Inverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editing

Abstract

With over one million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alus that are in opposite orientation gives rise to double-stranded RNA (dsRNA). Although the presence of dsRNA in the cell was previously thought to only occur during viral infection, it is now known that cells express many endogenous small dsRNAs, such as short interfering RNA (siRNAs) and microRNA (miRNAs), which regulate gene expression. It is possible that long dsRNA structures formed from Alu elements influence gene expression. Here, we report that human mRNAs containing inverted Alu elements are present in the mammalian cytoplasm. The presence of these long intramolecular dsRNA structures within 3'-UTRs decreases translational efficiency, and although the structures undergo extensive editing in vivo, the effects on translation are independent of the presence of inosine. As inverted Alus are predicted to reside in >5% of human protein-coding genes, these intramolecular dsRNA structures are important regulators of gene expression.

Figure 1.

Cytoplasmic localization of endogenous mammalian mRNAs with inverted Alu 3′-UTR structures. (A) Northern blots were performed on total (T), cytoplasmic (C) and nuclear (N) HeLa RNA and hybridized with probes specific to the indicated RNAs. (B and C) Quantification of endogenous mRNA levels present in nuclear (light gray) and cytoplasmic (dark gray) fractions as determined by northern blot (B) or qRT-PCR (C) for three independent fractionations. Error bars represent SEM. (D) Total, cytoplasmic and nuclear mRNA from fractionation experiments shown in (A) was reversed transcribed, PCR amplified and sequenced. In addition, HeLa genomic DNA was PCR amplified and sequenced to serve as a negative control. One editing event for the PSMB2 and BPNT1 3′-UTRs is shown, but editing was measured across the 3′-UTR (Supplementary Tables S1 and S2).

Figure 2.

Inverted Alus affect gene expression independent of RNA editing. (A) Schematic of PSMB2 3′-UTR reporters. The wild-type (WT) PSMB2 3′-UTR was cloned immediately downstream of firefly luciferase. Arrows indicate location and orientation of Alu sequences. Control firefly reporters have individual Alus replaced with their corresponding reverse complement (RC) sequence. Thus, the Alus in the controls are in the same orientation. (B, C and G) Bar height represents luciferase activity of the indicated firefly PSMB2 3′-UTR reporters relative to a co-transfected Renilla luciferase plasmid, normalized to WT. Error bars show SEM for at least three independent biological replicates. (B) In HeLa cells, the mean of ten independent biological replicates indicates significant differences in luciferase activity between WT and 1st (P < 0.0001) and 2nd (P < 0.0001) Alu RC controls. (C) In HEK293 cells, significant differences between WT and 1st (P = 0.0009) and 2nd (P = 0.04) Alu RC controls were observed. (D) Reporter plasmid DNA and reporter cDNA was amplified and sequenced. One editing event is shown, but editing was observed at multiple sites. (E) HeLa cells stably transfected with no (−), scrambled (scr.) or an ADAR1 shRNA were subjected to SDS–PAGE and western blotting for ADAR1 and Tubulin. (F) Control DNA and cDNAs from endogenous and reporter PSMB2 were amplified from the indicated cells. Three editing events are shown, but editing was measured across the 3′-UTR (Supplementary Tables S3 and S4). (G) In ADAR1^k.d. cells, significant differences in luciferase activity of WT PSMB2 3′-UTR and 1st (P = 0.014) and 2nd (P = 0.019) Alu RC controls were observed.

Figure 3.

Double-stranded 3′-UTRs do not affect mRNA export or stability. (A) Northern blots of total RNA from HeLa cells transfected with the indicated firefly PSMB2 3′-UTR reporters and a control Renilla luciferase reporter, hybridized with indicated probes. (B) Quantification of five independent biological replicates of experiment in (A). Height of the bar represents levels of firefly mRNA relative to Renilla mRNA, normalized to WT. (C) Northern blots of equal cell equivalents of cytoplasmic (C) and nuclear (N) RNA from HeLa cells transfected with the indicated firefly PSMB2 reporters and Renilla luciferase, hybridized with specific probes. (D) Translational efficiency of a firefly luciferase reporter with the indicated PSMB2 3′-UTR in HeLa cells. Each bar represents the mean and SEM of five biologically independent experiments. In each experiment, protein and RNA were isolated at the same time and levels measured by luciferase assay and northern blot, respectively. Efficiency is reported as the ratio of the normalized luciferase activity to the normalized mRNA level and is reported relative to the efficiency of the WT PSMB2 3′-UTR. Both luciferase activity and RNA levels are normalized to the co-transfected Renilla control reporter. Significant differences in translational efficiency between the PSMB2 3′-UTR (WT) and 1st (P = 0.02 and 2nd (P = 0.01) Alu RC controls were observed.

Figure 4.

Some, but not all intramolecular Alu dsRNA structures affect gene expression. (A) Schematic of MPST 3′-UTR reporters. (B) Bar height represents luciferase activity of the indicated Renilla reporters relative to a co-transfected firefly luciferase, normalized to WT. Significant differences in luciferase activity between the MPST 3′-UTR (WT) and 1st (P = 0.04) and 2nd (P = 0.04) Alu RC controls were observed. (C) Schematic of BPNT1 3′-UTR reporters. (D) HeLa cells transfected with BPNT1 reporters described in (C). Bar height represents luciferase activity of the indicated firefly reporters relative to co-transfected Renilla luciferase, normalized to WT. (E) Northern blots of total RNA from the same HeLa cells assayed for luciferase activity in (D), hybridized with indicated probes.

Figure 5.

Distance of inverted Alus from stop codon affects translation efficiency. (A, B) HeLa cells were transfected with firefly reporters bearing shortened PSMB2 3′-UTRs. Protein and RNA were isolated from the same cells and measured by luciferase assay (A) and Northern blot (B), respectively. (A) Bar height represents luciferase activity of the indicated firefly reporters relative to co-transfected Renilla luciferase, normalized to WT. Error bars show SEM for three independent biological replicates. (B) Northern blots of total RNA from HeLa cells transfected with the indicated shortened PSMB2 3′-UTR reporters and Renilla luciferase, hybridized with indicated probes. (C) Plasmid DNA and the indicated reporter cDNAs were amplified and sequenced. One editing event is shown, but editing was observed at multiple sites. (D and E) HeLa cells were transfected with firefly reporters bearing expanded BPNT1 3′-UTRs. Protein and RNA were isolated from the same cells and measured by luciferase assay (D) and Northern blot (E), respectively. (D) Bar height represents luciferase activity of the indicated firefly reporters relative to co-transfected Renilla luciferase, normalized to WT. Error bars show SEM for five independent biological replicates. In HeLa cells, significant differences in luciferase activity between the expanded BPNT1 3′-UTR (WT) and expanded 1st (P = 0.002) and 2nd (P = 0.0018) Alu RC controls were observed. (E) Northern blots of total RNA from HeLa cells transfected with the indicated expanded BPNT1 3′-UTR reporters and Renilla luciferase, hybridized with indicated probes.