Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N–terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N–terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N–terminal domain is reduced within the full-length molecule, indicating that the C–terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility

Exon, intron and splice site locations in the spliceosomal B complex

In recent years, electron microscopy (EM) has allowed the generation of three-dimensional structure maps of several spliceosomal complexes. However, owing to their limited resolution, little is known at present about the location of the pre-mRNA, the spliceosomal small nuclear ribonucleoprotein or the spliceosome's active site within these structures. In this work, we used EM to localise the intron and the 5′ and 3′ exons of a model pre-mRNA, as well as the U2-associated protein SF3b155, in pre-catalytic spliceosomes (i.e. B complexes) by labelling them with an antibody that bears colloidal gold. Our data reveal that the intron and both exons, together with SF3b155, are located in specific regions of the head domain of the B complex. These results represent an important first step towards identifying functional sites in the spliceosome. The gold-labelling method adopted here can be applied to other spliceosomal complexes and may thus contribute significantly to our overall understanding of the pre-mRNA splicing process

Proximity of conserved U6 and U2 snRNA elements to the 5′ splice site region in activated spliceosomes

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA–RNA interactions, little is known about the tertiary structure of this RNA–RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5′ end of a pre-mRNA intron, to map RNA–RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step

An SP1-like transcription factor Spr2 acts downstream of Fgf signaling to mediate mesoderm induction

Fgf signaling, mediated in part by the transcription factor Brachyury/Xbra/Ntl, plays important roles in mesoderm formation during the early development of vertebrate embryos. We have identified a zebrafish gene, spr2, which encodes a member of the Sp1-like transcription factor family. spr2 is expressed in both hypoblast and epiblast cells during late blastulation/early gastrulation, and in some mesodermal and neural tissues at later stages. Injection with spr2 mRNA enhances ntl expression and alleviates the inhibitory effect on ntl of XFD, a Xenopus dominant-negative FGF receptor. In contrast, morpholino- mediated knockdown of Spr2 activity inhibits ntl expression and reduces the inductive effect of Fgfs on ntl. We also demonstrate that Fgf signaling relays mesoderm induction activity of Nodal signaling and Spr2 is involved in this signal relay process. Furthermore, the correct spatial expression of spr2 requires Nodal, Fgf and Wnt signals. We suggest that expression of spr2 is an immediate-early response to mesoderm induction by Fgfs, which in turn regulates the expression of effector genes involved in the development of mesodermal tissues