1. Nonsense-Mediated mRNA Decay in Mammalian Cells Involves Decapping, Deadenylating, and Exonucleolytic Activities 
Fabrice Lejeune, Xiaojie Li, Lynne E. Maquat 
Molecular Cell 
Volume 12, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Figure 1
SiRNA-Mediated Downregulation of Dcp2, PM/Scl100, or PARN Increases the Level of Nonsense-Containing Gl mRNA, which Is Subject to Nucleus-Associated NMD, as well as the Level of Nonsense-Containing GPx1 mRNA, which Is Subject to Cytoplasmic NMD
(A) HeLa cells (2 × 106) were transiently transfected with 180 ng of a polIII promoter-containing PCR fragment that directs the synthesis of Dcp2 siRNA in vivo, or 600 pmol of in vitro-synthesized PM/Scl100 siRNA or a nonspecific Control siRNA. Cells were retransfected 4 days later with the same PCR fragment or siRNA, 3 μg of a pmCMV-Gl test plasmid, and 3 μg of a GPx1 test plasmid, either nonsense-free (Norm) or nonsense-containing (39Ter or 46Ter, respectively), and 1 μg of the reference phCMV-MUP plasmid. Cells were harvested after an additional 2 days. Protein was purified from half of the cells, and RNA was purified from nuclear and cytoplasmic fractions of the remaining cells. Upper: Western blot analysis of total-cell protein (20 μg) demonstrates that siRNA downregulated the level of Dcp2 or PM/Scl100 to ∼35% of normal when Upf3X was used to control for differences among lanes in protein loading. The three or four leftmost lanes, which consist of 2-fold dilutions of cellular protein, indicate that the analysis is semiquantitative. Middle: RT-PCR analysis of nucleus-associated Gl and MUP mRNAs. The leftmost four lanes, which analyze decreasing amounts of RNA, demonstrate that the conditions of RT-PCR were quantitative. Numbers below the figure represent the level of Gl mRNA normalized to the level of MUP mRNA, where the normalized level of nonsense-free (Norm) mRNA in the presence of each siRNA is defined as 100%. Lower: RT-PCR analysis of cytoplasmic GPx1 and MUP mRNAs, essentially as described in middle.
(B) HeLa cells (2 × 106) were transiently transfected with 600 ng of a polIII promoter-containing PCR fragment that directs the synthesis of PARN siRNA in vivo or a nonspecific Control siRNA. Upper: As in ([A], upper), except that the level of PARN was shown to be downregulated by siRNA to ∼15% of normal. Middle and lower: As in ([A], middle and upper). All results are representative of at least three independently performed experiments.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
SiRNA-Mediated Downregulation of Dcp2 or PM/Scl100 Decreases the Rate at which fos-Gl 39Ter mRNA Undergoes NMD
L cells (4 × 106) were transiently transfected as described in the legend to Figure 1. However, the test plasmid was either pfos-Gl Norm or pfos-Gl 39Ter. Furthermore, serum was removed 12 hr after the second transfection. After an additional 24 hr, serum was added to 15%. Protein was purified from the cytoplasmic fraction, and RNA was prepared from the nuclear fraction at the specified times thereafter.
(A) Western blot analysis of cytoplasmic protein (10 μg) at 0 min after serum addition demonstrates that siRNA downregulated the level of Dcp2 or PM/Scl100 to, respectively, <10% or ∼40% of normal. For each analysis, the level of Upf3X was used to control for differences between samples in protein loading.
(B) RT-PCR analysis of levels of nucleus-associated fos-Gl and MUP mRNAs demonstrates that downregulation of Dcp2 or PM/Scl100 increases the half-life of fos-Gl 39Ter mRNA. The three or four leftmost lanes analyzing decreasing amounts of RNA demonstrate that the RT-PCR conditions are quantitative. For each time point, the level of fos-Gl mRNA was normalized to the level of MUP mRNA. Normalized levels were calculated as a percentage of the normalized level of either fos-Gl Norm mRNA (for both Norm and Ter mRNAs) or fos-Gl 39Ter mRNA (for Ter mRNA) at time 0, which was defined as 100, and is specified below each RT-PCR analysis. To the right of each RT-PCR analysis, normalized levels are plotted as a function of time after serum addition.
(C) Comparison of the relative rates of fos-Gl Norm mRNA (left) or fos-Gl 39Ter mRNA decay (right) in the presence of Control, Dcp2, or PM/Scl100 siRNA using data obtained in (B).
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
PM/Scl100 Localizes to Both Nuclei and Cytoplasm of HeLa and 293T Cells Using Indirect Immunofluorescence and Confocal Microscopy
(A) Western blotting demonstrating that the immunoreactivity of HeLa cell and 293T cell extracts with each antibody used for immunolocalization is specific to the protein of interest. The molecular weights of standards are shown to the left of each Western blot.
