HIV-1 suppressive sequences are modulated by Rev transport of unspliced RNA and are required for efficient HIV-1 production.

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3' untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3' untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.

Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells.

BACKGROUND: The group I intron, a ribozyme that catalyzes its own splicing reactions in the absence of proteins in vitro, is a potential target for rational engineering and attracted our interest due to its potential utility in gene repair using trans-splicing. However, the ribozyme activity of a group I intron appears to be facilitated by RNA chaperones in vivo; therefore, the efficiency of self-splicing could be dependent on the structure around the insert site or the length of the sequence to be inserted. To better understand how ribozyme activity could be modulated in cultured mammalian cells, a group I intron was inserted into a short hairpin RNA (shRNA), and silencing of a reporter gene by the shRNA was estimated to reflect self-splicing activity in vivo. In addition, we appended a theophylline-binding aptamer to the ribozyme to investigate any potential effects caused by a trans-effector. RESULTS: shRNA-expression vectors in which the loop region of the shRNA was interrupted by an intron were constructed to target firefly luciferase mRNA. There was no remarkable toxicity of the shRNA-expression vectors in Cos cells, and the decrease in luciferase activity was measured as an index of the ribozyme splicing activity. In contrast, the expression of the shRNA through intron splicing was completely abolished in 293T cells, although the silencing induced by the shRNA-expressing vector alone was no different from that in the Cos cells. The splicing efficiency of the aptamer-appended intron also had implications for the potential of trans-factors to differentially promote self-splicing among cultured mammalian cells. CONCLUSIONS: Silencing by shRNAs interrupted by a group I intron could be used to monitor self-splicing activity in cultured mammalian cells, and the efficiency of self-splicing appears to be affected by cell-type specific factors, demonstrating the potential effectiveness of a trans-effector.

Expression of shRNA using intron splicing.

RNA is considered a highly promising candidate for several applications such as gene knock-down, gene repair and gene therapy, where double-stranded RNA and RNA with catalytic activity are key players. A group I intron, a ribozyme catalyzing its own splicing reactions in the absence of any proteins, has generated interest for its potential utility in gene repair using trans-splicing. On the other hand, the induction of small interfering RNA, via double-stranded RNA cleavage in short hairpin RNA (shRNA) by the RNase* *enzyme DICER is a convenient and powerful mechanism for gene silencing. We constructed shRNA expression vectors directed against Firefly luciferase, in which the loop region of the shRNA was interrupted by an intron. The decreased levels of luciferase activity were measured in cultured cells as an index of the ribozyme splicing activity.

Bhc-cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation.

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6-bromo-7-hydroxycoumarin-4-ylmethyl esters of cNMP (Bhc-cNMP) and their acetyl derivatives (Bhc-cNMP/Ac) as new caged second messengers. Irradiation of Bhc-cNMPs quantitatively produced the parent cNMPs with one-photon uncaging efficiencies (Phiepsilon) of up to one order of magnitude better than those of 2-nitrophenethyl (NPE) cNMPs. In addition, two-photon induced photochemical release of cNMP from Bhc-cNMPs (7 and 8) can be observed with the two-photon uncaging action cross-sections (delta(u)) of up to 2.28 GM (1 GM=10(-50) cm(4) s photon(-1)), which is the largest value among those of the reported Bhc-caged compounds. The wavelength dependence of the delta(u) values of 7 revealed that the peak wavelength was twice that of the one-photon absorption maximum. Bhc-cNMPs showed practically useful water solubility (nearly 500 microM), whereas 7-acetylated derivatives (Bhc-cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP-mediated transduction channels in newt olfactory receptor cells and cAMP-mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Nuclear translocation of mouse polycomb m33 protein in regenerating liver.

Immunoblots probed with an antibody to M33 protein, a homolog of Drosophila Polycomb, revealed that most M33 in adult mouse liver had a higher electrophoretic mobility than that in F9 embryonal carcinoma cells. High-mobility 60-kDa M33 localized in the cytoplasmic fraction of liver homogenates, and two less abundant 66- and 70-kDa species were detected in the nuclear fraction. Immunocytochemistry of freeze-substituted tissues showed a punctate pattern of immunofluorescence in the cytoplasm of hepatic parenchymal cells. Nuclear M33 isoforms treated with alkaline phosphatase had increased mobilities corresponding to cytoplasmic M33. In partially hepatectomized mice, nuclear M33 isoforms appeared after 48 h, near the time of maximum DNA synthesis as measured by bromodeoxyuridine incorporation. By 60 h, most M33 was in the form of these low-mobility species, and the pattern of immunofluorescence suggested the existence of chromatin-bound and free states of the protein in the nucleus. Thereafter, high-mobility 60-kDa M33 reappeared. The data are consistent with a phosphorylation-associated translocation mechanism that is a cell cycle-dependent.