lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements - PubMed (original) (raw)

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements

Chenguang Gong et al. Nature. 2011.

Abstract

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).

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Figures

Figure 1

Figure 1. lncRNA_AF087999 (½-sbsRNA1) binds to and reduces the abundance of specific SMD targets

a, Predicted base-pairing between the Alu element within the SERPINE1 or FLJ21870 3'UTR and the Alu element within ½-sbsRNA1, where 1 was defined as the first transcribed nucleotide of each mRNA or ½-sbsRNA1(S). b, Left: Western blotting (WB), using the designated antibody (α), of lysates of HeLa cells treated with the specified siRNA, where Calnexin serves as a loading control. Right: Representation of RT-sqPCR analyses of ½-sbsRNA1, SERPINE1 or FLJ21870 mRNA from the same lysates, where the normalized level of each transcript in the presence of Control siRNA was defined as 100. c, Representation of RT-sqPCR analyses of FLUC-SERPINE1 3'UTR or FLUC-FLJ21870 3'UTR SMD reporter mRNA in cells that had been transiently transfected with the specified siRNA, where the normalized level of each transcript in the presence of Control siRNA was defined as 100. d, Diagrams of expression vectors encoding ½-sbsRNA1(S)R, which is siRNA-resistant, ½-sbsRNA1(S) or FLUC with or without 12 copies of the MS2 coat protein binding site (MS2bs). e, Western blot (upper) or RT-sqPCR (lower) before (−) or after immunoprecipitation (IP) using anti-FLAG or, as a control for nonspecific IP, mouse(m) IgG of lysates of formaldehyde-crosslinked HeLa cells that had been transiently transfected with pFLAG-MS2-hMGFP and either the denoted ½-sbsRNA1(S) expression vector or pFLUC_MS2bs. f, As in e, except cells were treated with Control or STAU1 siRNA. Left: Western blotting. Right: RT-sqPCR, where the co-IP efficiency indicates the level of each mRNA-derived product after IP relative to before IP. Each ratio in the presence of Control siRNA was defined as 100%. See Supplementary Fig. 5 for phosphorimages and evaluations of RT-sqPCR data shown here as histograms. Error bars indicate s.e.m. Single asterisk, n = 6, P < 0.01; double asterisks, n = 3, P < 0.05.

Figure 2

Figure 2. ½-sbsRNA1 co-immunoprecipitates with STAU1 and is required for STAU1 binding to specific SMD targets

a, Western blotting (upper) or RT-sqPCR (lower) of lysates of formaldehyde-crosslinked HeLa cells that had been transiently transfected with the specified siRNA and either empty vector (−) or p½-sbsRNA1(S)R (+) before or after IP with anti-HA or rat IgG. After IP, each sample was spiked with _in vitro_-synthesized E. coli LACZ mRNA. The co-IP efficiency provides the level of each mRNA RT-sqPCR product after IP relative to before IP, where each ratio in the presence of Control siRNA was defined as 100%. b, Diagrams of pFLUC-SERPINE1 3'UTR FL, which contains the full-length (FL) SERPINE1 3'UTR, and 3'UTR deletion variants. Yellow boxes, FLUC sequences; blue bars, SERPINE1 3'UTR sequences; Δ, deletion; pale green boxes, 3'UTR of FLUC No SBS, which does not bind STAU1. The 5'-most pale green box ensures that ribosomes translating to the FLUC termination codon do not displace STAU1 that had been recruited to the ½-sbsRNA1-binding site (BS, which is 86 nucleotides as shown in Supplementary Fig. 1a). c, Western blotting (upper) and RT-sqPCR (middle and lower) of lysates of HeLa-cells that had been transiently transfected with the noted pFLUC-SERPINE1 3'UTR test construct and the phCMV-MUP reference plasmid. Lower: The normalized level of each FLUC mRNA in the presence of Control siRNA was defined as 100%. See Supplementary Fig. 5 for phosphorimages and evaluation of RT-sqPCR data shown here as histograms. Error bars indicate s.e.m. Single asterisk, n = 6, P < 0.01; double asterisks, n = 3, P < 0.05.

Figure 3

Figure 3. Evidence that ½-sbsRNA2, ½-sbsRNA3 and ½-sbsRNA4 base-pair with particular mRNA 3'UTRs and decrease mRNA abundance, as do STAU1 and UPF1

a, Predicted base-pairing between the 3'UTR Alu element of CDCP1 mRNA (Acc#: NM_022842) and ½-sbsRNA2, or MTAP mRNA (Acc#: NM_002451) and ½-sbsRNA3 as well as ½-sbsRNA4, where 1 was defined as the first nucleotide listed in the NCBI data base for each mRNA or lncRNA. b, Essentially as in Fig. 1b. See Supplementary Fig. 5 for phosphorimages and evaluation of RT-sqPCR data shown here as histograms. Error bars indicate s.e.m. Asterisk, n = 6, P < 0.01. c, Model for how an Alu element-containing ½-sbsRNA that is polyadenylated and largely cytoplasmic (red) base-pairs with a partially complementary Alu element, i.e., a half-STAU1 binding site (½-SBS), within the 3'UTR of a particular mRNA (blue) to trigger SMD. Base-pairing forms a functional SBS. The STAU1-bound SBS triggers SMD in a UPF1-dependent mechanism when translation terminates sufficiently upstream of the SBS so that translating ribosomes do not remove bound STAU1. The ½-sbsRNA is not destroyed in the process. N, nucleus; C, cytoplasm; AUG, translation initiation codon; Ter, termination codon (which is generally, but not necessarily, a normal termination codon).

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