Cis-acting influences on Alu RNA levels - PubMed (original) (raw)

Cis-acting influences on Alu RNA levels

C Alemán et al. Nucleic Acids Res. 2000.

Abstract

The human short interspersed repeated element (SINE), Alu, amplifies through a poorly understood RNA-mediated mechanism, termed retroposition. There are over one million copies of Alu per haploid human genome. The copies show some internal variations in sequence and are very heterogeneous in chromosomal environment. However, very few Alu elements actively amplify. The amplification rate has decreased greatly in the last 40 million years. Factors influencing Alu transcription would directly affect an element's retroposition capability. Therefore, we evaluated several features that might influence expression from individual Alu elements. The influence of various internal sequence variations and 3' unique flanks on full-length Alu RNA steady-state levels was determined. Alu subfamily diagnostic mutations do not significantly alter the amount of Alu RNA observed. However, sequences containing random mutations throughout the right half of selected genomic Alu elements altered Alu RNA steady-state levels in cultured cells. In addition, sequence variations at the 3' unique end of the transcript also significantly altered the Alu RNA levels. In general, sequence mutations and 3' end sequences contribute to Alu RNA levels, suggesting that the master Alu element(s) have a multitude of individual differences that collectively gives them a selective advantage over other Alu elements.

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Figures

Figure 1

Alu construct used for transfections and sequence alignment of mutations made. (A) Schematic diagram of the basic construct used for the transient transfections in NIH 3T3 cells. The 7SL upstream (117 bases of the human 7SL RNA gene accession no. M20910) contains _cis_-acting enhancer elements (white). The Alu body sequence (black) is divided into the left- and right-half by the middle A-rich region. The RNA polymerase III promoter is in the left half of Alu. The BC1 unique region from the BC1 master gene (47) (gray) contains the sequence complementary to the oligo used for RNA detection. The Alu body sequence was changed to represent the different subfamilies and the different right half sequences obtained from the GenBank database (B). Residues were changed in the BC1 unique region to represent the different 3′ end flanking sequences (C). (B) Sequence alignment of four Alu subfamilies (Sx, Sg1, Y and Ya5) that show the diagnostic mutations and the Alu body sequence from elements found in the GenBank databases (Sx5368, Sx3959, Sx0115, Sx0453, Sx84472 and Sx98047). Ya5#223 represents clone pYa5-31274223). The initial 5′ sequences of Sx5369, Sx3959, Sx0115, Sx0453, Sx84472 and Sx98047 is not shown since they are identical to their consensus counterpart. (C) The mutations incorporated into the BC1 unique region of the construct are shown. The sequence alignment contains the 3′ end flank sequences from the B1 gene, 5S RNA gene, SxBC1–tm (BC1 tm), Ya5-31274223 (Ya5#223) and Ya5-31274TTT (Ya5#TTT). Dots represent sequence identity, dashes represent lack of sequence and letters represent the nucleotide change from the consensus sequence on the top. The asterisk represents the rest of the sequence downstream of the terminator sequence.

Figure 1

Figure 2

Northern blot of Alu subfamily RNA expression. Comparison of the expression from transient transfection in NIH 3T3 of the different subfamilies: lane 1, p7SLSxBC1–tm; lane 2, p7SLSg1BC1–tm; lane 3, p7SLYBC1–tm; lane 4, p7SLYa5BC1–tm; lane 5, pTAblue only (empty vector control); lane 6, p7SLBC1BC1 only. Arrows indicate positions of Alu RNA and BC1 RNA (internal control). No bands were detected in the mock transfection with no DNA (data not shown).

Figure 3

Alu RNA expression from constructs with altered pol III terminator regions. Each experiment consists of a co-transfection of the individual plasmid with the internal control, p7SLBC1BC1. The Alu/BC1 ratios (n ≥ 7) were calculated by dividing the amount of Alu RNA by the amount of BC1 RNA detected and expressed as Phosphoimager (PI) units. The bars represent the means ± the standard error of the mean (SEM). Asterisks indicate P < 0.05 from ANOVA tests when compared to p7SLSxBC1 (1) or p–416Ya5-31274223 (5). (A) The effect of different pol III terminator sequences on Alu expression. The following constructs were evaluated: 1, p7SLSxBC1–tm (n = 12) SEM ± 0.118; 2, p7SLSxBC1 (n = 12) SEM ± 0.176; 3, p7SLSxBC1–B1 (n = 7) SEM ± 0.188; 4, p7SLSxBC1-5S (n = 9) SEM ± 0.103. The numbers on top of the bars represent the stability values (kcal/mol) as determined from mfold analyses (45,46). (B) Effect of the endogenous 3′ end on RNA expression from a specific genomic Alu element. The following constructs were evaluated: 5, p–416Ya5-31274223 (n = 8) SEM ± 0.032; 6, p–416Ya5-31274TTT (n = 7) SEM ± 0.072; 7, p7SLYa5-31274223 (n = 8) SEM ± 0.090; 8, p7SLYa5-31274TTT (n = 8) SEM ± 0.086. No secondary structure formation was detected by mfold analyses.

References

1. Ausubel F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1996) Current Protocols In Molecular Biology. John Wiley & Sons, Inc., Canada.
1. Rogers J.H. and Willison,K.R. (1983) Nature, 304, 549–552. - PubMed
1. Weiner A., Deininger,P. and Efstradiatis,A. (1986) Annu. Rev. Biochem., 55, 631–661. - PubMed
1. Batzer M.A., Deininger,P.L., Hellmann-Blumberg,U., Jurka,J., Labuda,D., Rubin,C.M., Schmid,C.W., Zietkiewicz,E. and Zuckerkandl,E. (1996) J. Mol. Evol., 42, 3–6. - PubMed
1. Shen M., Batzer,M. and Deininger,P. (1991) J. Mol. Evol., 33, 311–320. - PubMed

Cis-acting influences on Alu RNA levels - PubMed (original) (raw)