Endonuclease-Mediated mRNA Decay Involves the Selective Targeting of PMR1 to Polyribosome-Bound Substrate mRNA  Feng Yang, Daniel R Schoenberg  Molecular Cell  Volume 14, Issue 4, Pages 435-445 (May 2004) DOI: 10.1016/j.molcel.2004.05.001

Figure 1 Polysome Binding of PMR1 and Albumin mRNA (A) Cos-1 cells were transfected with vector (pcDNA3) or plasmid expressing a catalytically inactive form of PMR1 (PMR60°) with a c-myc epitope tag. Cytoplasmic extracts were analyzed by Western blot with a monoclonal antibody to the c-myc epitope tag (left) or a polyclonal antibody to Xenopus PMR1 (right). (B) Cos-1 cells were transfected with plasmid expressing PMR60° and albumin mRNA. Postmitochondrial extract from transfected cells was separated on a 10%–40% sucrose density gradient containing 5 mM MgCl2, and fractions were analyzed by absorbance at 260 nm, for distribution of PMR60° by Western blot with antibody to the c-myc tag and by Northern blot for albumin mRNA. The direction of sedimentation is indicated with an arrow. The identity of polysome-containing fractions was confirmed by Western blot with a polyclonal antibody to ribosomal protein S6. (C) Postmitochondrial extract from transfected cells was treated with 10 mM EDTA, separated on a 10%–40% sucrose density gradient containing 10 mM EDTA, and individual fractions were analyzed for the distribution of RNA, PMR60°, and 40S ribosomal subunits as in (B) and (D). Transfected cells were treated with puromycin for 30 min prior to harvest and sedimentation on a gradient containing 5 mM MgCl2. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 2 Cytoplasmic Distribution of PMR1 (A) Cos-1 cells were transfected plasmid expressing myc-PMR60° and a plasmid expressing a Dbp5-EGFP fusion protein. Fixed cells were stained with monoclonal antibody to the c-myc epitope and DAPI and examined by confocal microscopy for Dbp5-EGFP (Aa), PMR60° (Ab), and DAPI (Ac). The merged images are presented in (Ad). Scale bar, 10 μm. (B) Cells were transfected as above with plasmid expressing myc-PMR60° and Dcp1-EGFP. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 3 N- and C-Terminal Domains of PMR1 Required for Targeting to Polysomes (A) Sequential 50 amino acid deletions were prepared from the N and C termini of PMR60° in a manner that retained the c-myc epitope tag on the N terminus. (B) Expression of each of the individual PMR60° deletions in transfected Cos-1 cells is shown by Western blot using the monoclonal antibody to the c-myc epitope tag. (C) Each of the N- and C-terminal deletions were cotransfected with full-length PMR60° into Cos-1 cells, and cytoplasmic extracts from transfected cells were separated on discontinuous 10% and 35% sucrose density gradients containing either 5 mM MgCl2 or 10 mM EDTA. The top, middle (mRNP), and bottom (polys) fractions were collected and analyzed by Western blot using the monoclonal antibody to the c-myc epitope tag. Full-length PMR60° is indicated on each blot with a diamond. The sedimentation of polysomes was confirmed by Western blot with antibody to ribosomal protein S6. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 4 Impact of a Targeting Domain Deletion on PMR1 Binding to Polysomes (A) Postmitochondrial extract of Cos-1 cells transfected with a plasmid expressing the Δ50C deletion of PMR60° was separated on linear 10%–40% sucrose gradients containing 5 mM MgCl2 (upper panels). (B) Cells transfected as in (A) were treated with puromycin for 30 min prior to harvest. (C) Extract from transfected cells was separated on a gradient containing 10 mM EDTA. Fractions were analyzed by Western blot for PMR60° using antibody to the c-myc epitope tag or for ribosomal protein S6. The arrow indicates the direction of sedimentation. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 5 Domains of PMR1 that Can Target GFP to Polysomes (A) A schematic is shown of constructs where the indicated amino acid residues from the N- or C-terminal portions of PMR60° were fused to the N or C termini of GFP, respectively. (B) Cos-1 cells were transfected with equal amounts of each of the GFP fusion proteins indicated in (A) and GFP. Postmitochondrial extracts were separated on discontinuous sucrose density gradients, and fractions collected from the top, interface (mRNP), and bottom (polys) were analyzed by Western blot using a polyclonal antibody to GFP. GFP is indicated with a filled circle. (C) Cos-1 cells transfected with PMR60°, Δ50C, GFP-50C, GFP-100C, or GFP alone were treated with puromycin for 30 min prior to harvest. Postmitochondrial extracts were separated on 10%–40% glycerol gradients, and individual fractions were analyzed by Western blot for PMR60° and Δ50C (upper panel) or for GFP fluorescence (lower panel). Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 6 PMR1 Forms a Specific Complex with Its Substrate mRNA (A) Full-length PMR60° and each of the deletion constructs from Figure 3 were fused at the C terminus with a tandem affinity (TAP) tag. These constructs or vector (pcDNA3) were transfected into Cos-1 cells together with plasmids expressing albumin mRNA and luciferase. The relative expression level of each of the fusion proteins in total cytoplasmic extract (input) was determined by Western blot using antibody to the c-myc epitope tag. These are denoted as PMR-TAP. Beneath this is a Western blot for luciferase (luc) expression. The relative amount of albumin and luciferase mRNA in each sample was determined by quantitative RT-PCR, and the products were separated on a denaturing 6% polyacrylamide/urea gel and quantified by phosphorimager analysis. The results were compared to a standard curve of serial dilutions of in vitro transcripts for each mRNA (indicated with a triangle). In this particular experiment, albumin mRNA yielded a doublet product. (B) Each of the cytoplasmic extracts in (A) was bound to IgG-Sepharose and eluted by Tev protease cleavage (bound). Because this step generates products with a C-terminal calmodulin binding protein tag these are denoted PMR-CBP. Recovered protein was analyzed by Western blot using the monoclonal antibody to the c-myc epitope tag, and albumin and luciferase mRNA recovered in each sample were analyzed by quantitative RT-PCR and phosphorimager analysis. The relative amount of PMR1-containing construct in each of the input and bound fractions was determined by scanning densitometry, and these data were used to determine the relative recovery of albumin mRNA by each of these. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 7 Endonuclease-Mediated Decay Requires Polysome Binding of Both PMR1 and Substrate mRNA (A) Cos-1 cells were transfected with catalytically active PMR60 or Δ50C PMR1, together with plasmids expressing luciferase and albumin mRNA, or albumin mRNA with a 24 bp 5′ stem-loop to block translation (SL-albumin). The relative expression of each protein in cytoplasmic extracts was determined by Western blot in the upper panels, and albumin and luciferase mRNA were quantified by RNase-protection assay (lower panel). (B) To test whether Δ50C PMR1 retained the activity of the parent protein, Cos-1 cells were transfected with C-terminal TAP fusions of both proteins or with GFP-TAP. Cells were treated with puromycin for 30 min prior to harvest and TAP-tagged protein was recovered from postmitochondrial extract (input) by binding to IgG-Sepharose and elution with Tev protease (bound). Western blots of input and bound protein are in the upper panel. Catalytic activity of the recovered proteins was assayed by incubation with 5′-[32P]labeled albumin mRNA substrate transcript for 0, 5, 10, and 30 min, followed by electrophoresis on a 6% polyacrylamide/urea gel. Input RNA is in lane 1, and lane 2 is RNA recovered following degradation by PMR1 present in Xenopus liver polysome extract. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)

Figure 8 Model for Endonuclease-Mediated mRNA Decay We propose that mRNAs are specifically targeted for degradation by exonuclease or endonuclease-mediated mRNA decay by their complement of specific mRNA binding proteins. mRNAs targeted for exonuclease-mediated mRNA decay first undergo deadenylation, which triggers the loss of ribosome binding and degradation either by the cytoplasmic exosome or by decapping and 5′-3′ decay in P bodies. In contrast, mRNAs that are targeted for endonuclease-mediated mRNA decay are bound by one or more proteins (shown in black) that recruit PMR1 to the ∼680 kDa polysome-bound substrate mRNP complex. Activation of the endonuclease in this context (lightning bolt) results in cleavage within the mRNA body without the need for prior deadenylation. The resulting decay intermediates are then most likely degraded by the exosome, with the scavenger decapping enzyme DcpS (Liu et al., 2002) acting to remove the cap from the 5′ limit digest. Molecular Cell 2004 14, 435-445DOI: (10.1016/j.molcel.2004.05.001)