Showing papers on "RNA-induced transcriptional silencing published in 1981"

Sequence-specific contacts between the RNA polymerase of vesicular stomatitis virus and the leader RNA gene

Abstract: Methylation-protection studies with nucleocapsids from vesicular stomatitis virus indicate that the viral polymerase (L and NS proteins) contacts the genomic RNA template in the middle of the leader gene, 16-30 nucleotides from the 3' terminus. The data suggest that the NS protein binds to the sequence (formula, see text) and may function as an initiator protein for transcription.

RNA processing comes of age

Abstract: During the past two decades, an awareness of the importance of RNA processing has evolved as part of the quest to understand how living cells express the information encoded in their genes . As the knowledge of gene expression has expanded, we have come to realize that the old central dogma of DNA --+ RNA -+ protein is embellished with elegant and intricate design features, many of which are revealed in the processing of primary gene transcripts into the functional forms of RNA. The production of the two large structural RNA components of the ribosome (rRNAs), the synthesis of transfer RNA (tRNA), and the formation of messenger RNA (mRNA) in higher organisms all involve rather elaborate processing reactions, including nucleolytic cleavages, ligations, terminal additions, and nucleoside modifications. A raison d'etre for processing is readily apparent in the case of the coordinate production of rRNA components from a single transcriptional unit and the synthesis of mRNA from noncontiguous genetic elements . However, the purpose of the polyadenylate and methylated cap structures that are added to the termini of mRNA and the modification of internal nucleotides in most RNA species is less clear . These structural alterations may serve to improve the stability of the RNA and the efficiency of its function, but they might also be implicated in more subtle forms of discriminative regulation that are yet to be discovered . In any event, it is clear that RNA processing constitutes a major cellular activity and an integral part of the mechanism ofgene expression. In this essay I shall try to trace the evolution of our concepts of RNA processing in relation to the contemporary issues of cellular and molecular biology and to the introduction of key experimental tools which were critical to the development of these concepts . My idea for treating the subject in this way came from an engaging article on the nucleolus by a former colleague and source of inspiration, Jack Schultz (1). It is my intention to provide both a historical and a reasonably up-todate overview of the subject without the burden of extensive detail . Fortunately, there are several recent reviews to which the reader can refer for a more comprehensive coverage of particular aspects of RNA processing (2-7) .

Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene

Abstract: The RNA of a unique long defective interfering particle (DI-LT2) derived from the heat-resistant strain of vesicular stomatitis virus (VSV) contains 70 nucleotides at its 3' end that are complementary to the 5' end of the VSV RNA. Following this region of terminal complementarity, there is a precise copy of the 3' end of the nondefective VSV RNA. The sequence homology between the DI-LT2 RNA and the 3' end of VSV RNA extends for at least 60 bases and probably for most of the length of the DI-LT2 RNA. The DI-LT2 particle is capable of transcription in vitro but produces only a short RNA [defective interfering (DI) particle product], which is encoded by the extreme 3' terminus of the DI RNA. Neither leader RNA nor capped VSV mRNAs are synthesized by DI-LT2, although competent templates for these are present. These data suggest that the 3'-terminal initiation is a prerequisite of the production of competent transcripts and that the sequence coding for leader RNA is not, by itself, sufficient for initiation. We propose a model for the origin of this DI particle, involving specific termination and resumption of replication, which is similar to that described previously for another class of DI particle RNAs.

Structure and origin of a snapback defective interfering particle RNA of vesicular stomatitis virus.

Abstract: The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.

Monocistronic transcription is the physiological mechanism of sea urchin embryonic histone gene expression.

Abstract: We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.

Localization of RNA polymerase B and histones in the nucleus of primary spermatocytes of Drosophila hydei, studied by immunofluorescence microscopy.

Abstract: By means of indirect immunofluorescence microscopy, we have studied the distribution of RNA polymerase B, of the nucleosomal histones H2b, H3, and H4 and of histone H1, in nuclei of primary spermatocytes of Drosophila hydei. RNA polymerase B and histones, including H1, are found to be present on the loop structures of the Y chromosome. The nucleolus stains only for the histones, but not for RNA polymerase B. Various mutants deficient for some of the loops or altering their morphology, were used to identify the individual chromosomal segments. In growing spermatocytes of the genetic constitution X/0, autosomes and the chromosome X react strongly with antibodies against RNA polymerase B, but not with antibodies against histones. The results suggest that the autosomes, the chromosome X and the Y chromosomal loop structures, with the exception of the nucleolus, are transcribed mostly by RNA polymerase B.