export of the unspliced MLV RNA. We review here the current understanding of the export mechanism and highlight the determinants that influence MLV export. As the molecular mechanism of MLV export is elucidated, we will gain insight into the contribution of the export pathway to the cytoplasmic fate of the viral RNA.

Introduction

Murine leukemia viruses (MLV) were among the first mammalian retroviruses discovered more than 60 years ago and are today among the best characterized retroviruses. The study of retroviruses has a broad impact on diverse domains of biology, medicine and biotechnologies. Since most of the retroviruses are oncogenic, study of their pathogenic effects at the cellular and molecular levels has led to a better understanding of fundamental mechanisms involved in cellular growth control and carcinogenesis. [START_REF] Braoudaki | Tumorigenesis related to retroviral infections[END_REF][START_REF] Wolff | A Moloney murine leukemia virus-based retrovirus with 4070A long terminal repeat sequences induces a high incidence of myeloid as well as lymphoid neoplasms[END_REF] MLVs induce leukemia in mice and provide a useful model to investigate the genetic basis of hematopoietic cancer contributing to a better understanding of hematopoiesis. Like many retroviruses, MLVs induce tumors by proviral insertional mutagenesis, corresponding to the integration of viral DNA near cellular protooncogenes and subsequent alteration of their expression. Analysis of integration sites led to the identification of many genes that regulate cell growth and differentiation and are involved in tumor formation. [START_REF] Suzuki | New genes involved in cancer identified by retroviral tagging[END_REF][START_REF] Lund | Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice[END_REF][START_REF] Kim | Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas[END_REF][START_REF] Mikkers | High-throughput retroviral tagging to identify components of specific signaling pathways in cancer[END_REF] Further understanding of the retrovirus life-cycle has brought new concepts in molecular biology and provided new powerful tools for biotechnology, cancer research and gene therapy. The discovery of the reverse transcription, the copying of RNA to DNA, in retrovirus replication cycle had a strong impact on biotechnology. Indeed, the use of MLV reverse transcriptase in the laboratory to generate DNA copies from RNA provided a remarkable tool for analysis of RNA molecules. Finally, in-depth knowledge of retrovirus molecular structure and full replication cycle allowed to use them as backbone for vectors to generate efficient and specific gene delivery systems. Vectors derived from MLV are widely used for cell transduction in laboratory [START_REF] Keswani | Design of hybrid lipid/retroviral-like particle gene delivery vectors[END_REF] and their use in gene therapy trials has been successful in most of the patients. However, adverse effects related to insertional mutagenesis were observed in 10% of the patients and work is in progress to improve their biosafety. [START_REF] Cavazzana-Calvo | Gene therapy for severe combined immunodeficiency: are we there yet?[END_REF][START_REF] Aiyer | Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction[END_REF][START_REF] El Ashkar | BET-independent MLV-based Vectors Target Away From Promoters and Regulatory Elements[END_REF] Considered as a prototypic retrovirus, MLV has been extensively studied and has a major impact on understanding the molecular organization and life-cycle of retroviruses. The genome of all retroviruses is a capped and polyadenylated RNA about 8-kb long and flanked by two long terminal repeats (LTR) containing regulatory elements for transcription and polyadenylation. Like all simple retroviruses, MLV genomic RNA (gRNA), also called full-length RNA (FL RNA), contains only the three major coding domains common to all retroviruses: gag, pol and env encoding for structural, enzymatic and receptor binding viral proteins, respectively (Fig. 1), whereas complex retroviruses, such as the human immunodeficiency virus (HIV), contain additional coding domains corresponding to accessory and regulatory proteins. In addition to coding capacity, cis-acting elements present on the FL RNA are directly involved in many steps of the virus replication cycle, such as splicing, packaging or reverse transcription, through their interaction with cellular or viral trans-acting factors. Finally, only a subset of the FL RNA will be used to produce viral spliced RNAs. Simple retroviruses are characterized by only one spliced RNA resulting from the removing of a single intron and generating the env mRNA, whereas complex retroviruses produce many other spliced RNA coding for accessory proteins, with at least 46 spliced RNA for HIV. [START_REF] Purcell | Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity[END_REF] However, although MLV belongs to simple retroviruses, a second MLV singly spliced mRNA called SD' RNA and generated from an alternative splice donor site was discovered. Unlike env mRNA, SD' RNA can be packaged, reverse transcribed and integrated in the host genome. [START_REF] Houzet | A new retroelement constituted by a natural alternatively spliced RNA of murine replication-competent retroviruses[END_REF] In contrast to cellular RNA, the replication cycle of a retrovirus requires unspliced introncontaining viral RNA to be exported from the nucleus. Once in the cytoplasm, this FL RNA will be either translated to provide the viral Gag and Gag-Pol polyproteins, or packaged as a genome into assembling virions. To this dual fate correspond two distinct pools of unspliced viral RNA in the cytoplasm of MLV infected cells, while a unique pool of a bifunctional RNA is used for translation and packaging in the case of HIV. 13 As opposed to retroviral FL RNA, the presence of unspliced cellular RNA in the cytoplasm is rare since splicing is a prerequisite for nuclear export of mRNA and intron retention targets RNA to degradation by cellular quality control mechanisms. Thus, retrovirus FL RNA needs to escape from degradation and then find a strategy to reach the cytoplasm. For example, HIV Rev regulatory protein binds the Rev response element (RRE) present in FL RNA and interacts with the nuclear export receptor CRM1 to exit the nucleus via the nuclear pore complex (NPC). The simple Mason Pfizer Monkey virus (MPMV) contains a cis-acting RNA motif, the constitutive transport element (CTE), which mediates the export of the FL RNA through the direct binding of the nuclear export factor NXF1, also called Tap. Studies of retroviral mechanisms to export their unspliced gRNA have also led to a better understanding of their host nuclear export pathways. Surprisingly, whereas MLV molecular organization and lifecycle have been extensively studied, the issue of the export of its unspliced FL RNA was resolved only very recently by several teams simultaneously, closing a major gap in the knowledge of MLV replication cycle. Here we will focus on the main similarities and differences between the cisacting elements and trans-acting factors reported as contributing to MLV export and we will discuss the apparent complexity of MLV export pathway.

