Isolation of two Drosophila melanogaster genes abundantly expressed in the ovary during vitelline membrane synthesis

Several genomic clones were isolated from a Drosophila library screened with cDNA prepared from abundant adult female mRNA. Cytoplasmic dot hybridizations have shown that the genes in all of these clones are expressed only in posteclosion (stages 8-14) follicles. One set of overlapping clones (lambda 20, lambda 28, and lambda 30) was localized by in situ hybridization to 66D, a previously described locus for chorion genes. Restriction mapping demonstrated that these clones contained chorion genes which had been isolated previously. Another clone, lambda 7, was mapped to chromosomal region 26A. This clone carries genes that hybridized to mRNA species similar or identical in size to the known chorion genes encompassed by lambda 28. Furthermore, one of these genes shows homology to the 66D chorion locus, apparently with the s18-1 gene. R-loop and S1-nuclease mapping indicated that lambda 7 contains two genes of 700-800 base pairs in length. Dot hybridization of cytoplasmic RNA from egg chambers demonstrated that these genes are expressed predominantly during stages 9 + 10, the time of vitelline membrane synthesis. Analysis of DNA extracted from embryos and various female tissues by dot hybridization showed that lambda 7 sequences are not amplified in the mature ovary. These results suggest that the two genes carried by lambda 7 and derived from region 26A may code for protein components of the vitelline membrane. In addition it appears that some evolutionary relatedness exists between one of these genes and a member of the chorion multigene family.     

Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form.

Leishmania sp. protozoa contain an abundant surface protease (gp63) that is important for the virulence of the parasite. We found that the average amount of gp63 expressed by Leishmania donovani chagasi promastigotes increases 6-11-fold as they develop from a less infectious form in logarithmic phase to a highly infectious form during stationary phase of cultivation in vitro. The predominant gp63 RNA switches from a 2.7 to a 3.0 kilobase (kb) RNA during the transition from log to stationary phase. Sequence analysis of gp63 cDNAs reveals that three different classes of gp63 RNAs, containing unique 3'-untranslated regions (3' UTRs), are expressed during growth to stationary phase. The predominant 2.7-(log) and 3.0-kb (stationary) class gp63 RNAs possess nearly identical coding regions, but they diverge in their 3' UTRs. A third class, consisting of 3.1- and 2.6-kb (constitutive) gp63 RNAs, is expressed at low levels throughout cultivation. This latter class encodes a gp63 with an additional 41 amino acids at its C terminus, replacing a potential signal for attachment of a glycolipid membrane anchor with a sequence that could be a transmembrane region. These findings are consistent with the regulated expression of different gp63 genes, resulting in different amounts of gp63 protein, during the promastigote's in vitro development to an infectious form.     

Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training

We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory.     

The nuclear 5S RNAs from chicken, rat and man. U5 RNAs are encoded by multiple genes

Preparations of chicken, rat and human nuclear 5S RNA contain two sets of molecules. The set with the lowest electrophoretic mobility (5Sa) contains RNAs identical or closely related to ribosomal 5S RNA from the corresponding animal species. In HeLa cells and rat brain, we only detected an RNA identical to the ribosomal 5S RNA. In hen brain and liver, we found other species differing by a limited number of substitutions. The results suggest that mutated 5S genes may be expressed differently according to the cell type. The set with the highest mobility corresponds to U5 RNA. In both rat brain and HeLa cells, U5 RNA was found to be composed of 4 and 5 different molecules respectively (U5A, U5B1-4) differing by a small number of substitutions or insertions. In hen brain, no U5B was detected but U5A' differing from U5A by the absence of the 3'-terminal adenosine. All the U5 RNAs contain the same set of modified nucleotides. They also have the same secondary structure which consists of two hairpins joined together by a 17 nucleotide long single-stranded region. The 3' half of the molecule has a compact conformation. Together, the results suggest that U5 RNAs are transcribed from a multigene family and that mutated genes may be expressed as far as secondary structure is conserved. The conformation of U5 RNA is likely to be related to its function and it is of interest to mention that several similarities of structure are found between U5 and U1A RNA.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.     

Suppression of viral RNA recombination by a host exoribonuclease

RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5'-3' exoribonuclease, whose absence led to an approximately 10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5'-truncated viral RNAs in xrn1delta mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1delta mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5'-truncated viral RNAs were expressed in the parental and xrn1delta mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5'-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5'-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5'-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.