The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.

The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.

Inhibition of gene expression in human cells through small molecule-RNA interactions

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from d-and l-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC50 of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

LvsA, a Protein Related to the Mouse Beige Protein, Is Required for Cytokinesis in DictyosteliumV⃞

We isolated a Dictyostelium cytokinesis mutant with a defect in a novel locus called large volume sphere A (lvsA). lvsA mutants exhibit an unusual phenotype when attempting to undergo cytokinesis in suspension culture. Early in cytokinesis, they initiate furrow formation with concomitant myosin II localization at the cleavage furrow. However, the furrow is later disrupted by a bulge that forms in the middle of the cell. This bulge is bounded by furrows on both sides, which are often enriched in myosin II. The bulge can increase and decrease in size multiple times as the cell attempts to divide. Interestingly, this phenotype is similar to the cytokinesis failure of Dictyostelium clathrin heavy-chain mutants. Furthermore, both cell lines cap ConA receptors but form only a C-shaped loose cap. Unlike clathrin mutants, lvsA mutants are not defective in endocytosis or development. The LvsA protein shares several domains in common with the molecules beige and Chediak–Higashi syndrome proteins that are important for lysosomal membrane traffic. Thus, on the basis of the sequence analysis of the LvsA protein and the phenotype of the lvsA mutants, we postulate that LvsA plays an important role in a membrane-processing pathway that is essential for cytokinesis.

Laser-mediated, site-specific inactivation of RNA transcripts

The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.

A 17-amino acid arginine-rich peptide from the bovine immunodeficiency virus Tat protein has been shown to bind with high affinity and specificity to bovine immunodeficiency virus transactivation response element (TAR) RNA, making contacts in the RNA major groove near a bulge. We show that, as in other peptide-RNA complexes, arginine and threonine side chains make important contributions to binding but, unexpectedly, that one isoleucine and three glycine residues also are critical. The isoleucine side chain may intercalate into a hydrophobic pocket in the RNA. Glycine residues may allow the peptide to bind deeply within the RNA major groove and may help determine the conformation of the peptide. Similar features have been observed in protein-DNA and drug-DNA complexes in the DNA minor groove, including hydrophobic interactions and binding deep within the groove, suggesting that the major groove of RNA and minor groove of DNA may share some common recognition features.