The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing

Myotonic dystrophy type 1 (DM1) is a genetic disorder linked to a (CTG)n repeat expansion in the 3′ untranslated region of the DMPK gene. Upon transcription in the nucleus, the CUG repeats form a stable RNA stem-loop that sequesters the RNA-binding protein MBNL1 from its normal function in the cell. MBNL1 regulates the alternative splicing of many pre-mRNAs, and upon MBNL1's sequestration, the alternative splicing of many genes is mis-regulated, leading to disease symptoms. MBNL1 is known to bind directly to at least 3 of the pre-mRNAs that it regulates, but how MBNL1 binding mechanistically regulates alternative splicing is unclear. Here, we demonstrate that MBNL1 controls the splicing of exon 5 in the cardiac troponin T (cTNT) pre-mRNA by competing directly with the essential splicing factor U2AF65 for binding at the 3′ end of intron 4. When U2AF65 is prevented from binding to the pre-mRNA, the U2 snRNP can no longer be recruited and the following exon is skipped. Furthermore, MBNL1 and U2AF65 appear to compete by binding to mutually exclusive RNA structures. When bound by splicing factors, the 3′ end of intron 4 can form either a stem-loop or a single-stranded structure. MBNL1 binds a portion of the intron as a stem-loop, whereas U2AF65 binds the same region in a single-strand structure. Mutations that strengthen the stem-loop decrease U2AF65 binding affinity and also repress exon 5 inclusion, independently of MBNL1. Thus, U2AF65 binding can be blocked either by MBNL1 binding or by the stabilization of RNA secondary structure

Magnetic textures and singularities in ferri ferromagnetic multilayers

The stacking of ferrimagnetic and ferromagnetic films leads to a competition between magnetic interactions that can stabilize Bloch points and other singularities at the interfaces. In this work, GdxCo1 x NdCo5 GdyCo1 y tri layers, with different thicknesses, have been prepared. By tuning the stoichiometry of the Gd Co outer layers, the Co magnetic sublattice dominates in one layer, whereas the opposite side is Gd dominated. In this way, in depth magnetization domain walls can be created due to the balance between exchange and magnetostatic interactions. In addition, the weak perpendicular magnetic anisotropy of the Nd Co central layer induces a stripe domain pattern, supporting the formation of Bloch points, meron like textures, and vortex antivortex pairs. The interaction between both types of structures, in depth domain walls and magnetic singularities, has been studied by a combination of macroscopic Magneto Optical Kerr Effect and Vibrating Sample Magnetometry measurements with microscopic Magnetic Force Microscopy and element selective magnetic Transmission soft X ray Microscopy imaging. The results confirm marked changes in the magnetic properties of the trilayers, as compared with those single NdCo5 films, and the formation of complex magnetic textures, associated with partial magnetization reversal, where magnetic singularities are forme

Transportin-SR2 mediates nuclear import of phosphorylated SR proteins

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin β family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains

Imp3p and Imp4p mediate formation of essential U3–precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA

In eukaryotes, formation of short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor rRNA (pre-rRNA) at multiple sites is a prerequisite for three endonucleolytic cleavages that initiate small subunit biogenesis by releasing the 18S rRNA precursor from the pre-rRNA. The most likely role of these RNA duplexes is to guide the U3 snoRNA and its associated proteins, designated the small subunit processome, to the target cleavage sites on the pre-rRNA. Studies by others in Saccharomyces cerevisiae have identified the proteins Mpp10p, Imp3p, and Imp4p as candidates to mediate U3–pre-rRNA interactions. We report here that Imp3p and Imp4p appear to stabilize an otherwise unstable duplex between the U3 snoRNA hinge region and complementary bases in the external transcribed spacer of the pre-rRNA. In addition, Imp4p, but not Imp3p, seems to rearrange the U3 box A stem structure to expose the site that base-pairs with the 5′ end of the 18S rRNA, thereby mediating duplex formation at a second site. By mediating formation of both essential U3–pre-rRNA duplexes, Imp3p and Imp4p may help the small subunit processome to dock onto the pre-rRNA, an event indispensable for ribosome biogenesis and hence for cell growth

Bendamustine as part of conditioning of autologous stem cell transplantation in patients with aggressive lymphoma: a phase 2 study from the GELTAMO group

We conducted a phase 2 trial to evaluate the safety and efficacy of bendamustine instead of BCNU (carmustine) in the BEAM (BCNU, etoposide, cytarabine and melphalan) regimen (BendaEAM) as conditioning for autologous stem-cell transplantation (ASCT) in patients with aggressive lymphomas. The primary endpoint was 3-year progression-free survival (PFS). Sixty patients (median age 55 [28-71] years) were included. All patients (except one who died early) engrafted after a median of 11 (9-72) and 14 (4-53) days to achieve neutrophil and platelet counts of >0.5 x 10(9) /l and >20 x 10(9) /l, respectively. Non-relapse mortality at 100 days and 1 year were 3.3% and 6.7%, respectively. With a median follow-up of 67 (40-77) months, the estimated 3-year PFS and overall survival (OS) were 58% and 75%, respectively. Patients in partial response at study entry had significantly worse PFS and OS than patients who underwent ASCT in complete metabolic remission, and this was the only prognostic factor associated with both PFS (Relative risk [RR], 0.27 [95% confidence interval {CI} [0.12-0.56]) and OS (RR, 0.40 [95% CI 0.17-0.97]) in the multivariate analysis. BendaEAM conditioning is therefore a feasible and effective regimen in patients with aggressive lymphomas. However, patients not in complete metabolic remission at the time of transplant had poorer survival and so should be considered for alternative treatment strategies