Intra-nuclear RNA trafficking: insights from live cell imaging

Despite recent advances, the mechanisms of RNA movements and targeting within the nucleus are still mysterious. While diffusion appears to play a crucial role in nuclear dynamics and RNA transport, some data argue for a model in which diffusion is controlled, at least in part, by the organization of the nucleus in well-defined compartments. Much of the recent progress is based on imaging technologies, and this review will first present them in some detail. We will then summarize studies that analyzed nuclear movements of both polyadenylated RNA and box C/D snoRNP. Indeed, this latter model has already brought a number of interesting results. We will finally present some of our original results on box C/D snoRNA transport.     

The influence of females on the initiation of female-to-male sex change in a coral reef fish

In the protogynous coral reef fish Anthias squamipinnis (Peters), all males are sex-reversed females. A sexually mature female can be induced to change sex by removing a male from her social group. The influence of non-sex-changing females on the initiation of sex change was evaluated in 109 social groups in the Gulf of Eilat. When the male and largest female were removed from each of 12 single-male groups, the second-largest female changed sex in 9 groups. This result distinguished between two behavioral hypotheses suggested by previous work and made it tenable that a particular behavioral measure, the profile of behavior-received, that depends on adult females, is critical to the initiation of sex change. This species forms all-female groups as well as bisexual groups. All-female groups can be expected to have some mechanism for the production of a male. The removal of the largest female from each of 8 all-female groups failed to induce sex change in any group. The dominant female in these groups thus does not function in the same way as does the male in bisexual groups, at least in terms of the initiation of sex change. Following the removal of the male from each of 8 bisexual groups containing five or fewer adult females, a female changed sex in only 4 groups. This 50% incidence of sex reversal was lower than the 77–80% incidence in control groups containing more than five adult females. Data suggest that a minimum of four adult females is probably required for the probability of sex change after male removal to equal 75%.     

The packaging signal of MLV is an integrated module that mediates intracellular transport of genomic RNAs

Packaging of MLV genomes requires four cis-acting stem-loops. Stem-loops A and B are self-complementary and bind Gag in their dimeric form, while the C and D elements mediate loop-loop interactions that facilitate RNA dimerization. Packaging also requires nuclear export of viral genomes, and their cytoplasmic transport toward the plasma membrane. For MLV, this is mediated by Gag and Env, and occurs on endosomal vesicles. Here, we report that MLV Psi acts at several steps during the transport of genomic RNAs. First, deletion of stem-loop B or C leads to the accumulation of genomic RNAs in the nucleus, suggesting that these elements are involved in export. Second, in chronically infected cells, mutation of the C and D loops impairs endosomal transport. This suggests that RNA dimerization is essential for vesicular transport, consistent with its proposed requirement for Gag binding. Surprisingly, deletion of stem-loop A blocks vesicular transport, whereas removal of stem-loop B has no effects. This suggests that stem-loop A has unique functions in packaging, not predicted from previous in vitro analyses. Finally, in packaging cells that do not express any Psi-containing RNA, endosomal RNA transport becomes sequence-independent. This non-specific activity of Gag likely promotes packaging of cellular mRNAs.     

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.     

Mass spectrometry-based immuno-precipitation proteomics - the user's guide

Immuno-precipitation (IP) experiments using MS provide a sensitive and accurate way of characterising protein complexes and their response to regulatory mechanisms. Differences in stoichiometry can be determined as well as the reliable identification of specific binding partners. The quality control of IP and protein interaction studies has its basis in the biology that is being observed. Is that unusual protein identification a genuine novelty, or an experimental irregularity? Antibodies and the solid matrices used in these techniques isolate not only the target protein and its specific interaction partners but also many non-specific 'contaminants' requiring a structured analysis strategy. These methodological developments and the speed and accuracy of MS machines, which has been increasing consistently in the last 5 years, have expanded the number of proteins identified and complexity of analysis. The European Science Foundation's Frontiers in Functional Genomics programme 'Quality Control in Proteomics' Workshop provided a forum for disseminating knowledge and experience on this subject. Our aim in this technical brief is to outline clearly, for the scientists wanting to carry out this kind of experiment, and recommend what, in our experience, are the best potential ways to design an IP experiment, to help identify possible pitfalls, discuss important controls and outline how to manage and analyse the large amount of data generated. Detailed experimental methodologies have been referenced but not described in the form of protocols.     

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.