Chem. 268:6423-28, 1993). The relationship between the -120-and -300-ntAlu transcripts had not been determined. However, a Bi SINE produces scBl RNA by posttranscriptional processing, suggesting a similar pathway for scAla. An Ala SINE which recently transposed into the neurofibromatosis 1 locus was expressed in microinjected frog oocytes. This neurofibromatosis 1 Alu produced a primary transcript followed by the appearance of the scAl species. 3' processing of a synthetic -300-ntAla RNA by HeLa nuclear extract in vitro also produced scAla RNA. Primer extension of scAla RNA indicates synthesis by RNA polymerase HI. HeLa-derived scAla cDNAs were doned so as to preserve their 5'-terminal sequences and were found to correspond to polymerase Im transcripts of the left monomeric components of three previously identified Ala SINE subfamilies. Rodent x human somatic cell hybrids express Ala RNAs whose size, heterogeneous length, and chromosomal distribution indicate their derivation from SINEs. The coexpression of dimeric and monomeric Ala RNA in several hybrids suggests a precursor-product relationship.

The number of Alu transcripts that accumulate in HeLa and other human cells is normally very low; however, infection with adenovirus type 5 increases the expression of Alu elements dramatically, indicating that the potential for polymerase III (pol III)-dependent Alu transcription in vivo is far greater than generally observed (B. Panning and J. R. Smiley, Mol. Cell. Biol. 13:3231-3244, 1993). In this study, we employed nuclear run-on in combination with a novel RNase H-based assay to investigate transcription from uninfected and adenovirus type 2-infected nuclei, as well as genomic DNAs from uninfected and infected cells. When performed in the presence of excess uninfected nuclear extract, such assays revealed that (i) the vast majority of transcriptionally competent Alu elements in nuclei are masked from the pol III transcriptional machinery and (ii) the induction of Alu expression upon adenovirus infection can be largely accounted for by an increased availability of these elements to the pol III transcription machinery. We also investigated the role of H1 histone for silencing of Alu genes and, in comparison, mouse B2 repetitive elements. Depletion of H1 led to an Ϸ17-fold activation of B2 repetitive elements but did not change Alu transcription relative to that of constitutively expressed 5S rRNA genes. These results are consistent with the view that Alu repeats are efficiently sequestered by chromatin proteins, that such masking cannot be accounted for by nonspecific H1-dependent repression, and that adenovirus infection at least partially overrides the repressive mechanism(s).Within the human genome reside at least 500,000 copies of the Alu interspersed repetitive gene family (9, 45). These repeat elements possess bipartite RNA polymerase III (pol III) promoters and serve as active templates in vitro, yet despite their high copy number seldom give rise to high levels of pol III-transcribed RNAs in vivo. Important exceptions to this rule are provided by the recent findings that pol III-dependent Alu transcription can be strongly induced in HeLa cells following high-titer adenovirus or herpesvirus infection (36, 37). These findings confirm that Alu repeats have the potential to function as active templates in vivo under certain conditions but leave unanswered why such low Alu small-RNA levels accumulate under normal circumstances. One explanation which has been offered is that the vast majority of Alu repeat elements, by virtue of their insertion into the genome via retroposition, lack 5Ј-flanking sequences requisite for efficient capture of transcription factors (43). According to this idea, since Alu elements are unable to compete for pol III transcription factors (which are presumed to be present in limiting amounts within the cell), they are progressively packaged into chromatin structures with concomitant transcriptional silencing. The differential developmental regulation of Xenopus somatic and oocyte 5S genes has served as a paradigm in this regard. It appears that oocyte 5S genes are subject to chrom...

Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs. Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted. SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.

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Purpose To evaluate the safety and efficacy of N-acetylmannosamine (ManNAc) in GNE myopathy, a genetic muscle disease caused by deficiency of the rate-limiting enzyme in N-acetylneuraminic acid (Neu5Ac) biosynthesis. Methods We conducted an open-label, phase 2, single-center (NIH, USA) study to evaluate oral ManNAc in 12 patients with GNE myopathy (ClinicalTrials.gov NCT02346461). Primary endpoints were safety and biochemical efficacy as determined by change in plasma Neu5Ac and sarcolemmal sialylation. Clinical efficacy was evaluated using secondary outcome measures as part of study extensions, and a disease progression model (GNE-DPM) was tested as an efficacy analysis method. Results Most drug-related adverse events were gastrointestinal, and there were no serious adverse events. Increased plasma Neu5Ac (+2,159 nmol/L, p < 0.0001) and sarcolemmal sialylation (p = 0.0090) were observed at day 90 compared to baseline. A slower rate of decline was observed for upper extremity strength (p = 0.0139), lower extremity strength (p = 0.0006), and the Adult Myopathy Assessment Tool (p = 0.0453), compared to natural history. Decreased disease progression was estimated at 12 (γ = 0.61 [95% CI: 0.09, 1.27]) and 18 months (γ = 0.55 [95% CI: 0.12, 1.02]) using the GNE-DPM. Conclusion ManNAc showed long-term safety, biochemical efficacy consistent with the intended mechanism of action, and preliminary evidence clinical efficacy in patients with GNE myopathy.