Overview of mammalian mRNA export

The nucleocytoplasmic transport of mRNA is closely related to its nuclear maturation (capping, splicing, cleavage and polyadenylation) since a mRNA maturation defect prevents its export and leads to RNA degradation. [START_REF] Hocine | RNA processing and export[END_REF] Interestingly, mRNA export is saturable, indicating the involvement of specific limiting factors. Export of the majority of the mature mRNA is dependent on the highly conserved Tap factor. Tap shuttles between nucleus and cytoplasm by interacting with the nucleoporins (NUP) in the nuclear pore via its C-terminus while its N-terminus binds the RNA cargo. Other factors are recruited to reinforce these weak interactions. The heterodimerization of Tap with the NTF2-related export protein-1 (NXT1), also called p15, induces a conformational switch that strengthens Tap-NUPs interaction within the nuclear pore. In parallel, the conserved transcription-export (TREX) complex, mainly including the THO complex, UAP56, Aly and CIP29, is recruited to the newly synthesized transcripts during splicing. This RNA/TREX complex finally uses the Tap/p15 heterodimer as an export factor to pass through the nuclear pores. [START_REF] Pante | Nuclear pore complex structure: unplugged and dynamic pores[END_REF] RNA helicases, such as Dbp5, help mRNAs to exit the pores on the cytoplasmic side (Fig. 2A). [START_REF] Montpetit | A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export[END_REF] Recent microscopy approaches focusing on single mRNA molecules in living cells have provided valuable insights into the mRNA nucleocytoplasmic translocation. The kinetic of mRNA export was spatially resolved and showed that mRNA molecules pass rapidly through the central channel of nuclear pores, and that docking on the nuclear side and then waiting for release into the cytoplasm are the most time consuming steps (totaling 180 +/-10 ms). Some mRNA molecules (10%) sit for seconds at nuclear pores without gaining exit, suggesting that RNA could be screened for quality at this point (for review see ref. [START_REF] Grunwald | Nuclear export dynamics of RNA-protein complexes[END_REF] ).

