Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells.

Abstract

We studied the distribution of repetitive sequence elements capable offorming double-stranded regions in nuclear RNA of HeLa, KB, and L cells. In human RNA populations, we called these regions duplex Alu family RNA (dAfRNA) because they represent transcripts of the highly reiterated family of DNA regions known as "Alu family DNA" (Rubin et al., Nature (London) 284:372-374, 1980). Although the dAfRNA populations of both human cell lines (HeLa and KB) have low sequence complexity, they represent 5% of the total heterogeneous nuclear RNA and have identical fingerprints; mouse L-cell dAf-like RNA (which has a similar complexity) represents only 2% of the total heterogeneous nuclear RNA and has an entirely different fingerprint. We utilized Escherichia coli RNase III as a highly specific reagent for the recognition of RNA:RNA duplex structure. This enzyme cleaves within the six characteristic RNase Tl-resistant oligonucleotides of HeLa-and KB-cell dAfRNA (Robertson et al., J. Mol. Biol. 115:571-589, 1977). In addition, the size of heterogeneous nuclear RNA from all three cell types is reduced from greater than 32S to about 15S after RNase III treatment. We conclude that this size shift is a result of cleavage within dAfRNA regions and that such regions are present in most or all of the large RNA transcripts of these cells.The heterogeneous nuclear RNA (hnRNA) of HeLa cells ranges in size up to at least 20,000 nucleotides. Roughly 5% of its total mass consists of highly repetitive, double-stranded regions approximately 300 base pairs in length. These structured regions can be analyzed by nuclease treatment (11,14,17,21,23, 28), electron microscopy (9), hybridiza- MATERIALS AND METHODSPreparation and characterization of 32P-labeled nuclear RNA species. Growth and labeling of cells were carried out as described by Robertson et al. (29) by an extensively modified version of the procedures described earlier (14,17) for preparation of nuclei and nuclear RNA. HeLa cells (type S3) and mouse L cells were from the collection of I. Tamm, The Rockefeller University, whereas KB cells (American Type Culture Collection) were the generous gift of S. Altman, Yale University. Cell densities were concentrated 10-fold to give a final titer of 5 x 106 cells per ml during the labeling periods (3 h in the presence of 1 to 2 mCi of 32p per ml). KB and HeLa cells were preincubated for 30 min with actinomycin D (0.05 ,ug/ml) before addition of 32p. In the case of L cells, however, this concentration of actinomycin D was insufficient to inhibit synthesis of rRNA prgcursors; a concentration of 0.12 ,ug of actinomycin D per ml was therefore used.Nuclear RNA was isolated as described previously (29) and subjected to preparative sucrose density gradient centrifugation in 15 to 30% sucrose gradients in NETS buffer (0.01 M Tris-hydrochloride [pH 7.5], 0.01 M NaCl, 0.01 M EDTA, and 0.2% sodium dodecyl sulfate). dAfRNA was prepared from pooled fractions of nuclear RNA sedimenting faster than 32S by a procedure involving (i) digestion by pancreatic DN...

RNA editing of non-coding RNA and its role in gene regulation

“…In transcripts, Alu sequences frequently occur in proximate pairs of opposite directions, which typically interact pairwise. The double strandedness of transcribed Alus was first discovered when Alu RNA was shown to be cleaved by the double strand specific ribonuclease, RNase III [36]. More recent transcriptome analyses have shown that inverted Alu repeats closer than 3500 nt, from each other, frequently hybridize to form stem loop structures, with the loop sometimes spanning an exon.…”

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

“…1C) (for reviews, see references 17, 18, 37, and 90). Alu elements have no known function (34,103); however, it has been suggested that they may provide cis-acting sequences that serve as modulators of chromatin structure (20), sites of initiation of cellular DNA replication (2,41), hotspots for recombination (14,38,69), inhibitors of gene conversion (30), stabilizers of cytoplasmic RNA (11,68), negative transcriptional regulators (72,85,100), or modulators of intranuclear processing of mRNA precursors (40). Alternatively, it has been proposed that they may encode the RNA component of a cytoplasmic ribonucleoprotein similar to the signal recognition particle (80).…”

Localization and characterization of members of a family of repetitive sequences in the goat βglobin locus

“…Alu-like elements contain the characteristic internal RNA polymerase III promoters defined by Fowlkes and Shenk (44), and it has been demonstrated that individual repetitive sequences can serve as RNA polymerase III templates in in vitro transcription assays (45)(46)(47)(48)(49)(50). In addition, these sequences are represented in hnRNA, both as short unit length transcripts and in the intervening sequences and 3' untranslated regions of the structural gene transcripts (51)(52)(53)(54)(55)(56)(57)(58)(59)(60). Finally, these repetitive elements share a short stretch of sequence homologous to the origin of replication of papovaviruses (8).…”