(B) Immunolocalization of PM/Scl100 and, as a control, Rrp4 in HeLa cells. For each antibody, panels represent the following: (a) differential interference contrast, (b) staining of nuclei by DAPI, (c) immunofluorescence of each antibody, and (d) overlay of (b) and (c). Vertical arrows specify exemplary nucleoli, and horizontal arrows specify exemplary cytoplasm. NRS, normal rabbit serum.
(C) Same as (B), only 293T cells were analyzed.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Upf1, Upf2, and Upf3X Coimmunopurify with Components of the 5′→3′ and 3′→5′ mRNA Decay Pathways
Proteins and RNP from 3 × 107 untransfected Cos cells were subjected to immunopurification (IP) using total-cell extract and anti-Upf1, anti-Upf2, or anti-Upf3/3X antibody or, as a control for nonspecific IP, normal rabbit serum (NRS). Proteins present in each IP were then analyzed by Western blotting using the antibody specified to the right of each panel. The three leftmost lanes analyze decreasing amounts of protein before IP and demonstrate that the conditions used for Western blotting are semiquantitative. IP efficiencies were 10% for Upf1, 10% for Upf2, and 15% for Upf3X. Notably, the comigration of Upf3 with antibody heavy chain precludes its detection. Results are representative of three independently performed experiments in that all degradative proteins that were assayed were invariably detected in all IPs, albeit with efficiencies that sometimes varied between IPs.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
The 5′ End of Transiently Induced fos-Gl 39Ter mRNA Is Degraded at a Rate that Is Comparable to the Decay Rate of Full-Length mRNA and Is Comparable or Slightly Faster Than the Decay Rate of the 3′ End
(A) The NMD of serum-induced fos-Gl 39Ter mRNA is evident after 30 min of serum addition to serum-deprived L-M(TK−) cells. L cells were transiently transfected and analyzed as described in the legend to Figure 2 except siRNA was omitted. Error bars specify the extent of deviation among three independently performed experiments.
(B) RT-PCR analysis of the relative rates of fos-Gl Norm and 39Ter mRNA 5′-end and 3′-end decay. As in (A), however, the levels of fos-Gl mRNA 5′ and 3′ ends were analyzed as 94 and 110 bp RT-PCR products, respectively, and data were plotted on a linear scale.
(C) Comparison of the relative rates of fos-Gl 39Ter 5′- and 3′-end decay using data obtained in (B).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
The NMD of fos-Gl 39Ter mRNA Is Not Accompanied by Detectable Deadenylated Intermediates
L cells were transfected as in Figure 5. However, cells were harvested at different times after serum addition, and both nuclear and cytoplasmic RNA was prepared.
(A) Nuclear RNA was analyzed by RT-PCR as described in Figure 5A.
(B) Nuclear RNA (4 μg) was analyzed by Northern blotting. The lengths of the poly(A) tails on newly synthesized fos-Gl Norm and Ter mRNAs were assessed relative to the lengths of the poly(A) tail on of steady-state Gl Norm and Ter mRNAs (i.e., Gl mRNA driven by the mouse CMV promoter) in 2 μg total-cell RNA before (A+) and after (A−) deadenylation in vitro using 0.2 μg of oligo (dT) and 0.1 U of RNase H. A lighter Phosphorimager exposure is shown for lanes containing pmCMV-Gl mRNA (total RNA) relative to fos-Gl Norm and Ter mRNAs (nuclear RNA) even though all RNAs were analyzed in the same gel. MW standards are shown to the left.
(C) Cytoplasmic RNA (4 μg) was analyzed as in (B).
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Model for the Polarity and Enzymology of NMD in Mammalian Cells
NMD takes during what is called the “pioneer” round of translation (Ishigaki et al., 2001; Lejeune et al., 2002). The pioneer translation initiation complex involves mRNA bound by CBP80/20 at the 5′ cap, PABP2 at the 3′ poly(A) tail, and an exon junction complex (EJC) that is deposited as a consequence of pre-mRNA splicing at each exon-exon junction. The EJC recruits Upf3 or 3X, each of which is a mostly nuclear shuttling protein involved in NMD (Lykke-Andersen et al., 2000; Serin et al., 2001). Upf3/3X then recruits Upf2, another protein involved in NMD that appears concentrated on the cytoplasmic side of the nuclear envelope (Lykke-Andersen et al., 2000; Serin et al., 2001). Upf1, a group 1 RNA helicase that is mostly cytoplasmic but also nuclear (Mendell et al., 2002), is not detected as an integral mRNP protein but presumably triggers NMD by interacting with EJC-bound Upf2 when translation terminates at a premature termination codon (Pre Ter) that is located more than 50–55 nucleotides upstream of the corresponding exon-exon junction. Data indicate that NMD initiates from both the mRNA 5′ end so as to involve decapping followed by 5′→3′ exonucleolytic decay and the mRNA 3′ end so as to involve deadenylation followed by 3′→5′ exonucleolytic decay, which may or may not take place on the same molecule. It is currently not possible to determine the relative efficiencies of 5′→3′ and 3′→5′ decay during NMD.
Molecular Cell  , DOI: ( /S (03) )