Structural features in the 3' external transcribed spacer affecting intragenic processing of yeast rRNA.

A highly conserved extended hairpin structure in the 3' external transcribed spacer (3' ETS) region of nascent eukaryotic rRNA transcripts is essential for the maturation of the large ribosomal subunit RNAs (5.8 S and 25 to 28 S rRNAs). Systematic changes were introduced into this structure by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the structural features that are essential for rRNA maturation. Changes in the lower half of the stem or the large loop at the end had little or no effect on the maturation of either the 5.8 S or 25 S rRNA, but changes that disrupted secondary structure in the upper half of this stem had equal and dramatic effects on both RNAs. When the RNA stem was incubated with a cellular protein extract, gel retardation studies indicated that the stem forms a ribonucleoprotein complex, and a comparison with mutant RNA indicated that protein binding could be compromised by changes that were critical for rRNA maturation. Sequence comparisons with other spacer regions as well as snRNAs reveal some structural analogy, which, when taken together with the mutational studies, raise the possibility that this hairpin functions during RNA processing in a manner that may be analogous with that of free snRNPs.

In vivo analyses of RNA polymerase I termination in Schizosaccharomyces pombe.

Recent studies on the termination of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae and Schizosaccharomyces pombe have suggested a more complex mechanism then previously described in higher eukaryotes. Termination appears to occur when a DNA-bound Reb1 protein molecule induces polymerase to pause in the context of a release element [see Reeder,R.H. and Lang,W. (1994) Mol. Microbiol ., 12, 11-15]. Because these conclusions in yeast were based entirely on in vitro analyses, we have examined the same termination process in S.pombe by expressing targeted mutations in vivo . S1nuclease protection studies indicate three tandemly arranged termination sites with most transcripts very efficiently terminated at the first site, 267 nt after the 3' end of the mature 25S rRNA sequence. Termination at each site is mediated by conserved terminator elements which bear limited sequence homology with that of mouse and also can be identified in S.cerevisiae . Removal of the first terminator element transfers dominance to the second site and construction of a new single terminator element at +150 still results in efficient termination and rRNA processing without a need for an additional upstream element. Genomic 'footprint' analyses and gel retardation assays confirm a process mediated by a strongly interacting protein factor but implicate an alternate binding site. Removal of the 5' flanking sequence or structure also had no effect on the site or efficiency of termination. Taken together the results in vivo suggest that the termination process in this fission yeast more strongly resembles the single element-mediated mechanism initially reported in mouse and is not dependent on additional upstream sequence as first reported in S.cerevisiae and postulated to function in general.

Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro.

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.

Termination as a factor in "quality control" during ribosome biogenesis.

In eukaryotes, nascent rDNA and 5 S rRNA gene transcripts undergo 3'-end processing after termination. Mutations in which terminator sequences in these ribosomal RNA genes are deleted completely result in highly unstable transcripts, which are not properly processed and integrated into stable ribosome structure. Mutations that retard RNA processing by extending the 3' external transcribed spacer or by introducing additional secondary structure in the spacers have a similar effect on stable transcript integration. The results indicate that proper termination coupled with efficient rRNA processing acts as a "quality control" process, which helps to ensure that only normal rRNA precursors are effectively processed and assembled into active ribosomes.

Intragenic processing in yeast rRNA is dependent on the 3' external transcribed spacer.

The nucleotide sequence of the 3' external transcribed spacer (3' ETS) region in Schizosaccharomyces pombe rDNA was determined to define structural features which mediate the termination of RNA transcription and subsequent rRNA maturation. S1 nuclease protection studies suggest three alternative termination sites and four cleavage sites in the processing of the 3' ETS sequence. Each of the termination sites precedes a "Sal box"-like sequence which has been demonstrated to mediate the termination of rRNA transcription in mammalian cells. A highly conserved extended hairpin structure in the ETS sequence was deleted by PCR-mediated mutagenesis and the mutant rDNA was expressed in vivo to determine its role in rRNA maturation. Despite an efficient expression of the mutant gene, mature 5.8 S or 25 S rRNA was not observed. Labelling kinetics and S1 nuclease protection analyses indicate that the deletion not only fully inhibits the removal of the 3' ETS but also fully inhibits the processive excision of the second internal transcribed spacer (ITS2). Instead, a relatively stable 27 S nRNA precursor remains easily detectable in the whole cell RNA population. The results demonstrate a critical dependence of ITS processing on the 3' ETS raising the possibility that these sequences interact in a common processing domain.

Inhibition of protein synthesis by an efficiently expressed mutation in the yeast 5.8S ribosomal RNA.

Recent studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides strongly suggested that this RNA plays an important role in eukaryotic ribosome function. To evaluate this possibility further, a ribosomal DNA transcription unit from Schizosaccharomyces pombe was cloned into yeast shuttle vectors with copy numbers ranging from 2 to approximately 90 per cell; to allow direct detection of expressed RNA and to disrupt the function of the 5.8S rRNA molecule, a five base insertion was made in a universally conserved GAAC sequence. The altered mobility of the mutant RNA was readily detected by gel electrophoresis and analyses indicated that mutant RNA transcription reflected the ratio of plasmid to endogenous rDNA. The highest copy number plasmid resulted in about 40-50% mutant RNA. This mutant RNA was readily integrated into the ribosome structure resulting in an in vivo ribosome population which was also about 40-50% mutant; the rates of growth and protein synthesis were equally reduced by approximately 40%. A comparable level of inhibition in protein synthesis was demonstrated in vitro and polyribosomal profiles revealed a consistent increase in size. Subsequent RNA analyses indicated a normal distribution of mutant RNA in both monoribosomes and polyribosomes, but elevated tRNA levels in mutant polyribosomes. Additional mutations in alternate GAAC sequences revealed similar but cumulative effects on both protein synthesis and polyribosome profiles. Taken together, these results suggest little or no effect on initiation but provide in vivo evidence of a functional role for the 5.8S rRNA in protein elongation.