Characterization of human placental glycosaminoglycans and regional binding to VAR2CSA in malaria infected erythrocytes.

Placental malaria is a serious problem in sub-Saharan Africa. Young women are particular susceptible to contracting this form of malaria during their first or second pregnancy despite previously acquired immunity from past infections. Placental malaria is caused by Plasmodium falciparum parasites expressing VAR2CSA on the erythrocyte surface. This protein adheres to a low-sulfated chondroitin sulfate-A found in placental tissue causing great harm to both mother and developing fetus. In rare cases, the localization of infected erythrocytes to the placenta can even result in the vertical transmission of malaria. In an effort to better understand this infection, chondroitin sulfate was isolated from the cotyledon part of the placenta, which should be accessible for parasite adhesion, as well as two non-accessible parts of the placenta to serve as controls. The placental chondroitin sulfate structures and their VAR2CSA binding were characterized. All portions of human placenta contained sufficient amounts of the appropriate low-sulfated chondroitin sulfate-A to display high-affinity binding to a recombinant truncated VAR2CSA construct, as determined using surface plasmon resonance. The cotyledon is the only placental tissue accessible to parasites in the bloodstream, suggesting it is the primary receptor for parasite infected red blood cells.

Genome-wide activation of latent donor splice sites in stress and disease.

Sequences that conform to the 5' splice site (5'SS) consensus are highly abundant in mammalian introns. Most of these sequences are preceded by at least one in-frame stop codon; thus, their use for splicing would result in pre-maturely terminated aberrant mRNAs. In normally grown cells, such intronic 5'SSs appear not to be selected for splicing. However, under heat shock conditions aberrant splicing involving such latent 5'SSs occurred in a number of specific gene transcripts. Using a splicing-sensitive microarray, we show here that stress-induced (e.g. heat shock) activation of latent splicing is widespread across the human transcriptome, thus highlighting the possibility that latent splicing may underlie certain diseases. Consistent with this notion, our analyses of data from the Gene Expression Omnibus (GEO) revealed widespread activation of latent splicing in cells grown under hypoxia and in certain cancers such as breast cancer and gliomas. These changes were found in thousands of transcripts representing a wide variety of functional groups; among them are genes involved in cell proliferation and differentiation. The GEO analysis also revealed a set of gene transcripts in oligodendroglioma, in which the level of activation of latent splicing increased with the severity of the disease.

A potential role for initiator-tRNA in pre-mRNA splicing regulation.

The translation initiator-tRNA plays a crucial role in the initiation of protein synthesis in both prokaryotic and eukaryotic cells, by employing specific base pairing between its anticodon triplet CAU and the general initiation codon AUG in the mRNA. Here we show that the initiator-tRNA may also act, in a manner that is independent of its role in protein translation, as a pre-mRNA splicing regulator. Specifically, we show that alternative splicing events that are induced by mutations in the translation initiation AUG codon can be suppressed by expressing initiator-tRNA constructs carrying anticodon mutations that compensate for the AUG mutations. These mutated initiator-tRNAs appeared to be uncharged with an amino acid. Our results imply that recognition of the initiation AUG sequence by the anticodon triplet of initiator-tRNA in its unloaded state plays a role in quality control of splicing in the cell nucleus by a yet unresolved mechanism. Identifying the initiator-tRNA as a transacting splicing regulator suggests a novel involvement of this molecule in splicing regulation and provides a critical step toward deciphering this intriguing mechanism.

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.

Glycosaminoglycans in infectious disease.

Glycosaminoglycans (GAGs) are complex carbohydrates that are ubiquitously present on the cell surface and in the extracellular matrix. Interactions between GAGs and pathogens represent the first line of contact between pathogen and host cell and are crucial to a pathogen's invasive potential. Their complexity and structural diversity allow GAGs to control a wide array of biological interactions influencing many physiological and pathological processes, including adhesion, cell-to-cell communication, biochemical cascades, and the immune response. In recent years, increasing evidence indicates an extraordinary role for GAGs in the pathogenesis of viruses, bacteria and parasites. Herein, we examine the interface between GAGs and different pathogens, and address the divergent biological functions of GAGs in infectious disease. We consider approaches to use this understanding to design novel therapeutic strategies addressing new challenges in the treatment of infectious diseases.