In contrast to splicing-dependent mRNA export, little is known about how natural mRNAs with retained introns are exported in mammalian cells. A substantial majority of genes contains introns and over 90% of human genes are alternatively spliced. Expression of genes with persistence of introns has been reported in several cancers [START_REF] Katenkamp | mRNA expression and protein distribution of the unspliced tenascin-C isoform in prostatic adenocarcinoma[END_REF] and could participate in adaptive regulation. [START_REF] Vitulo | A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype[END_REF] In addition, natural intronless cellular mRNAs such as histone H2A, HSPB3, IFNα1 and IFNβ1 constitute model systems. Their export is blocked by knockdown of TREX components (UAP56 and THOC2) or Tap (for review see ref. [START_REF] Grzybowska | Human intronless genes: functional groups, associated diseases, evolution, and mRNA processing in absence of splicing[END_REF] ) but the mechanisms used to bypass nuclear export restrictions remain unclear. However, most of the studies done with intronless viral mRNAs (i.e. herpes simplex virus, influenza A virus), reveal that these mRNAs bind to specific cellular or viral proteins that recruit the classic mRNA export machinery (for review see ref. [START_REF] Harris | RNA export: insights from viral models[END_REF] ). The unspliced RNA genome of retroviruses also provides valuable information on intron-containing mRNA export. Especially, studies of HIV and MPMV led to the understanding of the function of cellular export factors, CRM1 and Tap, respectively. In addition to the role of the Tap/p15 complex in the export of bulk transcripts, it is now well established that Tap/p15 promotes export of intron-containing mRNA through binding to the wellconserved CTE motif present in both viral and cellular mRNAs. [START_REF] Wang | Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns[END_REF]

MLV requires Tap/NXF1-dependent pathway to export its viral RNA

Little is known about viral RNA localization in cells infected with replication-competent MLV. Localization of unspliced viral RNA in mouse NIH3T3 cells chronically infected with Mo-MLV was determined by fluorescence in situ hybridization (FISH) and by cell fractionation experiments. These results showed that the majority of the unspliced RNA remained within the nucleus, suggesting that the nucleus constitutes an MLV RNA reservoir. [START_REF] Smagulova | The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA[END_REF] Unspliced RNAs are divided into two distinct RNA pools according to its dual fate in the cytoplasm: it can be targeted to the ribosomes to be translated as mRNA or be driven at the plasma membrane (PM) to be packaged as gRNA in the newly formed viruses. These two RNA populations (mRNA, gRNA) can be distinguished by Actinomycin D treatment and measurement of their different half-lives. 13,[START_REF] Levin | Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D[END_REF][START_REF] Messer | Metabolism of viral RNA in murine leukemia virusinfected cells; evidence for differential stability of viral message and virion precursor RNA[END_REF] Indeed, the virion precursor gRNA has a half-life of 3-4 hours although the unspliced mRNA is stable for a period of 12-24 hours, [START_REF] Messer | Metabolism of viral RNA in murine leukemia virusinfected cells; evidence for differential stability of viral message and virion precursor RNA[END_REF] suggesting that the two pools of RNA could form different ribonucleoprotein complex (RNP) according to their fate. Whether the RNP are already formed in the nucleus and predetermine the cytoplasmic fate of the RNA remains to be demonstrated. The export pathway used by MLV RNA was identified very recently, in 2014. [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF][START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF][START_REF] Sakuma | Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts[END_REF][START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF][START_REF] Pilkington | Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element[END_REF] Different complementary approaches such as RNA silencing, dominant-negative mutant of Tap and RNA competitor harboring four copies of the CTE sequence have been used either in human or murine cells lines, infected or transfected with MLV. All results concur that the Tap pathway is strictly required for MLV expression (Fig. 2). Indeed, over-expression of Tap protein increased the expression of MLV chimeric mRNA [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] and blocking the Tap pathway induced a drastic decrease in Gag and Env protein levels. [START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF][START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] The effect of Tap pathway inhibition on RNA export was very difficult to monitor due to the rapid degradation of the RNA retained in the nucleus. [START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF][START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] Thus, detection of nuclear retention of blocked unspliced RNA was made only possible when using a very sensitive RNA imaging method with single molecule sensitivity. This microscopy analysis allowed to image unspliced RNA from 8 hours after Tap-inhibition. [START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF] Furthermore, the interaction between Tap and the MLV RNAs was observed by co-immunoprecipitation experiments. Spliced and unspliced RNA were complexed with the cellular Tap protein in infected human cells [START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] or transfected murine cell lines. [START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF] In conclusion, in contrast to HIV-1, spliced and unspliced MLV RNA take the same route to reach the cytoplasm in order to be translated. It is not known how MLV discriminates between spliced and unspliced viral RNAs. Also, whether the same pathway is used to export the two different pools of unspliced viral RNA (to be translated or packaged) remains to be determined.

