Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target

A sensitive cellular assay system for RNA interference was developed using the firefly luciferase gene as target. RNA interference was noted not only in Drosophila cultured cells but Chinese hamster cells (CHO-K1) as well, although double-stranded RNA required for the latter was 2500 times more than for the former. Cognate double-stranded RNA as short as 38 bp was found to be still capable of inducing RNA interference in Drosophila cultured cells.     

Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.     

Structure and evolution of the 7SL RNA component of the signal recognition particle

We have cloned and characterized cDNA copies of larval and adult Drosophila 7SL RNA. The Drosophila 7SL sequence shares 66.3% homology with that of human 7SL RNA. The homology is not evenly distributed along the sequence, but is concentrated in blocks in the central part of the molecule. We have analysed the secondary structure of Drosophila and human 7SL RNA free in solution by digestion with single and double strand specific nucleases. Similar experiments with the 7SL RNA bound to proteins within the signal recognition particle show essentially the same digestion pattern. A model of the secondary structure common to Drosophila and human 7SL RNA is presented.     

Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA

Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.     

Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing

U1 RNA from cultured Drosophila melanogaster cells (Kc) was identified by its ability to be recognized, as an RNP, by anti-(U1)RNP antibodies from human lupus patients. Its sequence was deduced largely from direct analysis of the RNA molecule and then confirmed by DNA sequence determinations on a genomic clone isolated by hybridization to Drosophila U1 RNA. The Drosophila U1 RNA sequence exhibits 72% agreement with human U1 RNA. Nucleotides 3-11, which are complementary to the entire consensus sequence for donor (5') splice junctions in hnRNA, and to part of the acceptor (3') consensus, are exactly conserved. However, nucleotides 14-21, postulated to interact only with acceptor junctions, differ. Comparison of the Drosophila U1 sequence with vertebrate U1 sequences allows a particular secondary structure model to be preferred over others. These results are consistent with the hypothesis that U1 snRNPs are involved in splicing, but suggest specific modifications of the model detailing molecular interactions between U1 RNA and hnRNA during the splicing reaction.     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".     

Complexity of RNA in Eggs of DROSOPHILA MELANOGASTER and MUSCA DOMESTICA

Comparative measurements are presented of the sequence complexity of the RNA stored in the eggs of two dipteran flies, Musca domestica and Drosophila melanogaster. The genome of Musca is about five times the size of the Drosophila genome and contains about 3.6 times as much single-copy sequence. As shown earlier, the interspersion of repetitive and single-copy sequence is of the short-period form in Musca, and is of the long-period form in Drosophila. The egg RNA complexities were determined by hybridization of excess RNA with radioactively labeled single-copy DNA. Complexity is expressed as the length (in nucleotides) of diverse single-copy sequence represented in the RNA. The complexity of the RNA of the Musca egg is about 2.4 x 10⁷ nucleotides, and that of the Drosophila egg is about 1.2 x 10⁷ nucleotides. The RNA of the Musca egg is similar to or very slightly lower in complexity than that of other egg RNAs, e.g., those of Xenopus and sea urchin. Compared to all previously measured egg RNAs, Drosophila egg RNA is low in sequence complexity.     

Low molecular weight RNA of Drosophila cells which is induced by heat shock--I. Synthesis and its effect on protein synthesis

When Drosophila cells were heat shocked, low molecular weight RNA of a novel type was synthesized and accumulated in the cytoplasm. This RNA, which is about 320 nucleotides long, inhibited the translation of Drosophila mRNAs in rabbit reticulocyte lysate. It remains to be clarified whether this RNA is involved in the regulation mechanism of heat shock at the translational level.     

Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.     

Transgenic Inhibitors of RNA Interference in Drosophila

RNA silencing functions as an adaptive antiviral defense in both plants and animals. In turn, viruses commonly encode suppressors of RNA silencing, which enable them to mount productive infection. These inhibitor proteins may be exploited as reagents with which to probe mechanisms and functions of RNA silencing pathways. In this report, we describe transgenic Drosophila strains that allow inducible expression of the viral RNA silencing inhibitors Flock House virus-B2, Nodamura virus-B2, vaccinia virus-E3L, influenza A virus-NS1 and tombusvirus P19. Some of these, especially the B2 proteins, are effective transgenic inhibitors of double strand RNA-induced gene silencing in flies. On the other hand, none of them is effective against the Drosophila microRNA pathway. Their functional selectivity makes these viral silencing proteins useful reagents with which to study biological functions of the Drosophila RNA interference pathway.     

Evidence that siRNAs function as guides,not primers,in the Drosophila and human RNAi pathways

In Drosophila, two features of small interfering RNA (siRNA) structure--5' phosphates and 3' hydroxyls--are reported to be essential for RNA interference (RNAi). Here, we show that as in Drosophila, a 5' phosphate is required for siRNA function in human HeLa cells. In contrast, we find no evidence in flies or humans for a role in RNAi for the siRNA 3' hydroxyl group. Our in vitro data suggest that in both flies and mammals, each siRNA guides endonucleolytic cleavage of the target RNA at a single site. We conclude that the underlying mechanism of RNAi is conserved between flies and mammals and that RNA-dependent RNA polymerases are not required for RNAi in these organisms.