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Small nuclear RNAs are involved in splicing pre-mRNA, while small nucleolar RNAs facilitate ribosome biogenesis. But these distinct particles may have more in common than was first apparent: some of their RNA components share a common RNA binding protein, a common RNA structure and perhaps a common origin.
The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.
In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. Here we show that AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, which are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. Our findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila.
A human small nucleolar RNA, identified previously in HeLa cells by anti-fibrillarin autoantibody precipitation and termed RNA X, has been characterized. It comprises two uridine-rich variants (148 and 146 nucleotides), which we refer to as snRNA U15A and U15B. Secondary structure models predict for both variants a U15-specific stem-loop structure, as well as a new structural motif that contains conserved sequences and can also be recognized in the other fibrillarin-associated nucleolar snRNAs, U3, U14, and RNA Y. The single-copy gene for human U15A has been found unexpectedly to reside in intron 1 of the ribosomal protein S3 gene; the U15A sequence appears on the same strand as the S3 mRNA and does not exhibit canonical transcription signals for nuclear RNA polymerases. U15A RNA is processed in vitro from S3 intron 1 transcripts to yield the correct 5' end with a 5'-monophosphate; the in vitro system requires ATP for 3' cleavage, which occurs a few nucleotides downstream of the mature end. The production of a single primary transcript specifying the mRNA for a ribosomal or nucleolar protein and a nucleolar snRNA may constitute a general mechanism for balancing the levels of nucleolar components in vertebrate cells.
Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.
We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.
We report here the isolation and primary structure of two genes encoding rat U3 small nuclear RNA. One of the genes encodes U3B RNA; the other encodes an RNA which is almost identical to U3A RNA. Both genes are expressed after microinjection into the nuclei of Xenopus laevis oocytes and can direct the accumulation of mature U3 RNA as well as longer transcripts which may be the U3 precursors. We have also isolated and sequenced four other regions of the rat genome homologous to U3 RNA. One of these almost certainly represents a second U3B gene; the other three are pseudogenes which appear to have been generated by the reverse flow of genetic information from U3 RNA back into the genome. Using genomic blotting techniques, we show that the rat U3 genes are present in only a few copies per haploid genome and are probably not closely linked to one another.
We have cloned a single copy gene that encodes a small nuclear RNA, designated snR3, from the yeast Saccharomyces cerevisiae. This RNA is highly conserved among fungi, and sequence and secondary structure analyses suggest that snR3 is analogous to mammalian U4 snRNA. To determine whether snR3 has an essential function in yeast, the gene (designated SNR3), was disrupted by replacing 35 nucleotides of coding sequences with 2.2 kb of yeast DNA containing the LEU2 gene. Since cells entirely lacking snR3 were expected to be inviable, the nonfunctional gene was used to replace one chromosomal copy in a diploid cell, and the diploid transformants were sporulated. Surprisingly, virtually all tetrads gave rise to four viable spores. Moreover, these haploid strains, which have been shown by DNA blot hybridization to lack an intact copy of the SNR3 gene, and which contain no detectable snR3 transcripts, are indistinguishable from their SNR3+ sister spores under a variety of growth conditions.
Although small interfering RNA (siRNA) is a potentially useful therapeutic approach to silence the targeted gene of a particular disease, its use is limited by its stability in vivo. For the liver parenchymal cell (PC)-selective delivery of siRNA, siRNA was complexed with galactosylated cationic liposomes. Galactosylated liposomes/siRNA complex exhibited a higher stability than naked siRNA in plasma. After intravenous administration of a galactosylated liposomes/siRNA complex, the siRNA did not undergo nuclease digestion and urinary excretion and was delivered efficiently to the liver and was detected in PC rather than liver non-parenchymal cells (NPC). Endogenous gene (Ubc13 gene) expression in the liver was inhibited by 80% when Ubc13-siRNA complexed with galactosylated liposomes was administered to mice at a dose of 0.29 nmol/g. In contrast, the bare cationic liposomes did not induce any silencing effect on Ubc13 gene expression. These results indicated that galactosylated liposomes/siRNA complex could induce gene silencing of endogenous hepatic gene expression. The interferon responses by galactosylated liposomes/siRNA complex were controlled by optimization of the sequence of siRNA. Also no liver toxicity due to galactosylated liposomes/siRNA complex was observed under any of the conditions tested. In conclusion, we demonstrated the hepatocyte-selective gene silencing by galactosylated liposomes following intravenous administration.
Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children (n=20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus (n=3) or astrovirus (n=3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites (Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria (Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. (n=1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3-2.6 and 1.5-1.9kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25-PicoB43 for Genogroup I and PicoB23-PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs (n=6) and Genogroup II PBVs (n=5), while some could not be amplified (n=3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries.
Small interfering RNA (siRNA) directs the targeted destruction of mRNA encoding a specific protein, in a process known as RNA interference (RNAi). This stops translation of the targeted mRNA into protein, effectively silencing the gene. RNAi is a recent discovery, identified in mammalian cells in 2001, but it has rapidly advanced into a practical technique and is being used increasingly to investigate mammalian gene function. Tools are available to induce RNAi in cell lines, intact tissue preparations and even in vivo. Depending on the method used, loss of gene expression may be transient or sustained, enabling a wide range of functions to be investigated. RNAi therefore offers a powerful technique that can be used to produce targeted knockout of ion channel genes in mammalian cells. Its applications potentially include identification of ion channel function in health and disease, identification of novel channel genes and drug target validation. This paper outlines our current understanding of siRNA and the experimental requirements for producing efficient RNAi and gene silencing. Effective RNAi requires an appropriate siRNA sequence to be designed and an efficient method for delivering the siRNA to the cells of interest. Since not all potential siRNA sequences are effective, it is also important to verify the loss of gene expression by measuring the level of channel protein remaining. Limitations of the methods available for delivering siRNA are one of the main obstacles to producing efficient RNAi, especially in intact tissue preparations. Here we describe an in vitro method for targeted RNAi against the TASK-1 potassium channel gene in an isolated vascular preparation, using a DNA construct to direct the expression of siRNA, along with a non-viral method for transfecting cells within the vessel. Successful silencing of the TASK-1 gene is verified by immunostaining with an antibody directed against the TASK-1 protein.