Tap-cofactors involved in MLV RNA export

The export of unspliced MLV RNA mediated by Tap seems to require several co-factors (Fig. 2). Sakuma et al. highlighted the requirement of the three factors UAP56, THOC5 and THOC7. [START_REF] Sakuma | Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts[END_REF] THOC5 and THOC7 are two proteins from the THO complex recruited onto the mRNA during splicing and able to recruit UAP56. [START_REF] Dufu | ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex[END_REF] UAP56 is an RNA helicase of the DEAD-Box family that plays a critical role in nuclear export via its ability to recruit the Aly/Ref protein onto the mRNA, which itself is needed to recruit the Tap-p15 heterodimer. [START_REF] Gatfield | The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila[END_REF][START_REF] Zhang | Crystal structure of the C-terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants[END_REF] Interaction of Aly/Ref and THOC5 with Tap induces a conformational change of Tap resulting in a better interaction with the mRNA. [START_REF] Viphakone | TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export[END_REF] Interestingly, the study by Sakuma et al. revealed that although UAP56, THOC5 and THOC7 are required for the export of MLV unspliced RNA, Aly/Ref is dispensable (Fig. 2). The involvement of other proteins of the THO complex (THOC1, THOC2, THOC6) as well as the Cip29 protein has been assessed and the results showed that these proteins are not involved in the export of MLV RNA. Interestingly, the involvement of the co-factors are different for the MLV spliced and unspliced RNA. Indeed, although the UAP56 is necessary for the export of both spliced and unspliced RNA, THOC5 and THOC7 are only needed for the export of unspliced RNA. [START_REF] Sakuma | Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts[END_REF] Thus, although the MLV spliced and unspliced RNA take the same route to be exported from the nucleus, the RNP formed are different suggesting that some partners could be involved in the balance between export and splicing.

It was also demonstrated that MLV unspliced RNA is sensitive to the presence of the cellular SRp20 protein. SRp20 shuttles between the nucleus and the cytoplasm and promotes nuclear export and translation of cellular mRNAs by recruiting Tap/p15 on mRNAs. [START_REF] Ote | Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway[END_REF][START_REF] Huang | Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA[END_REF][START_REF] Sanford | A novel role for shuttling SR proteins in mRNA translation[END_REF] SRp20 was shown to serve as a bridge between Tap/p15 and the MLV unspliced RNA. [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] Moreover, SRp20 increases the binding of polysomes on the Gag mRNA in a Tap-dependent manner, thus linking nuclear export and translation. [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] These results are apparently not in agreement with the requirement of THOC5 and THOC7 but different binding sites could be present on the MLV unspliced RNA and could independently recruit Tap/p15 via some adaptors like SRp20 or THOC proteins (Fig. 2). Moreover, it is not excluded that SRp20 and THOC act in concert in the same export pathway.

MLV cis-acting RNA sequences

Retroviral unspliced RNA harbors particular cis-regulatory RNA elements that contribute to the recruitment of cellular nuclear export factors. For instance, the RRE present in the env sequence of HIV-1 is promoting the nuclear export of the FL RNA via the CRM1-dependent pathway. For the simple retrovirus MPMV, the FL RNA export is mediated via the binding of the CTE motif to Tap. Altogether, these studies reveal that retroviral cis-export elements usually localize at the 3' end of the viral FL RNA. However, the identification of the nuclear export signals of MLV appears more complex since the presence of multiple cis-export elements have been reported throughout the RNA, from its 5' to 3' regions (Table 1). Note that the species context used to study MLV should be taken into account since mechanisms of cellular pathways may differ from human to murine cells ( [START_REF] Zheng | Human p32 protein relieves a post-transcriptional block to HIV replication in murine cells[END_REF][START_REF] Swanson | Retroviral mRNA nuclear export elements regulate protein function and virion assembly[END_REF][START_REF] Sherer | Matrix mediates the functional link between human immunodeficiency virus type 1 RNA nuclear export elements and the assembly competency of Gag in murine cells[END_REF] and personal data).

