Alu Exonization Events Reveal Features Required for Precise Recognition of Exons by the Splicing Machinery

Despite decades of research, the question of how the mRNA splicing machinery precisely identifies short exonic islands within the vast intronic oceans remains to a large extent obscure. In this study, we analyzed Alu exonization events, aiming to understand the requirements for correct selection of exons. Comparison of exonizing Alus to their non-exonizing counterparts is informative because Alus in these two groups have retained high sequence similarity but are perceived differently by the splicing machinery. We identified and characterized numerous features used by the splicing machinery to discriminate between Alu exons and their non-exonizing counterparts. Of these, the most novel is secondary structure: Alu exons in general and their 5′ splice sites (5′ss) in particular are characterized by decreased stability of local secondary structures with respect to their non-exonizing counterparts. We detected numerous further differences between Alu exons and their non-exonizing counterparts, among others in terms of exon–intron architecture and strength of splicing signals, enhancers, and silencers. Support vector machine analysis revealed that these features allow a high level of discrimination (AUC=0.91) between exonizing and non-exonizing Alus. Moreover, the computationally derived probabilities of exonization significantly correlated with the biological inclusion level of the Alu exons, and the model could also be extended to general datasets of constitutive and alternative exons. This indicates that the features detected and explored in this study provide the basis not only for precise exon selection but also for the fine-tuned regulation thereof, manifested in cases of alternative splicing.     

Meiotic Recombination in Human Oocytes

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of “vulnerable” crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.     

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10⁻⁶) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.     

Applying unmixing to gene expression data for tumor phylogeny inference

Background  

While in principle a seemingly infinite variety of combinations of mutations could result in tumor development, in practice it appears that most human cancers fall into a relatively small number of "sub-types," each characterized a roughly equivalent sequence of mutations by which it progresses in different patients. There is currently great interest in identifying the common sub-types and applying them to the development of diagnostics or therapeutics. Phylogenetic methods have shown great promise for inferring common patterns of tumor progression, but suffer from limits of the technologies available for assaying differences between and within tumors. One approach to tumor phylogenetics uses differences between single cells within tumors, gaining valuable information about intra-tumor heterogeneity but allowing only a few markers per cell. An alternative approach uses tissue-wide measures of whole tumors to provide a detailed picture of averaged tumor state but at the cost of losing information about intra-tumor heterogeneity.     

Branch length estimation and divergence dating: estimates of error in Bayesian and maximum likelihood frameworks

Background  

Estimates of divergence dates between species improve our understanding of processes ranging from nucleotide substitution to speciation. Such estimates are frequently based on molecular genetic differences between species; therefore, they rely on accurate estimates of the number of such differences (i.e. substitutions per site, measured as branch length on phylogenies). We used simulations to determine the effects of dataset size, branch length heterogeneity, branch depth, and analytical framework on branch length estimation across a range of branch lengths. We then reanalyzed an empirical dataset for plethodontid salamanders to determine how inaccurate branch length estimation can affect estimates of divergence dates.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

Fluorescent non-imidazole histamine H3 receptor ligands with nanomolar affinities

Omega-piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH(3) receptor affinities with K(i) values ranging from 178 to 11nM. The new compounds may act as tools for identification and understanding of the binding site on the histamine H(3) receptor.     

Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.     

Detoxification of Multiple Heavy Metals by a Half-Molecule ABC Transporter,HMT-1, and Coelomocytes of Caenorhabditis elegans

Background

Developing methods for protecting organisms in metal-polluted environments is contingent upon our understanding of cellular detoxification mechanisms. In this regard, half-molecule ATP-binding cassette (ABC) transporters of the HMT-1 subfamily are required for cadmium (Cd) detoxification. HMTs have conserved structural architecture that distinguishes them from other ABC transporters and allows the identification of homologs in genomes of different species including humans. We recently discovered that HMT-1 from the simple, unicellular organism, Schizosaccharomyces pombe, SpHMT1, acts independently of phytochelatin synthase (PCS) and detoxifies Cd, but not other heavy metals. Whether HMTs from multicellular organisms confer tolerance only to Cd or also to other heavy metals is not known.

Methodology/Principal Findings

Using molecular genetics approaches and functional in vivo assays we showed that HMT-1 from a multicellular organism, Caenorhabditis elegans, functions distinctly from its S. pombe counterpart in that in addition to Cd it confers tolerance to arsenic (As) and copper (Cu) while acting independently of pcs-1. Further investigation of hmt-1 and pcs-1 revealed that these genes are expressed in different cell types, supporting the notion that hmt-1 and pcs-1 operate in distinct detoxification pathways. Interestingly, pcs-1 and hmt-1 are co-expressed in highly endocytic C. elegans cells with unknown function, the coelomocytes. By analyzing heavy metal and oxidative stress sensitivities of the coelomocyte-deficient C. elegans strain we discovered that coelomocytes are essential mainly for detoxification of heavy metals, but not of oxidative stress, a by-product of heavy metal toxicity.

Conclusions/Significance

We established that HMT-1 from the multicellular organism confers tolerance to multiple heavy metals and is expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are cell types that are affected by heavy metal poisoning in humans. We also showed that coelomocytes are involved in detoxification of heavy metals. Therefore, the HMT-1-dependent detoxification pathway and coelomocytes of C. elegans emerge as novel models for studies of heavy metal-promoted diseases.