Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.

In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions in vivo, creating complex regulatory networks where cooperation with regulatory proteins is necessary. We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.

The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific splicing decisions most likely result from differences in the concentration and/or activity of these proteins. However, large-scale data to systematically address this issue have just recently started to become available. Here we show that splicing factor gene expression signatures can be identified that reflect cell type and tissue-specific patterns of alternative splicing. We used a computational approach to analyze microarray-based gene expression profiles of splicing factors from mouse, chimpanzee and human tissues. Our results show that brain and testis, the two tissues with highest levels of alternative splicing events, have the largest number of splicing factor genes that are most highly differentially expressed. We further identified SR protein kinases and small nuclear ribonucleoprotein particle (snRNP) proteins among the splicing factor genes that are most highly differentially expressed in a particular tissue. These results indicate the power of generating signature-based predictions as an initial computational approach into a global view of tissue-specific alternative splicing regulation.

After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that Rce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmtdeficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 deficiency, with virtually all of the knockout embryos (Icmt؊/؊) dying by mid-gestation. An analysis of chimeric mice produced from Icmt؊/؊ embryonic stem cells showed that the Icmt؊/؊ cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt؊/؊ embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-L-cysteine). In addition, Icmt؊/؊ cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.After isoprenylation, Ras and other proteins that terminate with a CAAX 1 sequence undergo two additional C-terminal modifications (1). First, the last three amino acids of the protein (i.e. the -AAX) are released by an endoprotease associated with the endoplasmic reticulum (1-3). Second, the carboxyl group of the newly exposed isoprenylcysteine is methylated (4, 5). These post-isoprenylation processing steps may help target CAAX proteins to membrane surfaces within cells (1).The endoprotease and methyltransferase steps have attracted interest because they offer a potential means for modulating the activity of CAAX proteins, many of which participate in cell signaling (1). Several groups have hypothesized that inhibiting the endoprotease or the methyltransferase might retard the growth of tumors caused by mutation-activated Ras proteins (1, 2, 6, 7). At this point, however, testing such hypotheses appears to be a few years away. No specific high affinity inhibitors suitable for animal testing have been developed, either for the endoprotease or for the methyltransferase. Just as importantly, neither the spectrum of substrates nor the physiologic importance of the two processing steps has been explored fully. This is particularly the case for the methyltransferase.To define the physiologic relevance of the post-isoprenylation processing steps, our laboratory generated and characterized mice lacking the endoprotease Rce1 (8). Membranes from Rce1-deficient embryos and cells were completely unable to carry out the endoproteolytic processing of Ras and a host of other CAAX proteins. Surprisingly, the consequences of knocking out Rce1 in the mouse were relatively mild. Although most of the Rce1 knockout mice died bef...

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27 − γδ T cells) or interferon-γ (IFN-γ) (CD27 + γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory frames of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27 + γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27 − γδ T cells displayed permissive chromatin configurations at loci related to both the T H 17 and T H 1 subsets of helper T cells and differentiated into cells producing both IL-17 and IFN-γ in a tumor microenvironment.γδ T cells have emerged as key providers of interleukin 17 (IL-17) in various models of infection, inflammation and autoimmunity [1][2][3][4][5][6] . Antibody-mediated or genetic depletion of γδ T cells greatly reduces disease severity in IL-17-driven models of chronic inflammation [1][2][3][4]7 . Those results notwithstanding, many reports have made a compelling case for γδ T cells as the main producers of interferon-γ (IFN-γ) in both mice and humans 8 , which has been a major foundation for clinical trials targeting these lymphocytes in cancer immunotherapy 9 . Given the dual ability of γδ cells to produce IL-17 and IFN-γ, published work has aimed to contributed to designing the study and writing the manuscript; A.Q.G. helped to design and supervise the study; and B.S.-S. designed and supervised the study and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://www.nature.com/reprints/index.html. Europe PMC Funders GroupAuthor Manuscript Nat Immunol. Author manuscript; available in PMC 2016 April 18. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts identify markers that associate with functional attributes of mouse γδ T cells [10][11][12][13] . Expression of the costimulatory receptor CD27 segregates IL-17-producing (CD27 − ) γδ T cells and IFN-γ-producing (CD27 + ) γδ T cells in both naive and Plasmodium-infected C57BL/6 mice 10 . Moreover, the chemokine receptor CCR6, which is expressed exclusively on CD27 − γδ T cells, constitutes an additional marker for IL-17 + γδ T cells 13,14. Both CD27 + and CD27 − γδ T cell subsets show spontaneous cytokine secretion after activation, in contrast to the delayed differentiation of conventional CD4 + T cells of the T H 1 or T H 17 subset of helper T cells 15 . That finding is highlighted by the observation that 30-40% of peripheral γδ T cells freshly isolated from naive mice produce either IL-17 or IFN-γ after 3 h of restimulation in vitro 10 . Those functionally mature γδ T cell subsets are also found in the thymus, as early as the embryonic stages of mouse development [10][11][12]16 . Moreove...

On the path to successful immunotherapy of hematopoietic tumors, ␥␦ T cells offer great promise because of their human leukocyte antigen (HLA)-unrestricted targeting of a wide variety of leukemias/ lymphomas. However, the molecular mechanisms underlying lymphoma recognition by ␥␦ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to ␥␦ T cell-mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all V␥9 ؉ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by ␥␦ T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to ␥␦ T cell-based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials. (Blood. 2010;115:2407-2411)

Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.

Abundant interferon-g secretion, potent cytotoxicity, and major histocompatibility complex-independent targeting of a large spectrum of tumors make gd T cells attractive mediators of cancer immunotherapy. However, a better understanding of the molecular mechanisms involved in tumor cell recognition and gd T-cell activation is required to improve the limited success of gd T-cell-mediated treatments. Here, we review key advances in basic knowledge made over the past 3 years, and summarize the results of gd T-cell-based clinical trials concluded to date. We also highlight new research directions on the basis of the modulation of receptors that control the function of gd T cells.