-The R stem-loop. The importance of the R sequence present in the 5' UTR of the MLV SL3 strain was first reported by Cupelli and colleagues. By using a construct containing a chloramphenicol acetyltransferase (CAT) reporter placed under the control of both SL3 U3 and the first 32 nt of the R region, they showed that the presence of R enhanced CAT activity in mouse cell lines whereas this CAT activity appeared more variable in human cell lines. [START_REF] Cupelli | The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression[END_REF][START_REF] Cupelli | Transcriptional initiation and postinitiation effects of murine leukemia virus long terminal repeat R-region sequences[END_REF] The first 28 nt of R is predicted to form a stem loop, called RSL. Maintaining the secondary structure of the RSL appears to be important to enhance the CAT activity of the construct. [START_REF] Cupelli | The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression[END_REF] The RSL stemloop is present in both unspliced and spliced RNA. Importantly, RSL is highly conserved among type-C retroviruses [START_REF] Cupelli | The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression[END_REF][START_REF] Mougel | Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization[END_REF] and can also form inside the R sequence of the Moloney strain (Mo-MLV) (Fig. 3A). [START_REF] Mougel | Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization[END_REF] Indeed, MoMLV RSL can substitute the SL3 RSL activity. [START_REF] Cupelli | The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression[END_REF] Then, the role of this RSL was studied in the presence or the absence of an intronic and exonic sequence by using the same CAT reporter assays and revealed the importance of the R sequence in the expression of introncontaining reporter RNA. [START_REF] Cupelli | The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression[END_REF] The role of the RSL was later supported by experiments conducted with the full-length SL3 MLV. Deletion or mutation of the RSL in this viral context impaired the cytoplasmic accumulation of unspliced gRNA in murine and human cells lines. [START_REF] Trubetskoy | R region sequences in the long terminal repeat of a murine retrovirus specifically increase expression of unspliced RNAs[END_REF] Interestingly, all these studies showed that mutation or deletion of the RSL impacted Gag production, suggesting that the export driven by R targets the unspliced RNA to the translation machinery.

-The Psi region. The Psi region is mainly known for its role in RNA dimerization [START_REF] Tapia | Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374)[END_REF][START_REF] Prats | cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA[END_REF][START_REF] Rein | Take two[END_REF] and RNA packaging in new viral particles. [START_REF] Mougel | Cis-active structural motifs involved in specific encapsidation of Moloney Murine Leukemia Virus RNA[END_REF][START_REF] Mann | Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs[END_REF][START_REF] Mann | Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus[END_REF][START_REF] Rein | Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus[END_REF] Psi is located at the 5' end of the genome, 350nucleotide downstream of the 5' splice donor site (SD), and thus it is removed during the canonical splicing generating the non-packageable env RNA (Fig. 1). The Psi region forms an independent and highly structured domain including four stem-loops (called A, B, C and D) [START_REF] Tounekti | Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA[END_REF][START_REF] Miyazaki | An RNA structural switch regulates diploid genome packaging by Moloney murine leukemia virus[END_REF][START_REF] Souza | Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition[END_REF] (Fig. 3B). In 2005, two independent studies claimed a role for Psi in the nuclear export of FL RNA (Fig. 3). [START_REF] Smagulova | The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA[END_REF][START_REF] Basyuk | The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs[END_REF] The A-D stem-loops were deleted independently or in combination in Mo-MLV-based constructs and the RNA localization was monitored by subcellular fractionation followed by RT-qPCR as well as fluorescence microcopy (FISH). Both teams showed that ABCD hairpins promote the nuclear export of the viral transcripts in murine NIH3T3 cells and human HT-Fly cells. [START_REF] Smagulova | The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA[END_REF][START_REF] Basyuk | The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs[END_REF] The role of Psi in FL RNA export was confirmed by competition experiments in which the overexpressed Psi sequence competed with the unspliced MLV FL transcript for the nuclear export in MLV-infected cells. [START_REF] Smagulova | The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA[END_REF] Altogether, these studies revealed a new function for the four Psi stemloops in the transport of intron-containing viral RNAs from the nucleus to the cytoplasm.

-The pol coding sequence. Several studies have reported the contribution of coding sequences located in the pol gene. MLV or XMRV gag gene is poorly expressed under the control of the natural LTR or a CMV promoter whereas gag-pol gene is efficiently expressed, [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF][START_REF] Pilkington | Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element[END_REF] suggesting a putative role of the pol sequence in the expression of viral transcripts. Pilkington et al. worked with the complete genome of Mo-MLV or XMRV. [START_REF] Pilkington | Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element[END_REF] They deleted env or pol separately or env and pol together and observed in human cells that pol is required for Gag production. They defined a region of 1467nt in Mo-MLV (RT and IN) which was called post-transcriptional element (PTE). Insertion of PTE in the intron of poorly expressed reporter genes known to depend on post-transcriptional regulation (i.e. HIV Gag or CAT genes in respectively pNLgag and pDM138 plasmids), enhances the expression of the respective gene reporter. The PTE is a highly complex RNA structure with seven stem-loops (SL1 to SL7). A major role has been ascribed to SL1 and SL7 in the activity of the PTE, suggesting the presence of a bipartite signal for the PTE function (Fig. 3C). The fact that SL2 contains another cis-sequence identified by another group, [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] reinforces the importance of the PTE. Although SL2 is dispensable for the function of the XMRV PTE, [START_REF] Pilkington | Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element[END_REF] its role remains to be studied in the context of Mo-MLV.

