1. Nonsense-Associated Altered Splicing
Jun Wang, Yao-Fu Chang, John I. Hamilton, Miles F. Wilkinson 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1
NAS Occurs by a Mechanism Independent of mRNA Stabilization in the Nuclear Fraction of Cells
(A) Diagram of full-length TCRβ precursor mRNA and normally spliced (norm) and alternatively spliced (alt-SA/SD) mRNAs derived from it. The alt-SA/SD mRNA is generated by use of an alternative splice acceptor (alt SA) and alternative splice donor (alt SD) previously described (Wang et al., 2002b).
(B) Ribonuclease protection analysis (RPA) of nuclear RNA (10 μg) from HeLa cell lines transiently transfected with construct A− (PTC−) or A+ (PTC+; UAG at codon 51) described in the Experimental Procedures. The alt-SA/SD and norm bands protected by the TCRβ probe were ∼84 nt and ∼72 nt, respectively, which are the sizes expected based on the position of the splice sites in the alt-SA/SD and norm mRNAs. TCRβ mRNA levels were determined by normalizing against the levels of neomycin (neo) mRNA, which was assessed in the same RPA as TCRβ mRNA. The level of alt-SA/SD mRNA in the PTC− sample was arbitrarily given a value of 1. Note that while norm mRNA levels were only reduced ∼7-fold in the nuclear fraction, they were decreased by ∼50-fold in the cytoplasmic fraction (unpublished data).
(C) Alt-SA/SD mRNA half-life was determined by RPA of nuclear, nuclear wash, and cytoplasmic RNA (10 μg) from HeLa cell lines stably transfected with TCRβ constructs (BM− [PTC−] and B+ [PTC+]) and incubated with 5 μg/ml actinomycin D for the lengths of time shown. TCRβ mRNA levels were determined by normalizing against the levels of β-actin mRNA. The values represent the mean of three independent experiments; error bars indicate standard error.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 Depletion of hUPF2 Reverses NMD but Not NAS
RPA of total cellular RNA (10 μg) from HeLa cell lines transiently transfected with TCRβ constructs A− (PTC−) or C+ (PTC+) and an antisense-hUPF2 (as-hUPF2) expression plasmid (0, 0.2, 0.4, and 0.8 μg in lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8, respectively). TCRβ mRNA levels were determined by normalizing against the levels of β-globin mRNA, which was from an expression plasmid also cotransfected with the TCRβ plasmids.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Engagement of NAS by the Kozak AUG Upstream of the Alternatively Spliced Intron but Not by the AUG Downstream of the Intron
(A) Upper panel: schematic of TCRβ precursor mRNA showing the position of the two AUGs; AUG1 is the normal initiator that generates full-length TCRβ protein. Lower panel: RPA of total cellular RNA (10 μg) from HeLa cell lines transiently transfected with the TCRβ constructs shown. TCRβ mRNA levels were determined by normalizing against the levels of neo mRNA as described in Figure 1.
(B) Upper panel: the sequences surrounding AUG1 in the mutated constructs. Construct A− has the wild-type (Wt) sequence, and constructs F−/F+ and G−/G+ have mutations that improve or reduce the match with the Kozak consensus sequence (R = A or G), respectively. Lower panel: RPA of total cellular RNA (10 μg) performed and quantitated as in (A).
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
NAS versus NMD
(A) Schematic of precursor mRNA from the TCRβ gene and the alt-SD transcript derived from it. The alt-SD transcript has a frameshift (FS) that generates the in-frame nonsense codons shown.
(B) RPA of total cellular RNA (10 μg) from HeLa cell lines transiently transfected with TCRβ constructs with nonsense, missense, and silent mutations at the positions depicted in (A). The constructs used for lanes 1 to 9 are A−, D+, D+, DM−, C+, CM1−, CM2−, B+, and BM−, respectively. TCRβ mRNA levels were determined by normalizing against the levels of neo mRNA as described in Figure 1.
(C) RPA of total cellular RNA (10 μg) from HeLa cell lines stably transfected with TCRβ constructs A− (PTC−) or B+ (PTC+) and incubated with or without 10 μg/ml cycloheximide (CHX) for 6 hr. TCRβ mRNA levels were determined by normalizing against the levels of β-actin mRNA.
Molecular Cell  , DOI: ( /S (02) )