Showing papers on "RNA-induced transcriptional silencing published in 1988"

Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle.

Abstract: We have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe. Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane. First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure. Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus. Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures. Fourth, the particle containing S. pombe 7SL RNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose. Disruption of the single-copy gene, designated slr1+, reveals that the RNA is indispensable for growth in fission yeast. This result is not surprising, since secretion is an essential cellular process.

A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody

Abstract: An autoantibody from a patient with lupus-overlap syndrome was found to bind a specific region of U1 RNA. By using RNA sequence analysis, immunoprecipitation, and competition experiments with in vitro synthesized fragments of U1 RNA, a region of 40 nucleotides representing a stem-loop secondary structure was found to be an immunoreactive domain. This antibody recognized a conformational epitope because neither the RNA stem nor the RNA loop alone was immunoprecipitable. Antisense U1 RNA, U1 DNA, and other small RNAs were not reactive with the antibody. While the origins of nucleic acid-binding antibodies are unknown, this RNA-specific autoantibody probably originated by direct presentation to the immune system or as an anti-idiotype against a more common U1 small nuclear ribonucleoprotein-specific autoantibody. Thus, these findings have implications for the mechanisms of autoimmune recognition and provide an immunological approach to probing RNA structure and protein-RNA interactions.

3' processing of pre-mRNA plays a major role in proliferation-dependent regulation of histone gene expression

Abstract: A short histone-like fusion RNA, generated when the RNA 3' processing signal from a mouse histone H4 gene is inserted into a heterologous transcription unit, becomes correctly down-regulated in G1-arrested cells of a temperature-sensitive mouse mastocytoma cell cycle mutant (21-Tb; Stauber et al., EMBO J. 5, 3297-3303 [1986]), due to a specific deficiency in histone RNA processing (Luscher and Schumperli, EMBO J. 6, 1721-1726 [1987]). In contrast, inhibitors of DNA synthesis, known to stimulate histone mRNA degradation, have little or no effect on the fusion RNA. This RNA can therefore be used to discriminate between regulation by RNA 3' processing and RNA stability, respectively. The fusion RNA is also faithfully regulated in 21-Tb cells arrested in G1 phase by the drug indomethacin or in C127 mouse fibroblasts during a serum starvation experiment. Moreover, nuclear extracts from serum-starved C127 cells show a specific deficiency in a heat-labile component of the histone RNA processing apparatus, similar to that previously observed for temperature-arrested 21-Tb cells. These results suggest that RNA 3' processing is a major determinant for the response of histone mRNA levels to changes in cell proliferation.

Vectors for the synthesis of specific RNAs in vitro.

Abstract: Publisher Summary This chapter provides an overview of the vectors for the synthesis of specific RNA in vitro. RNA molecules play diverse roles in the expression of genetic information, not only as messengers between DNA and protein but also as components of the machinery for the processing of RNA itself, for its translation, and for the posttranslational modification of proteins. The realization that RNA molecules can also exhibit catalytic activities raises the likelihood that additional roles for the complement of RNA molecules found in nature are discovered. Despite their importance, most RNA species have not been studied as intensively as proteins. Three reasons account for this: (1) RNA molecules are inherently less stable than DNA given their sensitivity to alkali and to transition metals, (2) many RNA molecules display short half-lives in vivo , and (3) many RNAs are found in low abundance in a limited number of tissues. Recombinant DNA technology offers, in principle, the means to circumvent two of these intrinsic difficulties. The key problem is obtaining useful levels of expression of selected RNA molecules encoded by a recombinant DNA. Achieving both selectivity and yield is not a trivial undertaking. All vectors contain promoters essential for the expression of selectable markers and for replication. Thus, both in vivo and in vitro , transcription of recombinant plasmids or phages by the host's RNA polymerase directs the synthesis of many RNAs unrelated to the cloned sequence.

Functional domains of the RNA component of ribonuclease P revealed by chemical probing of mutant RNAs.

Abstract: The higher-order structure of the RNA component of ribonuclease P from Escherichia coli was analyzed using chemical probes. The secondary structure model which had been constructed from the comparative sequence analysis of the RNA was refined using the experimental data. In a mutant RNA (A89 RNA), which contains a G----A substitution at nucleotide 89, we detected a number of conformational alterations clustered between nucleotides 90 and 239. In view of the fact that A89 RNA is as catalytically active as wild-type RNA, but defective in association with the protein component, it is clear that the catalytic function of the RNA component resides on the structure which is not disrupted by the A89 mutation and that the structures altered by the mutation represent the region(s) interacting with the protein component. Another mutant (A329 RNA), which has a G----A substitution at nucleotide 329 and is defective in catalytic function, showed no detectable change in higher-order structure.