A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5 end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3 splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3 splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.T he vast majority of eukaryotic mRNAs are translated via a mechanism in which the 40S ribosomal subunit engages the mRNA at its methylguanosine-capped 5Ј end (1). Upon associating with the transcript, these subunits are believed then to scan in the 5Ј to 3Ј direction for an appropriately situated AUG at which to begin translation (2, 3). A smaller number of mRNAs are translated by a 5Ј end-and cap-independent mechanism wherein ribosomes are recruited to the transcript at an interior location through an internal ribosome entry site (IRES).IRESs were first discovered in the picornaviruses encephalomyocarditis virus (EMCV) and poliovirus (4, 5). The RNA of these viruses possesses very long 5Ј UTRs bearing many unutilized upstream AUGs (uAUGs) and, unlike cellular mRNA, is uncapped (6). Soon after the identification of picornaviral IRESs, a number of cellular mRNAs were also reported to contain IRESs. To date, at least 85 cellular IRESs have been described (7). The experimental grounds on which proof of most cellular IRESs rest, however, has been the subject of dispute (8-10).A primary criticism of the data presented as establishing the existence of cellular IRESs concerns the plasmid-based dicistronic assay, the standard method of ascertaining IRES activity. In this assay, the candidate sequence is inserted between two reporter genes (5) so that both the upstream and downstream cistron are transcribed on the same RNA. If the test insert causes increased expression of the downstream cistron relative to the upstream cistron, the result is considered evidence for internal ribosome entry. However, the generation of even low levels of monocistronic RNAs from dicistronic constructs has the potential to falsely indicate IRES activity (8, 9, 11). One way that such RNAs could arise is through splicing of the dicistronic transcript due to the presence of a 3Ј splice site (ss) in the test sequence [see supporting information (SI) Fig....

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.Immune responses against donor (i.e, non-self) antigens are the primary cause of allogeneic transplant rejection resulting in graft failure. To date, the primary strategies for avoiding rejection have been to minimize antigenic differences between donor and recipient by matching HLA alleles and by subjecting the transplant recipient to potent immunosuppression. Of the several gene loci encoding HLA antigens, the most important for graft survival are the Class I antigens A and B and the Class II antigen DR. However, HLA is remarkably polymorphic, with more than 220, 460, 110, and 360 molecularly defined epitopes for HLA-A, B, C, and DR, respectively. Mismatching of the serological antigens is enough to increase the probability of graft failure in bone marrow transplantation, 1 and even when serology is matched, small NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular genetic differences may cause transplant rejection. 2 Similarly, recent studies have provided evidence that Class I HLA matching at the triplet level can benefit kidney transplant outcome, 3 and HLA mismatches are associated with a higher incidence of chronic rejection, a major cause...

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