Bartels and Luban identified two sequences in XMRV and MLV pol gene, which independently enhanced the expression of Gag (2232nt-3456nt and 4876nt-5199nt in XMRV) in human cells. [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] These sequences increased i) mRNA stability, ii) mRNA association to the polyribosomes and iii) mRNA nuclear export. From the first sequence, Luban's team predicted a hairpin of 100nt similar to the CTE cis-element present in MPMV, and named it γ-CTE (Fig. 3D). In particular, γ-CTE and CTE share a similar AAGACA loop that specifically recruits the cellular Tap factor to promote nuclear export of viral transcripts. [START_REF] Gruter | TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus[END_REF] The study also shows that both MLV gag and pol sequences are also sensitive to the presence of SRp20 protein. This cellular factor links nuclear export and translation by increasing the binding of polysomes to Gag mRNA in a Tap-dependent manner. [START_REF] Bartels | Gammaretroviral pol sequences act in cis to direct polysome loading and NXF1/NXT-dependent protein production by gag -encoded RNA[END_REF] More recently, another cis-element of 70nt was identified in the pol gene and called CAE for cytoplasmic accumulation element (Fig. 3). [START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] This CAE sequence is conserved among the gamma-retroviruses and is predicted to fold into 3 major stem-loops (Fig. 3E). Nine nucleotides (GGAAAGGAC) of XMRV CAE sequence are essential but not sufficient for the CAE function (Fig. 3E). Both MoMLV and XMRV CAE promote the nuclear export of viral transcript without affecting the stability, splicing or translation efficiency of the transcripts in human cell lines. Indeed, the expression of an HIV Gag construct became Rev independent when the CAE was inserted downstream the gag gene. In addition, the study revealed that MLV and XMRV CAE are both able to specifically interact with the nuclear export factor Tap triggering export of both spliced and unspliced viral RNAs. [START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] Finally, a sequence upstream the SA site in the integrase gene, formed by 237bp (5119-5355 in MLV), has been identified as a cis-element promoting the specific expression of the unspliced RNA in a position-independent manner. [START_REF] Hoshi | cis-Elements involved in expression of unspliced RNA in Moloney murine leukemia virus[END_REF] This cis-element may play a role in FL RNA export, but this hypothesis was not assessed in the study and remains to be elucidated.

-The U3 region. A deletion within the U3 region is commonly used in the design of MLVbased vectors to prevent the expression of the full-length RNA by the LTR promoter. [START_REF] Maetzig | Gammaretroviral vectors: biology, technology and application[END_REF] A recent study showed that the nuclear cytosolic levels of RNA transcripts decreased when most of the U3 region was deleted (381 nt). [START_REF] Volkova | The U3 region of Moloney murine leukemia virus contains position-independent cis-acting sequences involved in the nuclear export of full-length viral transcripts[END_REF] The U3 deletion was performed in constructs containing the 5' LTR, 3' LTR, and Psi domain of MLV and a reporter gene (GFP or neomycin). This effect was partly due to the incorrect 3' end RNA processing but also to a default in unspliced RNA nuclear export. This defect was complemented by transferring the U3 region to another position within the vector, highlighting the presence of a position independent cis-acting sequence within the U3 region (Fig. 3).

Discussion

Viral FL RNA metabolism and transport are highly regulated processes relying on cellular pathways. MLV does not encode regulatory proteins unlike complex retroviruses and requires specialized interactions with cellular machinery to ensure splicing, stabilization and export of its viral RNA. In particular, splicing and export are closely linked processes resulting in the presence of a significant amount of unspliced transcripts in the cytosol. Thus, it is important to study the nuclear export of viral RNA while maintaining the intron-containing feature of the RNA (i.e. functional splice sites). Note that several aberrant splice sites have been reported in MLV-based vectors in which splicing was shown to depend on sequences located in gag (1183-1649 nt) and pol (5140-5400 nt) genes [START_REF] Machinaga | Polyadenylation of Friend murine leukemia virus env-mRNA is affected by its splicing[END_REF][START_REF] Yamamoto | Friend murine leukemia virus A8 regulates Env protein expression through an intron sequence[END_REF] . None of these sequences are present in the different cis-elements described as facilitating export of the MLV unspliced RNA. However, it cannot be excluded that in native context of the complete virus, mutating export signals located in intronic region may deregulate splicing and indirectly affect RNA export. Furthermore, studies of HIV replication in murine cells have revealed species-specific defects at the level of splicing, [START_REF] Zheng | Human p32 protein relieves a post-transcriptional block to HIV replication in murine cells[END_REF] nuclear export or assembly efficiencies. [START_REF] Swanson | Retroviral mRNA nuclear export elements regulate protein function and virion assembly[END_REF][START_REF] Sherer | Matrix mediates the functional link between human immunodeficiency virus type 1 RNA nuclear export elements and the assembly competency of Gag in murine cells[END_REF] These studies reinforce the notion that post-transcriptional regulation of retroviral gRNA requires specific cellular factors that are not functionally equivalent in murine and human cells. Therefore, it would be important to study MLV nuclear export within murine cell lines (Table 1) and using reporter constructs harboring MLV splice sites. Unfortunately, it is difficult to monitor the transport of the entire replication-competent MLV, since the effects of any mutation/deletion in the sequence of this multifunctional gRNA would make the interpretation ambiguous.

