Antisense RNA in transgenic plants

The data on the effects of antisense RNA in plants is reviewed. Results of expression of the genes for selective markers, antisense reporter genes, functioning and viral genes are analyzed. The molecular mechanisms for inhibiting effects of antisense RNA and the potential use of the phenomenon in the plants biotechnology are discussed. The formation of long duplexes between the antisense RNA and messenger RNA are supposed to be irrelevant to suppression of gene expression in plants by the antisense RNA.     

Organization of spliceosomal U6 snRNA genes in the mouse genome

U6 RNA is an abundant small nuclear RNA (snRNA) required for splicing of pre-mRNAs. In mammalian cells, the genes for U1 to U4 snRNAs consist of multigene families ranging from 10 to 100 copies of real genes per haploid genome, and are transcribed by RNA polymerase II. In contrast, results obtained in this study indicate that U6 RNA, which is transcribed by RNA polymerase II and III, may be coded for in mouse cells by only two genes. These two U6 genes are at least 9 kb apart from each other, and the flanking sequences are highly conserved, indicating that the organization of U6 genes is similar to that observed for other mammalian U-snRNA genes.This investigation was supported by Grant GM 38320, awarded by the Department of Health and Human Services, United States Public Health Service.     

RNA interference in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. This organism has recently been developed as an experimental system, with initiation of a genome project among other molecular advances. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. We have shown that expression of double-stranded RNA corresponding to portions of the cryptococcal CAP59 and ADE2 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Specific modulation of protein expression through introduction of double-stranded RNA thus operates in C. neoformans, which is the first demonstration of this technique in a fungal organism. Use of RNA interference in Cryptococcus should allow manipulation of mRNA levels for functional analysis of genes of interest and enable efficient exploration of genes discovered by genome sequencing.     

Identification of conserved regulatory RNA structures in prokaryotic metabolic pathway genes

A combination of algorithms to search RNA sequence for the potential for secondary structure formation, and search large numbers of sequences for structural similarity, were used to search the 5'UTRs of annotated genes in the Escherichia coli genome for regulatory RNA structures. Using this approach, similar RNA structures that regulate genes in the thiamin metabolic pathway were identified. In addition, several putative regulatory structures were discovered upstream of genes involved in other metabolic pathways including glycerol metabolism and ethanol fermentation. The results demonstrate that this computational approach is a powerful tool for discovery of important RNA structures within prokaryotic organisms.     

Genome-Wide Analysis of Host Factors in Nodavirus RNA Replication

Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.     

Localization of 5 S RNA genes in Chironomus tentans

The genes for 5 S RNA in Chironomus tentans have been located to region 2A of chromosome II by cytological hybridization. RNA from individual chromosomes, nuclear sap and nucleoli of salivary gland cells hybridized with the identified 5 S RNA genes in region 2A of chromosome II. The results suggest a common origin of 5 S RNA in these different nuclear compartments.     

U1 small nuclear RNA genes are located on human chromosome 1 and are expressed in mouse-human hybrid cells.

The majority, and perhaps all, of the genes for human U1 small nuclear RNA (U1 RNA) were shown to be located on the short arm of human chromosome 1. These genes were mapped by Southern blot analysis of DNA from rodent-human somatic cell hybrids, using the 5' region of a human U1 RNA gene as a human-specific probe. This probe hybridized to DNA fragments present only in digests of total human DNA or to the DNAs of cell lines which contained human chromosome 1. The major families of human U1 RNA genes were identified, but some human genes may have gone undetected. Also, the presence of a few U1 RNA genes on human chromosome 19 could not be ruled out. In spite of the lack of extensive 5'-flanking-region homology between the human and mouse U1 RNA genes, the genes of both species were efficiently transcribed in the hybrid cells, and the U1 RNAs of both species were incorporated into specific ribonucleoprotein particles.     

Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

Specific RNA interference in psbP genes encoded by a multigene family in Nicotiana tabacum with a short 3'-untranslated sequence

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3'-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3'-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3'-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3'-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.