Repression of an essential nucleolar small nuclear RNA (snRNA) gene of Saccharomyces cerevisiae was shown to result in impaired production of 18S rRNA. The effect, observed for an snRNA species of 128 nucleotides (snR128), was evident within one generation after the onset of SNR128 gene repression and correlated well with depletion of the snRNA. The steady-state mass ratio of 18S RNA to 25S RNA decreased eightfold over the course of the analysis. Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor. This effect appears to result from impairment of processing of the 35S rRNA transcript at sites that define the 20S species coupled with rapid turnover of unstable intermediates. Possible bases for the effects observed are discussed. A common U14 designation is proposed for the structurally related yeast snRNA and 4.5S hybRNAs from amphibians and mammals.
My colleagues and I have reported that a small nuclear RNA, U5, can cause transformation of and chromosomal aberrations in rat fibroblast 3Y1 cells. In the study described here, I found that a partial U5 sequence, the 3' half of the first stem structure (designated RNA3S), transformed the cells morphologically at a high frequency when expressed with a poly(A) tail (RNA3S+). The transformation critically depended upon a polypurine sequence in RNA3S+. The expressed RNA3S+ was associated with polysomes. The transformed cells did not exhibit significant frequencies of chromosome aberrations when compared with control cells, suggesting that the transformation occurs without significant induction of chromosome damage. Heterogeneous subcolonies emerged in monolayers when the morphologically transformed cells were subcultured and maintained at postconfluence. Cells from subcolonies acquired the ability to form small colonies in soft agar and tumors in nude mice, suggesting that RNA3S+ can drive the cells into the neoplastic stage. RNA3S+ also conferred morphological alterations and a growth advantage and decreased levels of fibronectin protein synthesis in human HeLa cells, confirming the transforming potential of the RNA. RNA3S+ transformation may therefore be a useful model system for studying a transformation process.
A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.
U1, U2, U4, U5, and U6 small nuclear RNAs are essential for precursor mRNA splicing. We previously found one and four mRNA-type, or spliceosomal introns, in the U2 and U6 genes, respectively, of yeast Rhodotorula hasegawae (Erythrobasidium hasegawianum). We proposed that U2 and U6 RNAs form a catalytic core for precursor mRNA splicing and that the introns in those small nuclear RNA genes may have been acquired through reverse splicing of an intron from a precursor mRNA into a catalytic site in small nuclear RNAs. In the present study, we analyzed U1, U4, and U5 genes in R. hasegawae. One spliceosomal intron was found in the U1 region forming base-pairs with a 5' splice site of a precursor mRNA. The U5 gene has two spliceosomal introns in the region that interacts with 5' and 3' splice sites. In contrast, the gene for U4 RNA, which is released from the spliceosome prior to the first step of the splicing reaction, has no intron. These results lend a further support to the proposed relation between presence and position of an intron in an small nuclear RNA gene and the function of the encoded small nuclear RNA.
This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.
Six human genomic clones containing sequences homologous to the U3 small nuclear RNA (snRNA) were isolated and characterized. Four of these clones were real U3 snRNA genes because they were transcribed in frog oocytes and the DNA sequences corresponding to the U3 snRNA were identical to the U3 snRNA of HeLa cells. The nucleotide sequences of four true U3 snRNA genes, 537 nucleotides on the 5'-flanking region and 340 nucleotides on the 3'-flanking region, were found to be identical. In addition, the restriction patterns, upto 2 kb on the 5' side and 2.2 kb on the 3' side, appeared to be same. All the isolated U3 clones, containing 15-20 kb of genomic DNA, contained only one U3 snRNA gene, indicating that the human U3 snRNA genes are several kilobases apart. One of the U3 clones contained a full-length U3 pseudogene. Southern blot analysis of genomic DNA with cloned U3 DNA as probe indicated that human DNA contains two families of U3 genes which differ in their flanking sequences. In the 5' flanking region of human U3 snRNA genes, homology to U-gene promoter element, an octamer motif, the 'U3 box', SP1 binding sites and a consensus 3' box in the 3' flanking region, were observed. These data show that the genomic organization and the sequence motifs that control transcription of human nucleolar U3 snRNA genes are similar to those of human U1 and U2 snRNA genes and suggest common mechanism(s) in the evolution of snRNA genes.
Selective gene inhibition by antisense oligodeoxynucleotide (AS-ODN) or by small interference RNA (siRNA) therapeutics promises the treatment of diseases that cannot be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation has been developed in our lab. Human lung cancer cells often overexpress the sigma receptor and, thus, can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexing with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain LPD (liposome-polycation-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the postinsertion method. Nontargeted nanoparticles coated with DSPE-PEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of anisamide ligand. The uptake of oligonucleotide in targeted, PEGylated nanoparticles could be competed by excess free ligand. Our results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells for therapy.