While MLV is among the first retroviruses discovered, we waited until 2014 to find out that MLV recruits the Tap-dependent export pathway, mainly due to the technical difficulties associated with the detection of a nuclear retention of MLV RNA when inactivating the Tap pathway. In contrast, clear and convincing nuclear accumulation of MPMV [START_REF] Hull | Analysis of synergy between divergent simple retrovirus posttranscriptional control elements[END_REF] was reported when inactivating CTE. What makes MLV so special? Actually, blocking the Tap-pathway systematically and specifically resulted into a fast and drastic degradation accounting for undetectable levels of MLV RNA in the nucleus. [START_REF] Pessel-Vivares | MLV requires Tap/NXF1-dependent pathway to export its unspliced RNA to the cytoplasm and to express both spliced and unspliced RNAs[END_REF][START_REF] Sakuma | Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts[END_REF] In addition, while other retroviruses harbor an export signal at their 3'end, MLV has the particularity to contain cis-acting signals throughout the molecule, at its ultimate 3' end (U3), at its 5' end (R, Psi) as well as in the coding sequence of pol (PTE, γ-CTE, and CAE) (Table 1). This dispersed pattern of cis-acting signals could suggest the existence of a conformational determinant but the complete RNA structure has not been solved yet and this hypothesis would require further investigation. Moreover, it is noticeable that Psi and CAE are particularly close to SD and SA splicing sites, respectively (Fig. 3), suggesting that splicing and export, two competitive events, could be regulated in a spatial way. Such competition could be crucial for the stability of the unspliced RNA as shown in HIV and MPMV studies. [START_REF] Schambach | Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors[END_REF][START_REF] Hammarskjold | Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron[END_REF] It might induce the recruitment of different co-factors which would explain the difference of the RNP on the spliced and unspliced RNA. [START_REF] Sakuma | Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts[END_REF] The fact that the same highly structured 5' region, Psi, also has the particularity to serve for both export and packaging functions suggests that the two processes might be intimately related to each other and might influence the cytosolic RNA fate.

Since Psi and PTE determinants are only present in unspliced FL RNA (Fig. 3), they could be recognized by Tap cofactors to specifically export these RNA species into the cytosol. This could explain how MLV discriminates its spliced and unspliced RNAs that are all dependent of the Tap export pathway.

Thus, further studies are required to better understand the relationship between the different cis-export signals and the other post-transcriptional regulation steps. To our knowledge, there is no inhibitory sequences identified in MLV RNA (unlike HIV-1 RNA) that could mediate the nuclear retention of the unspliced MLV RNA. Altogether, a better understanding of this particular mechanism might unravel new lines in these complex processes. Finally, export of intron-containing RNA represents a key step of retroviral infection that provides an interesting model to better understand the post-transcriptional regulation of the mammalian genes with persistence of introns leading to their expression in some cancers. the γ-CTE (D) within the PTE region harbors a short sequence AAGACA (in red) also found in the MPMV CTE and CAE which folds in 3 stem-loops (E), contains a 9nt core sequence (in red) necessary but not sufficient for the function of this motif. At the 3' end the U3 region is required for the specific export of unspliced RNA however no precise motif has been highlighted in this region yet.

