Volume 24, Issue 6, Pages 943-953 (December 2006) Identification of a Rapid Mammalian Deadenylation-Dependent Decay Pathway and Its Inhibition by a Viral RNA Element  Nicholas K. Conrad, Stavroula Mili, Eleanor L. Marshall, Mei-Di Shu, Joan A. Steitz  Molecular Cell  Volume 24, Issue 6, Pages 943-953 (December 2006) DOI: 10.1016/j.molcel.2006.10.029 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The ENE Renders Transcripts Less Likely to Be Rapidly Degraded In Vivo (A) Schematic diagram of the Tet-responsive PAN constructs. Positions of the promoter (TetRP), the 79 bp ENE (light-gray box), and the 300 bp Δ4 deletion (dotted lines, Δ) are shown. The KSHV sequence extending 45 bp downstream of the cleavage and polyadenylation sites is also shown (black box). (B) Northern blot from a transcriptional pulse experiment. The lane designations are the times with respect to transcription shut off; the −3.5 lanes were taken prior to transcription induction. PAN RNA and control (actin) signals are shown. The same exposure time is shown for Tet-PAN and Tet-Δ4 signals. A0 designates the approximate mobility of deadenylated transcripts (Figure S2). (C) Graphs of the in vivo decay data for various transcription pulse times. The data are fit to two-component exponential decay curves, except for those of Tet-PAN after a 24 hr pulse, which are fit to a single-component decay curve. Each point on the curve represents the average of at least three experiments, and error bars represent standard deviation. (D) Bar graph showing the calculated fraction of transcripts undergoing rapid decay after different pulse times. Error bars show standard deviation (see Experimental Procedures). Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The ENE Does Not Affect the Decay of Transcripts with Nonpolyadenylated 3′ Ends (A) Schematic diagram of constructs containing the U7-snRNP-dependent 3′-processing signals from the mouse histone H4-12 gene. (B) Northern blot of a 3.5 hr transcriptional pulse experiment. Details are the same as for Figure 1B. (C) Graphical representation of data from decay experiments. See Figure 1C for details. Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 The ENE Is Sufficient to Decrease the Fraction of Intron-less β-Globin Transcripts Undergoing Rapid Decay (A) Schematic diagram of the intron-less β-globin constructs containing the tetracycline-responsive promoter. The promoter (TetRP), coding region (β-globin), five copies of the 79 nt ENE (5xENE), and bovine growth hormone poly(A) signals (BGH PA) are depicted for Tet-βΔ1,2 and Tet-βΔ1,2-E5. (B) Northern blot of a 3.5 hr transcription pulse experiment with β-globin constructs. The signal observed in the uninduced Tet-βΔ1,2-E5 lane is likely due to leaky transcription coupled with the long half-life of this transcript. Subtraction of this value as background from the time points did not alter the curves. The control probe shown in the bottom panel was 7SL, and the asterisk denotes the position of crosshybridization to 18S ribosomal RNA. The same blot and exposure time are shown for Tet-βΔ1,2 and Tet-βΔ1,2-E5 signals. A0 designates the approximate mobility of the deadenylated transcripts (Figure S2) and refers to the 6 hr lane. (C) Graph of the data from decay experiments. See Figure 1C for details. (D) Bar graph showing the percent of transcripts rapidly degraded. See Figure 1D for details. Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 The ENE Inhibits RNA Decay and Deadenylation in Nuclear Extracts (A) Schematic representation of substrates used in the in vitro decay assays. Boxes represent the 79 nt ENE (pBS-E-A) or its reverse complement (pBS-Er-A), and lines are pBluescript sequences. (B) Left panels: representative in vitro decay assays with A60 (top) or A0 (bottom) substrates. RNAs were harvested at the indicated times and gel fractionated. The A60 and A0 markers are fully adenylated and deadenylated substrates, respectively. dT lanes are described in the text. To monitor RNA recovery, a radiolabeled DNA fragment was included in the reactions (“Control”). The doublet in the dT lane for pBS-E-A60 likely arises from interference by the ENE with hybridization of dT40. Right panels: data from multiple decay experiments were fit to single-component exponential decay curves. Error bars show standard deviations for three independent experiments. (C) Schematic representation of deletion substrates used in the deadenylation assays. The entire PAN RNA is shown on the top; the nucleotide locations of the 57 or 58 nt deletions are shown below. The shaded region denotes the ENE. The 5′ cap has been shown to stimulate deadenylation in vitro (Dehlin et al., 2000; Gao et al., 2000; Martinez et al., 2001), but the effects of the ENE were similar whether GpppG or m7GpppG capped the substrates (data not shown). (D) Representative in vitro deadenylation assays of WT and the 57 or 58 nt deletion substrates, with times indicated. dT lanes are as in (B). The positions of the starting A60 and fully deadenylated substrates (A0) are shown on the right. Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 The 3′ Ends of Polyadenylated Transcripts Interact with the ENE to Inhibit Deadenylation (A) Predicted structure of the 79 nt ENE (left) and schematic model of the proposed ENE mechanism (right; the number of As depicted does not reflect a proposed number of base pairing hybridization contacts). (B) Representative data from a GST-MS2 pull-down assay with naked RNA. Above the gel is a representation (not to scale) of the transcripts indicating the locations of the ENE, MS2-binding sites, and complementarity to oligonucleotide NC289 used for RNase H-directed cleavage (arrow). The lengths of the 5′ and 3′ fragments (nt) are listed; values in parentheses indicate the deletions. Panels show the input and pellet of the 5′ and 3′ fragments, as indicated. After selection, samples were treated with RNase H and oligo dT to detect both the 3′ A60 and 3′ A0 fragments on the same gel; the A60 substrates are slightly longer due to incomplete deadenylation by RNase H. All substrates except WT-A0 (lanes 6 and 12) had a 60 nt poly(A) tail. Both panels are from the same gel, but the exposure for the top panel was shorter. (C) Representative data from a GST-MS2 pull-down assay of RNAs generated in vivo. The transcripts assayed are schematized above the gel as in (B). Panels show the input and pellet of the “Middle” and 3′ fragments, as indicated. The top and bottom panels are from the same gel, but they have different exposure times. The nature of the 3′ doublet is unknown, but it may be due to differential RNase H cleavage sites. Samples in lanes 8 and 9 and 17 and 18 were treated with the indicated oligonucleotides prior to GST-MS2 pull-down. The “Input” of Δ4c and Δ4d was higher because 8-fold more of these constructs were transfected to compensate for their lower steady-state RNA levels. (D) Naked RNA substrates (same as Figure 4C) were incubated with purified recombinant PARN for the indicated times and were gel fractionated. Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The ENE Increases the Steady-State Levels of β-Globin Pre-mRNAs with Mutant Splice Sites (A) Northern blot analysis of β-globin pre-mRNAs with either WT, 5′ GU-to-CC, or 3′ AG-to-GG splice-site mutations. Transfected constructs had either no ENE (−) or five copies of the 79 nt ENE in the forward (5F) or reverse complement (5R) orientation. The probe detected intronic sequence. Asterisks denote cryptic splice products. The control was a non-coding RNA, mgU2-19/30 (Tycowski et al., 2004), transcribed from a cotransfected plasmid. (B) Quantitation of the pre-mRNAs in (A) relative to the GU-to-CC mutant with no ENE. Error bars are standard deviations (n = 4). Molecular Cell 2006 24, 943-953DOI: (10.1016/j.molcel.2006.10.029) Copyright © 2006 Elsevier Inc. Terms and Conditions