tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control.

CCA addition to the 3' end is an essential step in tRNA maturation. High-resolution crystal structures of the CCA enzymes reveal primary enzyme contact with the tRNA minihelix domain, consisting of the acceptor stem and T stem-loop. RNA and DNA minihelices are efficient substrates for CCA addition in steady-state kinetics. However, in contrast to structural models and steady-state experiments, we show here by single-turnover kinetics that minihelices are insufficient substrates for the Escherichia coli CCA enzyme and that only the full-length tRNA is kinetically competent. Even a nick in the full-length tRNA backbone in the T loop, or as far away from the minihelix domain as in the anticodon loop, prevents efficient CCA addition. These results suggest a kinetic quality control provided by the CCA enzyme to inspect the integrity of the tRNA molecule and to discriminate against nicked or damaged species from further maturation.

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling.

Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose-/N-acetylgalactosamine-specific-lectin (mMGL)/CD301, identified by the monoclonal antibody ER-MP23, as well as other macrophage markers. As expression of mMGL is induced by IL-4 or IL-13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin-1. Yet, as this expression profile was similar in IL-4 receptor alpha knockout mice, neither IL-4 nor IL-13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up-regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up-regulation when cultured in standard medium, but whole skin-conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin-draining lymph nodes, quickly down-regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL-4/IL-13 signaling.

Dendritic cells in vivo and in vitro.

Dendritic cells (DC) are crucial cells of the immune system, and bridge the essential connection between innate and adaptive immunity. They reside in the periphery as sentinels where they take up antigens. Upon activation, they migrate to lymphoid organs and present there the processed antigens to T cells, thereby activating them and eliciting a potent immune response. Dendritic cells are bone marrow-derived cells, still big controversies exist about their in vivo development. In vitro, DC can be generated from multiple precursor cells, among them lymphoid and myeloid committed progenitors. Although it remains unknown how DC are generated in vivo, studying the functions of in vitro generated DC results in fundamental knowledge of the DC biology with promising applications for future medicine. Therefore, in this review, we present current protocols for the generation of DC from precursors in vitro. We will do this for the mouse system, where most research occurs and for the human system, where research concentrates on implementing DC biology in disease treatments.

Macrophages and dendritic cells constitute a major subpopulation of cells in the mouse dermis.

Macrophages and dendritic cells (DC) in tissues with close contact to the environment are of essential importance in host defense and are therefore present in sizeable numbers. Therefore, it is surprising that mononuclear phagocyte populations of the dermis have rarely been investigated in a quantitative manner. In this study, we examined mouse dermal skin immunophenotypically and related the observed numbers of observed cells to the total number of nucleated cells. These analyses show that about 70% of all dermal cells represent CD45+ leukocytes. The vast majority of these cells (approximately 60% of total) expresses the mononuclear phagocyte markers mMGL (ER-MP23), F4/80 and CD11b. In addition, these cells show avid phagocytic capacity and thus are identified as dermal macrophages. Different subpopulations can be defined using markers such as sialoadhesin, ER-HR3 and mSIGN-R1 (ER-TR9). Interestingly, MHC class II expression differs significantly between dermal cells from ear versus back skin. Moreover, we have identified small populations of dermal DC and migrating Langerhans cells (together approximately 10% of total). In summary, our findings show that mononuclear phagocyte populations form the majority of dermal cells and thus have been clearly underestimated so far.

UBA1 and UBA2, two proteins that interact with UBP1, a multifunctional effector of pre-mRNA maturation in plants.

Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)(+) RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3' untranslated region (3'-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3'-UTRs and contributing to the stabilization of mRNAs in the nucleus.