Pathway/Factors

Figure 1 :

 1 Figure 1: The MLV genome and subgenomic SD' and Env mRNAs.The primary transcript(8.3kb RNA), named FL RNA Gag-Pol , includes the three major genes: gag gene components, including the matrix (MA), p12, capsid (CA), and nucleocapsid (NC), the pol gene components, including the protease (PR), reverse transcriptase (RT) and integrase (IN) as well as the env sequence, coding for the surface (SU) and transmembrane (TM) components of viral envelope glycoprotein. A subset of this FL RNA goes through splicing and generates the SD' RNA p50 (4.4Kb) that derives from an alternative donor site (SD') within CA to the canonical splice acceptor site (SA) and codes for the p50 protein. A second fully-spliced RNA is generated, SD RNA Env (3Kb) by splicing between the donor site (SD at position 205) and the acceptor site (SA at position 5490) encoding Env proteins.

Figures 2 :

 2 Figures 2: MLV FL RNA is exported from the nucleus by Tap. (A) Export of cellular mRNA viaTap-dependent pathway. The major mRNA export factor Tap, coupled to its heterodimeric partner, p15, is required for formation of an export-competent mRNP including the major adaptors: the TREX/THO complex and the SRp20 protein recruited cotranscriptionally. For simplicity, not all adaptors have been depicted. The factors shared with the MLV RNA export are in red. (B) In contrast with MPMV FL RNA, the interaction between MLV FL RNA and Tap seems indirect and requires the intervention of cellular co-factors (blue pathway). Some subunits from the TREX complex (THOC7, THOC5 and UAP56) and SRp20 proteins (in red) have been identified to promote this interaction. In contrast, only UAP56 has been reported to be involved in export of MLV spliced SD RNA (green pathway). The crosses indicate the dispensable factors for viral RNA export. The question mark indicates that SRp20 contribution to the spliced viral RNA export has not been analyzed yet.

Figure 3 :

 3 Figure 3: cis-acting motifs on MLV genome. Different sequences throughout the Mo-MLV RNA have been reported to be involved in FL RNA export. From 5' to 3': The RSL (R stem-loop), a signal made of 28nt folding in one stem-loop as pictured in (A); the Psi encapsidation signal which folds in four stem-loops (B); PTE which covers a large region in pol gene and contains seven stem-loops with the first and seventh motif (SL1 & SL7 depicted in C) of special importance;

This work was supported by institutional grants from the CNRS and the University of Montpellier. LPV was supported by fellowships from La Ligue contre le Cancer and the Ministère de l'Enseignement Supérieur et de la Recherche. Many thanks to C. Swanson and M. Ferrer for critical reading of the manuscript.


Anaïs Gouin

Anthony Bretaudeau

Karine Labadie

Jean-Marc Aury

Emmanuelle D'alençon

Claire Lemaitre

Fabrice Legeai1

Motivation : Some heterozygous regions have a signif i cant divergence between the two haplotypes and the assembly process can lead to the construction of two different contigs, instead of one consensus sequence.

Objective : Set up a strategy to detect and correct false duplications in alreadybuilt assemblies.

Intersection of the annotation

New gene predictions

Alignments of impacted genes onto scaffolds

Case A Gene cannot be aligned

  smallest scaffold is deleted Borders : the scaffolds are linked by their extremities, keeping the allele present on the longest scaffold of the pair

  strains: the genome of the Spodoptera frugiperda "rice variant" has been sequenced and assembled with Platanus, and annotated with the help of Maker. The comparison of the two draft sequences leads to the identification of about 16,000 pairs of orthologous genes, and thousands of segmental rearrangements (deletions, duplications or inversions).

Improvement of the assembly of heterozygous genomes of non-model organisms, a case study of the genomes of two Spodoptera frugiperda host strains APPLICATION : Spodoptera frugiperda genomes

			Initial assembly Allpaths	Platanus assembly	Corrected assembly
		Total size (Mb)	526.0	470.1	437.9
		Nb. scaffolds	48 272	41 633	41 577
		N50 (bp)	39 593	75 578	52 781
		Total Gap length	13.6 Mb	56.1 Mb	11.4 Mb
	BUSCO* stats	No hit Single hit Multi hit	14 1497 782	9 2047 237	15 1885 393


Mahyar Shahsavari

Philippe Devienne

Pierre Boulet

N2S3, a Simulator for the Architecture Exploration of Neuromorphic Accelerators

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

• Using Address Event Representation (AER)

• Using Scala programming language

• Akka actor library for parallel processing and concurrency

• A synapse/neuron is presented by an Actor

• A spike is a message for the Actors

  memristor structure, b) current versus voltage diagram, sinusoidal input with an amplitude of 1.5v, different frequencies, R ON =100Ω, R OFF =15kΩ, D=10nm, μ v =10 -10 cm 2 s -1 V -1 .

LIF model for Neuron