RNA polymerase III promoter and terminator elements affect Alu RNA expression

Abstract

Promoter elements derived from the 7SL RNA gene stimulate RNA polymerase III (Pol III) directed Alu transcription in vitro. These elements also stimulate expression of Alus transfected into 293 cells, but transcripts from these same constructs are undetectable in HeLa cells. A terminator resembling the terminator for the 7SL RNA gene has no effect on in vitro Alu template activity, but increases expression in vivo in a position independent manner. Alu transcripts generated from templates with and without this terminator have identical half-lives, indicating that this terminator stimulates expression by increasing template activity. Together, these results show that Alu expression may be regulated at multiple levels and can respond to cis-acting elements. This new found ability to express Alu transcripts by transient transfection provides an opportunity to monitor their post-transcriptional fate. Primary Alu transcripts are not extensively adenylated or deadenylated following transcription, but are short-lived compared to 118 nt scAlu RNA. In addition to Alu RNA, transfected templates encode scAlu RNA, but very high levels of Alu RNA expression does not increase the abundance of scAluRNA. ScAluRNA is not merely a transient RNA degradation product, but is instead tightly regulated by factors other than the abundance of primary transcripts.

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

“…Human 293 cells were cultured and transfected by the calcium phosphate method as previously described and harvested either 24 or 48 h later for analysis (6,18). Clones for transfection included a luciferase reporter; a previously described flAlu RNA-overexpressing clone, XAT (6); and a clone, XAL, which overproduces left Alu RNA monomers (i.e., scAlu-like RNA).…”

“…RNA was analyzed by primer extension with reverse transcriptase and primers to detect 7SL RNA, flAlu RNA, 5S rRNA, and scAlu RNAs as previously described (6). The 7SL RNA primer (ATGCCGAACTTAGTGCGG) gives a 128-nucleotide (nt) primer extension product.…”

“…Pol III proceeds through an Alu template until a fortuitous terminator, consisting of four or more T residues, is encountered within the 3Ј flanking sequence. These primary full-length Alu (flAlu) RNAs contain the dimeric repeat structure that is typical of consensus Alu elements and have a cytoplasmic lifetime of about 30 min (6,39,44). A fraction of flAlu RNA is processed into more stable, small cytoplasmic Alu (scAlu) RNA, which corresponds to the left monomer of the dimeric structure (32).…”

Gene regulation by SINES and inosines: biological consequences of A-to-I editing of Alu element inverted repeats

“…[4][5][6] Figure 1A shows the structural organization of a typical Alu element. There are up to 1.4 x 10 6 copies of these 300 bp elements in the genome, corresponding to more than 1 Alu about for every 3,000 bp of genomic DNA (reviewed in ref.…”

Diverse cis factors controlling Alu retrotransposition: What causes Alu elements to die?

“…An Alu is a dimer of two nonidentical sequences ancestrally derived from the 7SL RNA gene separated by a middle A-rich region (Ullu and Tschudi 1984). The left monomer contains the internal RNA polymerase III (Pol III) promoter A and B boxes (Ullu and Tschudi 1984;Chu et al 1995;Batzer and Deininger 2002). The basic Alu dimer is flanked by an adenosine (A) rich section (A-tail) at its 39 end.…”